Nucleic Acid Based Products and Services K= Ordering Information

Online Catalog

2010 2010 Nucleic Acid Based Products and Services k= Ordering Information

800-863-6801 [email protected] www.trilinkbiotech.com

All oligonucleotide sequences must be submitted electronically, either by email or online. Prices do not include shipping charges or applicable taxes. TriLink reserves the right to modify prices at any time or refuse to accept any order.

Payment We accept payment by purchase order, wire transfer and credit card (MasterCard, Visa and American Express). Net payment is 30 days for open accounts in good standing. All first time customers are required to submit a credit application before receiving their first order.

Distributors For a complete list of distributors, please see inside back cover.

Delivery All products are shipped by overnight delivery to ensure quality. Within the US, shipments typically arrive by 10:30 am the following business day. Orders valued at $5000 or more are shipped with additional insurance. Products with stability concerns or in solution will be shipped on dry ice. F.O.B. San Diego.

Shipping Charges Within the US standard shipping is approximately $15. International shipments range from $40 to $60. If your order requires dry ice or insurance, additional charges will apply. Please inquire for pricing specific to your location and order.

Quotations Quotations for bulk syntheses, custom compounds and research services may be requested. To estimate oligonucleotide pricing see page 9. Direct inquires to [email protected].

Technical Support Our sales team is committed to fully supporting TriLink's products and services from initial customer inquiry to post delivery technical assistance. The answers to many common questions can be found online in our FAQ database. Our technical support team is available to speak with you Mon-Fri, 7am to 5pm PST. 800-863-6801 www.trilinkbiotech.com/faqs.asp

Signature TriLink Quality TriLink has over 12 years of experience in making specialty modified oligonucleotides and nucleoside triphosphates. We believe it is our responsibility to work with our customers to overcome technical barriers. Consequently, we often prepare compounds that have not been made before or are not well established in the literature. Our extensive experience with unique modifications and our strong commitment to quality result in the best products available. FSC Label – Portrait Artwork Matrix

FSC Label Artwork FSC_Labels_English FSC_Labels_Portrait

FSC_Labels_PPC FSC_Labels_PNC FSC_Labels_PPBW FSC_Labels_PNBW Portrait / Positive / Colour (PPC) Portrait / Negative / Colour (PNC) Portrait / Positive / Black & White (PPBW) Portrait / Negative / Black & White (PNBW) Nucleic Acid Based

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Cert no. XXX-XXX-XXX Cert no. XXX-XXX-XXX Cert no. XXX-XXX-XXX Cert no. XXX-XXX-XXX 2010

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XX% XX% XX% XX% FSC_MS_2_PPC.EPS FSC_MS_2_PNC.EPS FSC_MS_2_PPBW.EPS FSC_MS_2_PNBW.EPS FSC_MS_2_PPC.JPG FSC_MS_2_PNC.JPG FSC_MS_2_PPBW.JPG FSC_MS_2_PNBW.JPG FSC_MS_2_PPC.TIF FSC_MS_2_PNC.TIF FSC_MS_2_PPBW.TIF FSC_MS_2_PNBW.TIF

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Quick Guide FSC_MS_4_PPC.EPS FSC_MS_4_PNC.EPS FSC_MS_4_PPBW.EPS FSC_MS_4_PNBW.EPS FSC_MS_4_PPC.JPG FSC_MS_4_PNC.JPG FSC_MS_4_PPBW.JPG FSC_MS_4_PNBW.JPG • Oligonucleotides . . . 8 FSC_MS_4_PPC.TIF FSC_MS_4_PNC.TIF FSC_MS_4_PPBW.TIF FSC_MS_4_PNBW.TIF • Nucleotides . . . 50 TM Cert no. XXX-XXX-XXX Cert no. XXX-XXX-XXX Cert no. XXX-XXX-XXX Cert no. XXX-XXX-XXX • CleanAmp PCR Products . . . 82 • Technical Information . . . 102 • Index . . . 156

XX% XX% XX% XX% FSC_MS_5_PPC.EPS FSC_MS_5_PNC.EPS FSC_MS_5_PPBW.EPS FSC_MS_5_PNBW.EPS FSC_MS_5_PPC.JPG FSC_MS_5_PNC.JPG FSC_MS_5_PPBW.JPG FSC_MS_5_PNBW.JPG FSC_MS_5_PPC.TIF FSC_MS_5_PNC.TIF FSC_MS_5_PPBW.TIF FSC_MS_5_PNBW.TIF

Cert no. XXX-XXX-XXX Cert no. XXX-XXX-XXX Cert no. XXX-XXX-XXX Cert no. XXX-XXX-XXX Cover Image: La Jolla, California © istockphoto.com/hsandler Cover Image: La

FSC_RE_1_PPC.EPS FSC_RE_1_PNC.EPS FSC_RE_1_PPBW.EPS FSC_RE_1_PNBW.EPS FSC_RE_1_PPC.JPG FSC_RE_1_PNC.JPG FSC_RE_1_PPBW.JPG FSC_RE_1_PNBW.JPG FSC_RE_1_PPC.TIF FSC_RE_1_PNC.TIF FSC_RE_1_PPBW.TIF FSC_RE_1_PNBW.TIF

Cert no. XXX-XXX-XXX Cert no. XXX-XXX-XXX Cert no. XXX-XXX-XXX Cert no. XXX-XXX-XXX Dear Colleagues,

We have grown so much over the past year! We have added nearly 40 new members to the TriLink family, almost doubling our size. It is your continued success that made this possible. We are very appreciative of the opportunities you gave us and, of course, the second chances when needed – this is a difficult science we practice. When we started TriLink, one of our stated objectives was to become our customers’ de facto chemistry group. We wanted to be a member of your team. In many ways, we have succeeded in that goal for quite a number of you. I hope you have come to appreciate our commitment to overcoming your technical challenges, as much as we enjoy solving them.

Responding to our customer’s needs, TriLink has vastly increased our synthesis capacity. We added a number of large-scale synthesizers, substantially increasing our 1-5 mmole scale capacity. We also added several 10-100 µmole scale mid-scale instruments and we even added a high throughput small-scale instrument. Naturally these were matched with the required number of HPLC purification instruments, lyophilization devices and QC capacity. We are grateful to be in a position where expansion is required.

We have also made significant advancements in product development. Our CleanAmp™ Primers, which were introduced in March of 2008, are now joined by our CleanAmp™ dNTPs. We are very proud of these two products. We believe they will have a very positive impact on the PCR field and become useful products in the years to come. We have several CleanAmp™ technology publications already out, and more that will be published soon. More CleanAmp™ Product information can be found in the orange tab section of this catalog.

Please take the time to peruse all our products and capabilities. We included all the technical information you have come to expect from a TriLink catalog, adding a number of articles about our new CleanAmp™ technologies. Even if you do not need one of our products today, you may find a useful piece of information inside that will help your research. We will be here when you need us, probably even bigger than we are today, and possibly even better able to serve you. See you then!

San Diego, California © Tim Banham

Best regards,

Richard Hogrefe, Ph.D. CEO/President

TriLink was founded in 1996 to meet a growing need in the marketplace for quality modified oligonucleotides. Still owned and managed by two of the original founders, TriLink now leads the industry in unique modified nucleic acids and mid-scale oligonucleotide synthesis. 4 4 Table of Contents Table ofContents Stocked Randomer Oligonucleotide ProductsOligonucleotide Randomer Stocked Spacers Linkers...... Inverted Oligonucleotides Inverted Non-Fluorescent Conjugates Non-Fluorescent Quenchers...... Labeling ...... Dye Fluorescent Oligonucleotide Conjugates Oligonucleotide Oligonucleotides ...... RNA Fluoro 2' and Methylphosphonate Oligonucleotides ...... RNA Modifications Standard Oligonucleotide Standard DNA Oligonucleotides DNA Standard Kits ...... Nucleotide Monophosphates Nucleoside Labeled TriphosphatesNucleoside Modified Phosphate Alpha mCAP ...... and CAP Nucleosides ...... Bisphosphate TriphosphatesNucleoside Modified Sugar TriphosphatesRibonucleoside Modified Base TriphosphatesDeoxynucleoside 2' Modified Base Primer Grade DNA Oligonucleotides DNA PrimerGrade PricingBeacon Molecular Synthesis Oligonucleotide NTP Quick Guide Quick NTP CAP and mCAP and CAP TriphosphatesNucleoside Fluoro 2' TriphosphatesNucleoside Biotin ...... Overview Beacon Molecular Aminoallyl Nucleoside TriphosphatesNucleoside Aminoallyl FeaturedProducts Services ...... Synthesis Custom ProductsNucleotide ...... of Overview 3. Nucleotides Synthesis Oligonucleotide Mid-Scale Services ...... Oligonucleotide Therapeutic ValidatedNonamers RandomNimbleGen Roche Services Oligonucleotide Diagnostic/OEM FeaturedProduct ProductsOligonucleotide ...... of Overview 2. Oligonucleotides Services ...... Contract and Chemistry Custom TriLinkService ...... Signature and Quality Letter fromthePresident The TriLink Team 1. AboutTriLink BioTechnologies Dimers andTrimers ...... Modified Bases...... Terminal Phosphates ...... www.trilinkbiotech.com ...... 48 47 38 37 34 33 33 31 30 30 20 20 19 18 17 16 75 78 77 74 74 67 62 56 54 15 14 53 53 52 52 13 51 51 12 11 10 3 9 7 6 2

141 140 139 139 138 135 134 132 130 128 126 120 153 147 152 150 154 153 153 156 103 119 117 116 114 112 109 93 90 88 86 84 83 99

Table of Contents of Table Table of Contents of Table 5 5 6 6 About TriLinkTable BioTechnologies of Contents • Please contactustodiscuss yourspecificmanufacturingneeds. • • • Program GuidelinesInclude: • • • Benefits ofManufacturingunderQSR: (QSR) andtheISO1345standard. needs. product Wespecific your will for then materials manufacture and processes your validate product and under document develop, a to quality you system with based work TriLinkwill on the FDA's Quality System Regulations Manufacturing cGMP and QSR shipment. before correct be to verified and Specialist Assurance Quality a by reviewed is analysis of TriLinkcertificate Every and product. product each with provided analysis of certificate the in incorporated then are analyses QC The specifications. release our meets product the ensure to analysis, PAGEMS a and typically testing, analytical TriLinkfinal All undergo products Assurance Quality synthesis anddevelopthebestpossibleproducts. management, manufacturingandqualityassuranceteamsworktogethertounderstandthechallengesofeach ity toworkwithourcustomerspushpasttechnicalbarriersandcreatetheprogramstheyneed.Ourproduct Wesmall andmid-scales. operateaGMPlaboratorywithQSRenvironment.We believeitisourresponsibil- TriLink isanindustryleaderinmanufacturinghighqualityoligonucleotidesand nucleosidetriphosphatesat • Controlled accesstodedicatedlaboratories Supplier validation&traceablerawmaterial SOPs ¢ralrecordshandling All personneltrainedinQSRcompliance Ultra-filtration desaltingavailable Optimized purificationprotocols Lot tolotconsistency Multi-gram batches Signature TriLinkService Signature and Quality

www.trilinkbiotech.com support to the science that supports us. supports that science the to support TriLinkway giving more is one just is Program,which ResearchRewardsGrant our for 153 page see Please products. successful develop clients our helping experience of years many and department research a Wehave companies. other and researchers academic with efforts collaborative developing in interested very Weare Collaborations Nucleoside Triphosphates, phosphoramiditesandmore • Fixed weeklyratesavailable • Oneon one interactionwithascientist • Developmental workonchallengingcompounds • Contract researchanddevelopmentprojectfeatures: QSRcGMP laboratoryfacilitiesavailable • Bulkoligo andnucleosidesynthesis • Modification ofnucleotides • Synthesis ofspecialtyoligonucleotides • We provideawiderangeofservicesincluding: di-,andtriphosphates. and nucleosidemono-, oligonucleotide therapyanddiagnostics.We offercustomsmallmoleculesynthesisincludingphosphoramidites TriLink offerscuttingedgeservicestoresearchersinthefieldsofgenetherapy, nucleosidechemotherapy, Custom ChemistryandContractServices [email protected] 800 863-6801 La Jolla, California © istockphoto.com/Kazmaniac © California Jolla, La

Table of Contents of Table About TriLink BioTechnologies TriLink About 7 7 Overview of Oligonucleotide Products

Custom Oligonucleotide Synthesis

You Design. We Synthesize.

With our expertise and extensive selection of dyes, quenchers, modified bases, linkers, spacers and other Oligonucleotides conjugates, your creativity is the limit. TriLink specializes in challenging constructs such as oligonucleotides with numerous modifications, molecular beacons and probes with multiple modifications.

Consistently producing unique, superior grade oligonucleotides is an art form. Automated systems cannot recognize the nuances of distinct sequences and dye sets. Our skilled technicians isolate each compound using protocols designed to ensure we deliver the highest purity and yield for each oligonucleotide. By employing a hands-on approach, we offer a scalable process with unsurpassed quality and exceptional value.

All of these reasons make us a natural choice for oligonucleotide kit suppliers and testing facilities as well as pharmaceutical, biotech and academic researchers. Oligonucleotides

Simple Oligonucleotide Pricing

La Jolla, California © istockphoto.com/ddbell Estimating the price of your oligonucleotide is easy as 1, 2, 3. 1. Backbone Overview of Oligonucleotide Products ...... 9 Choose your backbone. See pages 16-19 for per base pricing at your desired scale. Featured Product Diagnostic/OEM Oligonucleotide Services...... 10 2. Modifications Roche NimbleGen Validated Random Nonamers ...... 11 Determine the modifications required. See pages 20-29 for fluorescent dye labeling, pages 38-46 Therapeutic Oligonucleotide Services ...... 12 for base modifications and pages 31-37 for other conjugates, linkers and spacers. Don't see the Mid-Scale Oligonucleotide Synthesis ...... 13 modification you need? Please inquire. TriLink also offers custom phosphoramidite synthesis, Molecular Beacon Overview...... 14 see page 51. Molecular Beacon Pricing ...... 15 Oligonucleotide Synthesis 3. Purification Primer Grade DNA Oligonucleotides...... 16 Decide on a purification method. See pages 16-19 for pricing corresponding to the backbone of your Standard DNA Oligonucleotides ...... 17 oligonucleotide. Not sure which one to choose? Simply request a quote and indicate "Best Method" or Standard RNA Oligonucleotides ...... 18 ask our technical support team. Methylphosphonate and 2' Fluoro RNA Oligonucleotides ...... 19 Oligonucleotide Modifications For a formal quotation, please email [email protected]. Should your construct require special Oligonucleotide Conjugates ...... 20 processing or an unusual chemistry, additional charges may apply. Mid-scale oligonucleotide and Molecular Fluorescent Dye Labeling ...... 20 Beacon pricing can be found on pages 13 and 15, respectively. TriLink reserves the right to change pricing at Quenchers ...... 30 any time. Non-Fluorescent Conjugates...... 31 Inverted Oligonucleotides...... 33 Terminal Phosphates...... 33 Linkers ...... 34 Spacers...... 37 Modified Bases ...... 38 Dimers and Trimers ...... 47 Stocked Randomer Oligonucleotide Products...... 48

8 www.trilinkbiotech.com [email protected] (800) 863-6801 99 Featured Product: Diagnostic/OEM Oligonucleotide Services Featured Product: Roche NimbleGen Validated Random Nonamers

Oligonucleotides for Diagnostics/OEM Roche NimbleGen Validated Random Nonamers

Designing and developing specifications for your diagnostic kit components can be an impossible challenge Randomers have become an essential tool in diagnostic research. They can help identify a gene that codes for without a nucleic acid chemist on your team. TriLink has over 13 years experience applying unique nucleic acid a protein of interest when the specific sequence is unknown or amplify a genetic target to generate a pool of chemistry expertise to our customers’ technical barriers. all available sequences. Commercial assays have been developed that use randomers to detect a single base mismatch polymorphism, as well as map chromosomal differences, most commonly deletions. These sensitive applications require oligos of random sequence, consistant base composition and minimal lot to lot variability. Your Perfect Partner TriLink has done extensive research and development to ensure our randomer oligos meet these crucial Oligonucleotides We will work closely with you to tailor the manufacturing process to specifications. See our Randomer Oligonucleotide article in the Technical Information Section. We stock the your specific program needs. Our extensive research in randomization more commonly requested randomers, as well as provide custom randomer synthesis. Please contact our sales optimization for the Roche NimbleGen platform is a great example of team for more information. how we can make your product even better. Our process development TM and scale-up research in support of the ABI SOLiD System Each and every lot of our validated products are tested by Roche NimbleGen prior to release. This is to ensure oligonucleotides is another example of what our technical expertise that they pass Roche NimbleGen's specifications for use in their array systems. TriLink is the only outside supplier can do for you. To learn how our ingenuity can work for you, please of Roche NimbleGen validated oligonucleotides at this time. We are also the only supplier of pack sizes above contact our technical support team. 2 OD260 units, including Roche NimbleGen.

Roche NimbleGen Validated Random Nonamers

Oligonucleotides 5' Cy Labeled Random Nonamers A Customized Program Sold as a set: 1 vial of 5' Cy3 and 1 vial of 5' Cy5 Use: 2-color array systems Whether you are ordering your first oligonucleotide or are ready to set up a full supply program, TriLink is the right supplier for you. Material can be ordered in nmoles all the way up to multiple grams. We offer several 1 OD260 units of each N46-0010-01 $100 2 OD260 units of each N46-0010-02 $150 levels of purification to meet your needs. Our crude material ranges from 60% - 80% pure. We will not ship any 25 OD260 units of each N46-0010-25 $800 purified material unless it meets our 85% purity minimum without your request and approval. Greater purity can 50 OD260 units of each N46-0010-50 $1500 be achieved with additional purification steps at your request. 100 OD260 units of each N46-0010-100 $2600 200 OD260 units of each* N46-0010-200 $4200

QC Analyses Available Additional Services Available • PAGE analysis & gel densitometry • Custom labeling & documentation Roche NimbleGen Validated Unmatched 5' Cy3 Labeled Random Nonamers • Mass spectroscopy • Label control • RP/AX-HPLC analysis • Research & development 5' Cy3 Labeled Random Nonamers • Synthesis report • Manufacturing process optimization Use: 1-color array systems • Other assays available • Custom phosphoramidite synthesis

1 OD260 units N46-0007-01 $50 2 OD260 units N46-0007-02 $75 25 OD260 units N46-0007-25 $400 50 OD260 units N46-0007-50 $750 100 OD260 units N46-0007-100 $1300 200 OD260 units* N46-0007-200 $2100 QSR Manufacturing

Every product made at TriLink is manufactured under QSR, whether it is for diagnostics, therapeutics or research. Roche NimbleGen Validated Random 7mers At TriLink our top priority has always been manufacturing the highest quality products for our customers. We will work with you to develop, document and validate processes and materials for your specific program. We will 5' Cy Labeled Random 7mers then manufacture your product under a quality system based on QSR and the ISO 1345 standard. Sold as a set: 1 vial of 5' Cy3 and 1 vial of 5' Cy5 Use: short labeling in CHiP-chip array systems Benefits of Manufacturing under QSR: Program Guidelines Include: • Manufacturing process validation 25 OD260 units of each N46-0011-25 $800 • All personnel trained in QSR compliance 50 OD units of each N46-0011-50 $1500 Optimized synthesis & purification protocols 260 • • SOPs & central records handling 100 OD260 units of each N46-0011-100 $2600 • Locked process & specification documentation • Supplier validation & traceable raw material 200 OD260 units of each* N46-0011-200 $4200 • Separate batch record for each compound • Controlled access to dedicated laboratories • Dedicated program review *Larger quantities available, please inquire.

10 www.trilinkbiotech.com [email protected] (800) 863-6801 1111 Featured Product: Therapeutic Oligonucleotide Services Featured Product: Mid-Scale Oligonucleotide Synthesis

Therapeutic Oligonucleotide Services Mid-Scale Oligonucleotide Synthesis

TriLink provides small-scale oligonucleotides for the discovery and pre-clinical stages of drug development to TriLink specializes in mid-scale synthesis of both DNA and RNA oligonucleotides. Quantities range from 50 mg customers exploring a wide variety of DNA or RNA based therapies. Service options include synthesis in a QSR to 10 g. If you require more than 10 g, we recommend our alliance partner Avecia. All products are analyzed environment and full document control. by PAGE, HPLC and MS. Turn around time varies based on sequence, purification and quantity requested. Additional services include modifications, process development and cGMP/QSR manufacturing. QSR & Document Control Oligonucleotides

We will work with you to develop, document and validate processes and materials for your specific product needs. DNA Pricing Your product will then be manufactured in our laboratories, which are dedicated to production under a quality 50 mg $1,500 1.0 g $5,500 system based on QSR and the ISO 1345 standard. 100 mg $1,825 1.25 g $6,600 Documentation under QSR includes: 150 mg $2,075 1.5 g $7,350 • Standard operating procedures specific to your compound and specifications 200 mg $2,350 2.0 g $9,150 • Central records handling and traceable batch records 250 mg $2,450 3.0 g $12,375 • Supplier validation and traceable raw material 300 mg $2,700 5.0 g $17,500 Conduct your pre-clinical studies with the confidence that your oligonucleotide will comply with FDA guidelines. 500 mg $3,500 10.0 g $30,000

Oligonucleotides 750 mg $4,450

Prices are for unmodified oligonucleotides between 15 and 25 bases. It includes purification and diafiltration. Please request a quotation for other lengths or different quantities. The backbone may consist of phosphorothioate or The TriLink-Avecia Alliance phosphodiester linkages, or a combination of the two. Prices may vary due to sequence composition. From discovery to the clinic

The TriLink-Avecia Alliance allows easy transition from R&D RNA Pricing and pre-clinical studies to clinical trials and commercial manufacturing of therapeutic oligonucleotides. Set pricing for mid-scale RNA oligonucleotide synthesis is not available. The yield and quality of these syntheses are highly dependent on sequence. Please contact our technical support team to request a custom quotation. Working with the alliance minimizes your challenges and risks associated with transitioning from a research-grade vendor to an oligonucleotide development service and manufacturing company. We have harmonized our processes, so you can enjoy a smooth changeover when scaling up for clinical trials. The analysis below shows an AX-HPLC run on a mid-scale phosphodiester DNA oligonucleotide. For more information on the alliance go to our websites: The oligonucleotide is 34mer, HPLC purified.

www.trilinkbiotech.com/alliance Multi-Chrom 1 (1 : 260nm, 4nm) Pump Gradient B www.aveciabiotech.com/oligomedicines Retention Time % mAU

Minutes

1 : 260 nm 4nm Results

Retention Time Area Area Percent

34.15 158009 3.70

34.57 4072665 95.47

35.17 35063 0.82

Totals 4265737 100.00

12 www.trilinkbiotech.com [email protected] (800) 863-6801 1313 Featured Product: Molecular Beacon Overview Featured Product: Molecular Beacon Pricing

Molecular Beacon Overview Molecular Beacon Pricing

In 1996 Tyagi and Kramer described a novel diagnostic assay utilizing oligonucleotides with a fluorescent Targets for Molecular Beacons (0.2 umole scale, crude preparations) are available at no extra charge upon request. dye at one terminus and a quenching dye at the other. These "molecular beacons" formed a hairpin when Limit one primer pair per Molecular Beacon. Prices are listed below for beacons up to 40 bases in length. Please not hybridized to a target, thus placing the 5' and 3' termini, and their attached dyes, in close proximity. inquire for (1) pricing regarding QSY quenchers (QSY-7®, QSY-9®, QSY-21®, QSY-35®), (2) larger scale syntheses or The fluorescent dye was quenched, yielding no signal. However, upon hybridization of the probe to the (3 )other fluorophores and quenchers. target, the dyes were separated and fluorescence was detected. (Tyagi, S., and Kramer, F.R. (1996) Nature Biotechnology, 14, 303-308.) Pricing Oligonucleotides In order for the assay to work correctly the oligonucleotide conjugate must be of the highest quality as is expected from TriLink. We analyze the purity of every intermediate and pool only the purest fractions for your final product. 5' Fluorophore 3' Quencher 0.2 umole scale 1.0 umole scale without a licence* with a license* expected yield without a licence* with a license* expected yield

Carboxy-X-RhodamineTM Dabcyl or BHQ-2® $600 $490 1-3 ODs $700 $575 3-15 ODs CoumarinTM Dabcyl or BHQ-1 or 2® $600 $490 1-3 ODs $700 $575 3-15 ODs Schematic of Multi-allele Detection System using Molecular Beacons Cy3TM Dabcyl or BHQ-2® $600 $490 1-3 ODs $700 $575 3-15 ODs G T A C Cy3.5TM Dabcyl or BHQ-2® $750 $615 1-3 ODs $850 $700 3-15 ODs

TM ® G Cy5 Dabcyl or BHQ-2 $600 $490 1-3 ODs $700 $575 3-15 ODs

Q Q Q Q Cy3 Cy5 CB Fl TM ® Oligonucleotides Dabcyl or BHQ-2 $750 $615 1-3 ODs $850 $700 3-15 ODs Cascade BlueTM Fluorescein Cy 3 TM Cy 5 TM Cy5.5 DNA Pool Containing One of 4 Alleles Em = 410 nm Em = 520 nm Em = 570 nm Em = 670 nm + with Single Mismatch in Gene PCR Primers Molecular Beacons for Detection 6-FAM Dabcyl or BHQ-1® $450 $370 3-5 ODs $550 $450 10-20 ODs ® Target HEX Dabcyl or BHQ-1 $450 $370 3-5 ODs $550 $450 10-20 ODs

TM ® Q Mix All Three Components Together Rhodamine Green Dabcyl or BHQ-1 $600 $490 1-3 ODs $700 $575 3-15 ODs Fl Rhodamine RedTM Dabcyl or BHQ-2® $600 $490 1-3 ODs $700 $575 3-15 ODs Quenched Molecular Beacon PCR Amplify Allele Present TAMRA Dabcyl or BHQ-2® $600 $490 1-3 ODs $700 $575 3-15 ODs Q Fl Q Fl ® C TET Dabcyl or BHQ-1 $450 $370 3-5 ODs $550 $450 10-20 ODs

® ® G Texas Red Dabcyl or BHQ-2 $600 $490 1-3 ODs $700 $575 3-15 ODs

Detect Allele with Color Specific Molecular Beacon Fluorescence Detected When Amplified Sufficiently

*Molecular Beacons are sold under license from The Public Health Research Institute. The purchase of Molecular Beacons by end users is accompanied by limited rights under PHRI patents to use the probes for Quencher Selection their research only. Commercial use or any other use of Molecular Beacons requires a license from PHRI, and no license rights for any such use are conveyed with the purchase of Molecular Beacons. No representation or TriLink offers a wide range of quenchers including TAMRA, DABCYL, Black Hole Quenchers® and QSY®. warranty accompanies the sale of these products that their use, alone or in combination, or in any process will Please refer to the Technical Information section of this catalog for more on quenchers. Additional information not infringe the claims of United States or foreign patents. TriLink will not be responsible for damages related to and structures can be found on page 30. this product beyond replacement cost for any reason. |

For licensing information and disclaimers, please see page 152.

DABCYL/6-FAM Molecular Beacon HO O O

N N O O O H H OH N P Oligonucleotide 5' P N O O 3' O O O O N O O

See Licensing Information section for the disclaimer of license statement pertaining to Molecular Beacon products.

14 www.trilinkbiotech.com [email protected] (800) 863-6801 1515 Oligonucleotide Synthesis: Primer Grade DNA Oligonucleotide Synthesis: Standard DNA

Primer Grade DNA Oligonucleotides Standard DNA Oligonucleotides

Our primer grade synthesis services include 40 nmole and 200 nmole scales. Per base price includes synthesis, Our standard DNA synthesis ranges from 0.2 mmole to 15 mmole, in both phosphodiester and deprotection, cartridge desalt, PAGE analysis and a certificate of analysis. Primers are phosphodiester DNA phosphorothioate backbones. We offer an extensive selection of oligo modifications, such as linkers, spacers, only and must be between 10 and 45 bases in length. If you require larger scale synthesis, modifications or dyes and modified bases, please see pages 20-47. additional purification, please see Standard Synthesis DNA Oligonucleotides on the opposite page. Primer grade services include: For larger synthesis scales please refer to our mid-scale services on page 13 or request a custom quotation. Oligonucleotides • No set up charge • Free desalting Per Base Pricing • PAGE analysis on every primer • Certificate of analysis with every shipment O B O B Phosphodiester O Phosphorothioate O 0.2 µmole $2.00 0.2 µmole $4.50 O O 1.0 µmole $3.00 P O 1.0 µmole $6.00 P S 15 µmole $15.00 O 15 µmole $20.00 O O B O B O O Synthesis Per Base Minimum Desalt Desalt Cartridge $40 minimum $50 minimum Scale Price Yield (>20mer) Purification Purification O O Oligonucleotides 40 nmole $0.99 3 ODs no charge $15.00

200 nmole $1.25 15 ODs no charge $15.00 Purification Minimum charge per oligonucleotide: 40 nmole scale $18.00; 200 nmole scale $20.00. Method* 0.2 - 1.0 µmole 15 µmole Double RP-HPLC $200 $375 Single RP-HPLC & RP-Cartridge $150 $325 AX-HPLC $225 $425 RP-Cartridge $50 Not available PAGE $150 Inquire Extra Conjugation Purification $50 $100

*If you are not sure which purification is best for your oligonucleotide and application, see When is my Oligonucleotide Pure Enough? in the Technical Information section of this catalog, ask our technical support team or simply indicate "Best Method" when requesting your quotation.

Did You Know?

Every oligonucleotide sold by TriLink, even primer grade DNA, is analyzed by PAGE and usually MS as well. Many are also assayed by either AX-HPLC or RP-HPLC. Our strong quality commitment and our extensive nucleic acid chemistry experience results in the best oligonucleotides possible. Expected Yields

In general, yields of HPLC purified (~90%) unmodified oligonucleotides are as follows:

0.2 µmole scale: 5 - 15 OD260 units (~0.15 mg - 0.5 mg)

1.0 µmole scale: 20 - 60 OD260 units (~0.66 mg - 2 mg)

15 µmole scale: 300-750 OD260 units (~10 mg - 25 mg)

Yield and purity differences can be caused by many factors, such as sequence and length. If you require a specific yield, please let us know when you place your order or request a quote.

16 www.trilinkbiotech.com [email protected] (800) 863-6801 1717 Oligonucleotide Synthesis: Standard RNA Oligonucleotide Synthesis: Methylphosphonate and 2' Fluoro RNA

Standard RNA Oligonucleotides Methylphosphonate and 2' Fluoro RNA Oligonucleotides

Our standard RNA synthesis services range from 0.2 µmole to 15 µmole scale, in both 2' OH RNA and Methylphosphonate 2' O-Methyl RNA backbones. Both 2' OH and 2' O-Methyl RNA oligonucleotides can be made with phosphorothioate linkages at no extra charge. We offer an extensive selection of oligo modifications, such as linkers, spacers, dyes and modified bases, please see pages 20-47. Per Base Pricing This molecule has a number of unusual properties. It is O B O one of the few neutrally charged backbones that is readily 0.2 µmole N/A For larger synthesis scales please request a custom quotation. Set pricing for mid-scale RNA oligonucleotide available. This modification is most commonly used to Oligonucleotides 1.0 µmole $35.00 O protect the termini of oligonucleotides from enzymatic synthesis is not available, because the yield and quality of these syntheses is highly dependent on sequence. 15 µmole $125.00 P CH3 degradation. One disadvantage is that the racemic form of O $150 minimum* O B O this modification reduces the ability of the oligonucleotide to hybridize to its target. *This minimum refers only to the portion of the oligonucleotide with the methylphosphonate modification. This is to cover the additional O 2' OH RNA deprotection processing required for this modification.

Per Base Pricing Purification O B O

0.2 µmole $10.50 2' Fluoro RNA OH Method 0.2 - 1.0 µmole 15 µmole Oligonucleotides 1.0 µmole $14.50 O 15 µmole $65.00 P O PAGE $200 Inquire O Inquire for Pricing O B AX-HPLC $250 $475 $100 minimum* O The main applications of these 2' Fluoro modifications are O B O ribozyme development (1,3), to increase Tm (2,6,7), aptamer *This minimum refers only to the portion of the Each 2' Fluoro RNA modified oligonucleotide with the 2' OH modification. selection (4,5) and nuclease resistance (7). Oligonucleotides O OH oligonucleotide must be quoted containing 2' Fluoros have been reported to increase DNA- individually. The price is effected O F by the total number of 2' Flouro P O DNA Tm by 1.3°C per insertion (6). O modifications and if the modifi- O B O 1. Fell, et al. (1997) Antisense and Nucleic Acid Development 7, 319-326. cations are pyrimidines, purines 2. Pagratis, et al. (1997) Nature Biotechnology 15, 68-73. or a combination of both. 3. Ito, et al. (1999) Nucl. Acids Symp. Ser 42, 277-278. 2' O-Methyl RNA O F 4. Pat. No. 5,475,096, Nexstar. 5. Kubik, et al. (1997) J. Immunol. 159, 259-267. 6. Sabahi, et al. (2001) Nucleic Acids Research 29, 2163-2170. Per Base Pricing Purification 7. Kawasaki, et al. (1993) J. Medicinal Chemistry 36, 831-841. O B O 0.2 µmole $8.00 Same as Standard DNA Synthesis. 1.0 µmole $12.00 See previous page. O OCH3 15 µmole $50.00 P O O Purification and Yields $50 minimum O B O Methylphosphonate and 2' Fluoro modified oligonucleotides can often be purified using the same methods listed for Standard DNA Synthesis. However, this depends on the design of the entire molecule. Yields are OCH O 3 extremely variable, affected by modifications and sequence. Please contact us for information regarding purification methods and expected yields for your specific construct.

Expected Yields

In general, yields of PAGE purified (~90%) unmodified 2' OH RNA oligonucleotides are as follows (2' O-Methyl yields are comparable to DNA yields as shown on the previous page):

0.2 µmole: 2 - 8 OD260 units (~0.07 mg - 0.27 mg)

1.0 µmole: 10 - 40 OD260 units (~0.33 mg - 1.33 mg)

15 µmole: 150-400 OD260 units (~4.45 mg - 13.33 mg)

Yield and purity differences can be caused by many factors, such as sequence and length. If you require a specific yield, please let us know when you place your order or request a quote.

Please see the Technical Information section for further information on oligonucleotide purification procedures.

18 www.trilinkbiotech.com [email protected] (800) 863-6801 1919 Oligonucleotide Modifications: Oligonucleotide Conjugates Oligonucleotide Modifications: Fluorescent Dye Labeling

Oligonucleotide Conjugates Violet/Blue (Emission Max. 375-491 nm) SO3H SO3H P130*

O Our selection of conjugates includes over sixty fluorescent dyes. We also offer non-fluorescent conjugates, such O H P H 3' 3' OligonucOligonucleotidleotide e 5' P N N O O O Abs. max: 340 nm as biotin, psoralen and cholesteryl. Most conjugates can be placed at the 5' terminus, and internally on any O SO3H 0.2 mmole $125 OO O O Em. max: 376 nm 2'-deoxy base. Many conjugates can also be placed at the 3' terminus. Separate pricing will be noted for these 1.0 mmole $150 ε at Abs. max: 43,000 conjugates. Our standard protocol includes purification before and after conjugation. 15 mmole $500

7-Methoxycoumarin*

SO H SO H Oligonucleotides Fluorescent Dye Labeling (7-methoxycoumarin-3-carboxy) 3 3 Abs. max: 358 nm

0.2 mmole $175 O O O Dyes with a range of wavelengths are available for oligonucleotide conjugation. Structures and list prices can be O Em. max: 410 nm 1.0 mmole $200 H H 3' 3' OligonucOligonucleotidleotide e 5'5' P N N O OO O SO H ε at Abs. max: 26,000 3 found on the following pages. Please refer to the selection chart in the Technical Information section for more 15 mmole $600 O information on available dyes and their spectra. For additional help please contact our technical support team. O O

Cascade BlueTM* SO3H SO3H SO H SO H Violet/Blue Green/Yellow Green/Yellow (Cont.) Orange Red 3 3 (Em 375-491 nm) (Em 492-585 nm) (Em 492-585 nm) (Em 586-647 nm) (Em 647-700 nm) Abs. max: 396 nm max max max max max 0.2 mmole $225 O Em. max: 410 nm 1.0 mmole $275 H H 3' 3' OligonucOligonucleotidleotide e 5'5' P N N P130 BODIPY 493/503 6-JOE CAL Fluor® Red 590 Alexa Fluor® 633 OO OO O SO H ε at Abs. max: 29,000 O 3 SO3H 15 mmole $700 O 7-Methoxycoumarin DTAF BODIPY 530/550 BODIPY 576/589 Alexa Fluor® 647 O O

Oligonucleotides Cascade Blue 6-FAM (Fluorescein) Alexa Fluor® 532 BODIPY 581/591 Quasar 670® TM Alexa Fluor® 405 Dansyl-X HEX Alexa Fluor® 568 Cy5 SO3H SO3H AMCA-X Oregon Green 500 Carboxyrhodamine 6G Texas Red-X Naphthofluorescein Alexa Fluor® 405* Alexa Fluor® 350 Alexa Fluor® 488 CAL Fluor® Orange 560 Cy3.5TM Alexa Fluor® 660 TM Pacific Blue dT-FAM Alexa Fluor® 555 ROX Cy5.5 OO O H P H O SO3H Abs. max: 400 nm 3' 3' OligonucleotideOligonucleotide 5'5' P N N N Marina Blue Oregon Green 488 BODIPY 558/568 CAL Fluor® Red 610 Alexa Fluor® 680 m OO O O 0.2 mole $395 O- O SO3H O O Em. max: 424 nm Dimethylaminocoumarin Oregon Green 514 PyMPO BODIPY-TR-X Alexa Fluor® 700 1.0 mmole $650 O ε at Abs. max: 35,000 Rhodamine Green-X Alexa Fluor® 546 Alexa Fluor® 594 Alexa Fluor® 750 15 mmole $2423

NBD-X BODIPY 564/570 Alexa Fluor® 610 HO3S SO3H TET Quasar 570® CAL Fluor® Red 635 TM SO H SO H Alexa Fluor® 430 Cy3 3 3 CAL Fluor® Gold 540 TAMRA-X 7-Aminocoumarin-X* Alexa Fluor® 514 dT-TAMRA (AMCA-X) O O O 2',4',5',7' Br4-sulfoneflu. Rhodamine Red-X O O Abs. max: 353 nm H H 0.2 mmole $175 3' Oligonucleotide 5' P N N 3' Oligonucleotide 5' O O O O N NH2 O H SO3H Em. max: 442 nm O 1.0 mmole $200 O O ε at Abs. max: 19,000 15 mmole $600

SO3H SO3H Linkers Alexa Fluor® 350*

Prices for 0.2 and 1.0 µmole scale conjugations include the 5' or 3' C6-Amino linker if needed. Specialty OO O O NH2 H H Abs. max: 346nm PP m 3' 3'OligonucleotideOligonucleotide 5'5' N N 0.2 mole $300 OO O O linkers specified by the customer are not included. See page 34-36 for pricing. Linkers for larger scales are O- O SO3H O Em. max: 442nm O SO3H 1.0 mmole $375 O also not included in the conjugate price. ε at Abs. max: 19,000 15 mmole $800

® SO H SO H Purification Pacific Blue * 3 3

F Some conjugates require only a single purification. See page 17-18 for pricing. The asterisked conjugates, O O OH Abs. max: 410 nm O 0.2 mmole $175 H H however, require an extra purification step for an additional $50 (0.2 or 1.0 µmole scale) or $100 (15 µmole 3' 3' OligonucleotOligonucide leotide 5'5' P N N Em. max: 455 nm OO OO F m O SO3H 1.0 mole $225 O O scale). Many of the dyes listed above are succinimidyl esters (SE), and require an amino labeled oligonucleotide O ε at Abs. max: 46,000 15 mmole $650 unless specifically noted otherwise. Thiol reactive conjugates are also available for some of these dyes for an additional cost. Please inquire for availability and pricing.

SO H SO H Marina Blue®* 3 3

F O O O OH Abs. max: 365 nm H H 0.2 mmole $175 3' 3' OligonucleotOligonucide leotide 5'5' PP N N OO OO O SO3H Em. max: 460 nm O F 1.0 mmole $225 O O ε at Abs. max: 19,000 15 mmole $650

ε is noted in L mmole-1 cm-1. *These compounds require an extra purification step. See page 20 for details.

20 www.trilinkbiotech.com [email protected] (800) 863-6801 2121 Oligonucleotide Modifications: Fluorescent Dye Labeling Oligonucleotide Modifications: Fluorescent Dye Labeling Violet/Blue (Emission Max. 375-491 nm) continued Green/Yellow (Emission Max. 492-585 nm) continued

® SO H SO H OH O O Dimethylaminocoumarin * 3 3 dT-FAM

Fluorescein linked to 5 position of thymidine. O

O O Abs. max: 376 nm H OH Abs. max: 492 nm OO O O N N 0.2 mmole $175 H 5' or Internal 3' Terminus NH N H H 3' 3' OligonucleotOligonucide leotide 5'5' PP N N Em. max: 468 nm Em. max: 520 nm O OO O 1.0 mmole $200 O SO3H 0.2 mmole $150 $100 O O N OO O O ε at Abs. max: 22,000 ε at Abs. max: 74,850 15 mmole $600 O 1.0 mmole $200 $125 15 mmole $650 $700 O

Oregon Green 488TM* Oligonucleotides

SO3H SO3H HO O O Abs. max: 495 nm 0.2 mmole $175 F F O Em. max: 521 nm m O 1.0 mole $225 H H OH 3' 3' OligonucleotOligonucide leotide 5'5' P N N ε at Abs. max: 76,000 OO OO 15 mmole $650 O SO3H Green/Yellow (Emission Max. 492-585 nm) O O O

TM TM BODIPY 493/503 * SO H SO H 3 3 Oregon Green 514 * SO3H SO3H HO O O

F Abs. max: 500 nm m N F F Abs. max: 506 nm 0.2 mole $225 OO B O F Em. max: 506 nm 0.2 mmole $175 F HH + O m P N N Em. max: 526 nm 1.0 mole $275 3'3' OligonucOligonucleotidleotide 5'5' P N H H O OO m P N OH O SO3H ε at Abs. max: 79,000 1.0 mole $225 3' 3' OligonucleotOligonucide leotide 5'5' N OO OO OO S F m O SO3H ε at Abs. max: 85,000 15 mole $750 O O O 15 mmole $650 O F O Oligonucleotides

HO O O TM DTAF* SO H + Rhodamine Green-X * (mixed isomers) SO3H 3 NH O NH SO H SO H 2 2 (4,6-Dichlorotriazinyl)aminofluorescein 3 3 O Abs. max: 492 nm O Abs. max: 503 nm m OH O 0.2 mole $150 0.2 mmole $225 O OH O Em. max: 516 nm H H Em. max: 528 nm H PP N N 1.0 mmole $175 H N NH m 3' 3' OligonucOligonucleotideleotide 5'5' 3' Oligonucleotide 5' P N N 1.0 mole $275 O OO O N SO H 3' Oligonucleotide 5' O O ε at Abs. max: 83,000 O H 3 O O O SO H O O ε at Abs. max: 74,000 m O 3 O 15 mole $600 O N N 15 mmole $750 O O Cl

6-FAM (Fluorescein) SO H NBD-X* SO3H 3 SO3H SO3H 6-Carboxyfluorescein HO O O Abs. max: 466 nm O Abs. max: 496 nm NO m 2 Amidite SE* 3' Terminus OO 0.2 mole $125 O H H Em. max: 516 nm H H Em. max: 535 nm 5' PP N OH PP N 0.2 mmole $100 $150 $1003' 3' OligonucleotOligonucide leotide 5' N 1.0 mmole $150 3' 3' OligonucOligonucleotidleotide e 5'5' N O OO OO OO N N O SO3H O O H SO3H OO ε at Abs. max: 83,000 O ε at Abs. max: 22,000 O O N O 1.0 mmole $125 $175 $125 O 15 mmole $500 O 15 mmole $500 $600 $700 Amidite/SE version shown

SO H SO H Dansyl-X* 3 3 TET SO3H SO3H 6-((5-Dimethylaminonaphthalene-1-sulfonyl)amino) hexanoate Tetrachlorofluorescein HO O O

Abs. max: 335 nm Cl Cl Abs. max: 521 nm O N m O O O 0.2 mole $125 HH Amidite SE* O Cl O 3'3' OligonucOligonuclleotideotidee 5' P NN S Em. max: 518 nm Em. max: 536 nm O O H H m O NSO3H P OH 1.0 mole $150 H 0.2 mmole $100 $150 3' 3' OligonuclOligonuceotide leotide 5'5' P N N O OO O OCl SO H ε at Abs. max: 4,200 3 ε at Abs. max: 86,000 m OO m O 15 mole $500 1.0 mole $125 $175 O O 15 mmole $500 $600

SO3H SO3H Oregon Green 500TM* Alexa Fluor® 430*

SO3H SO3H

OH O O O H SO3H H Abs. max: 433 nm Abs. max: 499 nm P N (H2C)5 N 3' 3'OligonucleotideOligonucleotide 5'5' P N m OO OO 0.2 mmole $175 0.2 mole $325 O- O SO3H O Em. max: 539 nm Em. max: 519 nm O O SO3H O 1.0 mmole $225 O 1.0 mmole $400 H P H N ε at Abs. max: 78,000 F ε at Abs. max: 15,000 3' 3' OligonucOligonucleotidleotide e 5'5' P N m OO O 15 mmole $1000 F 15 mole $650 O O SO3H O O O O F O

CAL Fluor® Gold 540 Alexa Fluor® 488* (mixed isomers) SO3H SO3H

Note: Impurities in this dye can affect the oligo conjugate product purity. NH

OH O Abs. max: 495 nm Abs. max: 522 nm SO3H m O 5' Terminus Structure not available 0.2 mole $375 O H Em. max: 517 nm Em. max: 541 nm P H O from manufacturer. 3' Oligonucleotide 5' P N N 0.2 mmole $250 1.0 mmole $550 3' Oligonucleotide 5'O O O O O SO3H O ε at Abs. max: 73,000 ε at Abs. max: 74,700 SO H m 15 mmole $1650 O O 3 1.0 mole $275 15 mmole $875 NH2

ε is noted in L mmole-1 cm-1. ε is noted in L mmole-1 cm-1. *These compounds require an extra purification step. See page 20 for details. *These compounds require an extra purification step. See page 20 for details. 22 www.trilinkbiotech.com [email protected] (800) 863-6801 2323 Oligonucleotide Modifications: Fluorescent Dye Labeling Oligonucleotide Modifications: Fluorescent Dye Labeling

Green/Yellow (Emission Max. 492-585 nm) continued Green/Yellow (Emission Max. 492-585 nm) continued

SO3H SO3H Alexa Fluor® 555* Alexa Fluor® 514* (mixed isomers) NH OH O SO3H O O H Abs. max: 555 nm P H O 3' 3' OligonucOligonucleotidleotide e 5'5' P N N Abs. max: 518 nm O OO m O SO3H 0.2 mole $405 Structure not available 0.2 mmole $385 OO SO H Em. max: 572 nm O 3 Em. max: 540 nm O 1.0 mmole $700 from manufacturer. 1.0 mmole $565 ε at Abs. max:155,000 NH ε at Abs. max: 80,000 15 mmole $1785 15 mmole $2750

BODIPY 558/568TM*

2',4',5',7'-Tetrabromosulfonefluorescein* Oligonucleotides Br SO HBr SO H 3 3 O OH O O H P + 3' Oligonucleotide 5' N Abs. max: 558 nm Abs. max: 529 nm O O N m O Br Br 0.2 mole $225 B 0.2 mmole $175 O F N Em. max: 569 nm SO H O 3 Em. max: 544 nm m 1.0 mmole $225 O 1.0 mole $275 F H H ε at Abs. max: 97,000 P 3' 3' OligonucOligonucleotidleotide e 5'5' N N ε at Abs. max: 89,000 OO OO 15 mmole $750 m O SO3H S 15 mole $650 O O O

6-JOE* PyMPO* SO3H SO3H Cl Cl SO3H SO3H 6-Carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein HO O O 1-(3-carboxybenzyl)-4-(5-(4-methoxyphenyl)oxazol-2-yl) pyridinium bromide Abs. max: 415 nm Abs. max: 520 nm N O O O O 0.2 mmole $200 O 0.2 mmole $150 O O Em. max: 570 nm O Em. max: 548 nm H H + O PP m H H m 3' 3' OligonucOligonucleotidleotide e 5'5' N N N 1.0 mole $250 P OH 1.0 mole $200 OO OO O SO H 3' 3' OligonucleotOligonucide leotide 5'5' P N N 3 ε at Abs. max: 26,000 O O ε at Abs. max: 75,000 OO O O O SO3H O 15 mmole $700 O 15 mmole $600 O O O Oligonucleotides

SO3H SO3H

TM BODIPY-530/550 * Alexa Fluor® 546* SO H SO H H 3 3 SO H SO H O N3 O 3 N H H P 3' 3' OligonucOligonuleotidcleoteide 5'5' P N N + O OO N Abs. max: 554 nm O SO3H Abs. max: 534 nm O F B O 0.2 mmole $225 O O N 0.2 mmole $395 Em. max: 554 nm Cl Em. max: 570 nm F OO OH 1.0 mmole $275 1.0 mmole $650 H H H Oligonucleotide PP N (H C) N ε at Abs. max: 77,000 3' 3' Oligonucleotide 5'5' N2 5 ε at Abs. max:112,000 O O O O S SOClH 15 mmole $750 15 mmole $2425 O- 3 O O O Cl O

SO3H SO3H SO3H SO3H Alexa Fluor® 532* BODIPY-564/570TM* N O O H H P H H 3' 3' OligonucOligonucleotideleotide 5'5' P N N + P Abs. max: 530 nm OO OO N 3' 3' OligonucleotideOligonucleotide 5'5' P N N SO3H O SO3H Abs. max: 565 nm OO OO O m O- O SO3H F B N 0.2 mole $385 O O 0.2 mmole $225 O O O Em. max: 555 nm F Em. max: 571 nm 1.0 mmole $565 O 1.0 mmole $275 SO H ε at Abs. max: 81,000 15 mmole $1785 3 m ε at Abs. max:142,000 NH 15 mole $750

® SO H SO H HEX Quasar 570 * 3 3 Cl Cl SO3H SO3H Hexachlorofluorescein (The SE dye is highly recommended for a quality product.) HO O O Abs. max: 535 nm Abs. max: 547 nm Cl Cl 0.2 mmole $200 OO N N+ Amidite SE* O H H O Cl P N Em. max: 570 nm O Em. max: 556 nm 3' 3' OligonucleotideOligonucleotide 5'5' P N m OO OO m H H 1.0 mole $250 O- O SO3H 0.2 mole $100 $150 P OH 3' 3' OligonucleotOligonucide leotide 5'5' P N N O ε at Abs. max:115,000 OO OO Cl ε at Abs. max: 96,000 O O SO3H m O 1.0 mmole $125 $175 O 15 mole $875 O 15 mmole $500 $600 O

TM SO H SO H Carboxyrhodamine 6GTM* Cy3 3 3 Cy3 is available as both the amidite and as the succinimidyl SO H SO H 3 H 3 H + O N ester. Our experience is that the SE is a better dye. N Abs. max: 550 nm Abs. max: 525 nm O + m Amidite SE* H N 0.2 mole $175 P O 3' 3' OligonucleotOligonucide leotide 5'5' P N N Em. max: 562 nm Em. max: 555 nm OO OO O SO3H 1.0 mmole $225 O 0.2 mmole $125 $200 O O ε at Abs. max:150,000 H H OH ε at Abs. max:108,000 HO O m 3' 3' OligonucOligonucleotidleotide e 5' PP N N 1.0 mmole $150 $250 15 mole $650 O OO O SO3H OO O 15 mmole $500 $875 Amidite version shown O SO3H SO3H

CAL Fluor® Orange 560 TAMRA-X* O 6-(Tetramethylrhodamine-5(6)-carboxamido) hexanoate O H O P H N 3' 3'OligonucleotidOligonuce leotide 5'5' P N OO OO NO SO3H O H OH Abs. max: 543 nm Abs. max: 540 nm O 5' Terminus Structure not available Single Isomer Mixed Isomers O Em. max: 561 nm O Em. max: 571 nm 0.2 mmole $250 from manufacturer. 0.2 mmole $175 $175 + at Abs. max: 92,000 ε at Abs. max: 94,000 N O N ε 1.0 mmole $275 1.0 mmole $200 $200 15 mmole $875 15 mmole $750 $600

ε is noted in L mmole-1 cm-1. ε is noted in L mmole-1 cm-1. *These compounds require an extra purification step. See page 20 for details. *These compounds require an extra purification step. See page 20 for details. 24 www.trilinkbiotech.com [email protected] (800) 863-6801 2525 Oligonucleotide Modifications: Fluorescent Dye Labeling Oligonucleotide Modifications: Fluorescent Dye Labeling

Green/Yellow (Emission Max. 492-585 nm) continued Orange (Emission Max. 586-647 nm) continued

SO H SO H TM 3 3 + dT-TAMRA N O N Cy3.5 6-Tetramethylrhodamine-5(6)-carboxamide attached to dT-C6 amine Cy3.5 is available as both the amidite and as the succinimidyl O ester. Our experience is that the SE is a better dye. O O O OH Abs. max: 546 nm O Abs. max: 588 nm H + 5' or Internal N Amidite SE* H N NH N 5' P N H 3' 3' OligonucleotOligonucide leotide 5' N Em. max: 576 nm OO OO Em. max: 604 nm O O SO3H m O O N O 0.2 mole $175 O 0.2 mmole $325 $300 ε at Abs. max: 95,000 OH O ε at Abs. max:150,000 1.0 mmole $200 1.0 mmole $350 $550 15 mmole $750 O 15 mmole $1375 $1950 Amidite version shown

TM Carboxy-X-RhodamineTM* Rhodamine Red-X * SO H SO H Oligonucleotides 3 + 3 + N O N N O N (X-Rhodamine, ROX) Abs. max: 580 nm Abs. max: 560 nm O Single Isomer Mixed Isomers 0.2 mmole $175 SO H O 3 O Em. max: 605 nm Em. max: 580 nm H OH 0.2 mmole $200 $150 P H 1.0 mmole $225 3' 3' OligonucOligonucleotidleotide e 5'5' P N N O OO OO O SO H at Abs. max: 78,000 ε at Abs. max:129,000 3 ε H O P O 1.0 mmole $225 $175 15 mmole $650 3' Oligonucleotide 5' N S O O O O N O H O m O 15 mole $650 $550 6-ROX structure shown

CAL Fluor® Red 610

Abs. max: 590 nm 5' Terminus Structure not available 0.2 mmole $250 from manufacturer. Em. max: 610 nm 1.0 mmole $275 ε at Abs. max:108,000 15 mmole $875 Oligonucleotides Orange (Emission Max. 586-647 nm)

TM CAL Fluor® Red 590 BODIPY-TR-X * SO H SO3H 3 Texas RedTM substitute Abs. max: 588 nm Abs. max: 566 nm 5' Terminus Structure not available + 0.2 mmole $250 O N Em. max: 616 nm Em. max: 588 nm H H from manufacturer. P F B 0.2 mmole $250 1.0 mmole $275 3' 3' OligonucOligonuleotidcleotiede 5'5' P N N N O OO O O SO H ε at Abs. max: 68,000 at Abs. max: 79,000 O F 3 1.0 mmole $275 ε O 15 mmole $750 O O

15 mmole $875 S

SO3H SO3H

SO3H SO3H TM HO3S + BODIPY-576/589 * Alexa Fluor® 594* (mixed isomers) N

OH O O H H SO3H PP N + 3' 3' OligonucOligonuleotidcleoteide 5'5' N N O OO O Abs. max: 576 nm O Abs. max: 590 nm O SO3H O H H m O F B N O 0.2 mole $225 O 0.2 mmole $390 3' 3' OligonucOligonucleotidleotide e 5'5' P N N O Em. max: 590 nm OO O Em. max: 617 nm O SO3H m F O 1.0 mole $275 1.0 mmole $600 SO H ε at Abs. max: 83,000 O O 3 ε at Abs. max: 92,000 15 mmole $750 NH 15 mmole $1950 N

SO3H SO3H HO3S

BODIPY-581/591TM* Alexa Fluor® 610* O O N SOOH SO H H 3 N+ 3 P H 3' 3' OligonucleotidOligonuecleotide 5'5' P N N + OO O O N O SO H O 3 O B O F N Abs. max: 584 nm Abs. max: 612 nm 0.2 mmole $225 O F 0.2 mmole $425 O Em. max: 592 nm HO3S Cl SO3H Em. max: 628 nm OO OH m H 1.0 mole $275 1.0 mmole $725 H Oligonucleotide P N ε at Abs. max:136,000 3' 3' Oligonucleotide 5'5' P N ε at Abs. max:144,000 OO OO S O Cl SO H 15 mmole $750 15 mmole $2850 O- 3 O O Cl O

SO H SO3H Alexa Fluor® 568* (mixed isomers) 3 CAL Fluor® Red 635 HO3S N

OH O SO H O 3 Abs. max: 616 nm O Abs. max: 578 nm H H PP O 5' Terminus Structure not available 0.2 mmole $385 3' 3' OligonucleotidOligonucleotide e 5'5' N N OO O O Em. max: 636 nm O SO3H Em. max: 602 nm OO O SO H 0.2 mmole $275 from manufacturer. 3 1.0 mmole $565 O ε at Abs. max: 88,000 m ε at Abs. max:112,000 15 mmole $1785 NH 1.0 mole $300 HO S 3 15 mmole $900

SO3H SO3H Texas-Red-XTM* (mixed isomers)

+ O N O O O H H PP N Abs. max: 583 nm 3' Oligonuc3' leotidOligonuce leotide 5'5' N S SO3H OO O O N O SO H 0.2 mmole $225 O H 3 O O O Em. max: 603 nm 1.0 mmole $250 O m ε at Abs. max:112,000 15 mole $700 N

ε is noted in L mmole-1 cm-1. *These compounds require an extra purification step. See page 20 for details.

26 www.trilinkbiotech.com [email protected] (800) 863-6801 2727 Oligonucleotide Modifications: Fluorescent Dye Labeling Oligonucleotide Modifications: Fluorescent Dye Labeling

Red (Emission Max. 647-700 nm) Red (Emission Max. 647-700 nm) continued

Alexa Fluor® 633* Alexa Fluor® 700*

Abs. max: 621 nm Abs. max: 702 nm 0.2 mmole $405 Structure not available 0.2 mmole $475 Structure not available Em. max: 639 nm Em. max: 723 nm 1.0 mmole $725 from manufacturer. 1.0 mmole $750 from manufacturer. ε at Abs. max:159,000 ε at Abs. max:196,000 15 mmole $2850 15 mmole $3000

Alexa Fluor® 647* Alexa Fluor® 750* Oligonucleotides

Structure not available Abs. max: 651 nm 0.2 mmole $425 from manufacturer. Abs. max: 749 nm Em. max: 672 nm 0.2 mmole $425 Structure not available m Em. max: 782 nm 1.0 mole $725 1.0 mmole $725 from manufacturer. m ε at Abs. max:270,000 ε at Abs. max:240,000 15 mole $2850 15 mmole $2850

Quasar 670® *

SO3H SO3H

Abs. max: 644 nm 0.2 mmole $200 OO N N+ Em. max: 670 nm 1.0 mmole $250 H P H 3' 3' OligonucleotideOligonucleotide 5'5' P N N ε at Abs. max:187,000 O O O 15 mmole $875 O- O SO3H O O O Oligonucleotides

TM SO H Cy5 SO3H 3 Cy5 is available as both the amidite and as the succinimidyl ester. Our experience is that the SE is a better dye. Abs. max: 649 nm O Amidite SE* O + H N Em. max: 670 nm 3' 3' OligonucleotOligonucide leotide 5'5' P N 0.2 mmole $125 $200 O O N O O O SO3H O ε at Abs. max:250,000 1.0 mmole $150 $250 HOO 15 mmole $500 $875 Amidite version shown

Carboxynaphthofluorescein* HO O SO3H SO3H (5 and 6 mixed esters) O Abs. max: 602 nm 0.2 mmole $125 O O O Em. max: 672 nm 1.0 mmole $150 H H 3' 3'OligonucleotOligonucide leotide 5'5' PP N N OH OO OO O SO H ε at Abs. max: 42,000 3 15 mmole $500 O O O

Alexa Fluor® 660*

Abs. max: 663 nm 0.2 mmole $395 Structure not available Em. max: 690 nm 1.0 mmole $650 from manufacturer. ε at Abs. max:132,000 15 mmole $2425

TM Cy5.5 SO3H SO3H Cy5.5 is available as both the amidite and as the succinimidyl ester. Our experience is that the SE is a better dye. Abs. max: 675 nm O Amidite SE* O + H N Em. max: 694 nm 3' 3'OligonucleotOligonucide leotide 5' 5' PP N 0.2 mmole $350 $395 O O N O O O SO3H OO ε at Abs. max:250,000 1.0 mmole $375 $650 HOO 15 mmole $1500 $1950 Amidite version shown

Alexa Fluor® 680*

Abs. max: 684 nm 0.2 mmole $395 Structure not available Em. max: 707 nm 1.0 mmole $650 from manufacturer. ε at Abs. max:183,000 15 mmole $2425

28 www.trilinkbiotech.com [email protected] (800) 863-6801 2929 Oligonucleotide Modifications: Quenchers Oligonucleotide Modifications: Non-Fluorescent Conjugates

Quenchers Non-Fluorescent Conjugates

SO3H SO3H SO H SO H DABCYL SE, (Carboxyamide linker)* 3 3 Biotin O We use 3' amine labeled support or 5' C6 amino linker and DABCYL succinimidyl ester to prepare this labeled product with superior properties. HN NH Abs. max: 453 nm O O 5' or Internal O H H P 0.2 mmole $125 3' 3' OligonucleotOligonucide leotide 5'5' P N N P H ε at Abs. max: 32,000 m OO O O S SO H P N 0.2 mole $100 3 3' 3' OligonucleotOligonucide leotide 5'5' N O OO OO H O SO H 1.0 mmole $150 O 3 O O N 1.0 mmole $125 O O N 15 mmole $500 m N 15 mole $425

SO3H SO3H SO H SO H 3'-DABCYL CPG Biotin Diol Linker 3 3 Oligonucleotides O O O HN NH Abs. max:H 453 nm 5' or Internal OO P 3'3' OligonucOligleotidoneucleotide 5' P 5' N 500Å 1000Å N O O O O O H O SO3H 5' PP H N N H O O 0.2 mmole $125 3' 3' OligonucleotOligonucide leotide 5' OH ε at Abs. max: 8,000 OO O N O SO H N N 3 0.2 mmole $40 $40 O O OO S 1.0 mmole $150 O O 1.0 mmole $50 $50 HO 15 mmole $500 15 mmole $400 $575

SO H SO H DABCYL-dT Biotin TEG 3 3

O O O H N N NH N NH NH N N Abs. max: 453 nm OH H O 0.2 mmole $140 O 5' Terminus 3' Terminus O O O N H H O ε at Abs. max: 22,700 P 3' 3' OligonucOligonucleotidleotide e 5'5' P O N O N m 0.2 mmole $150 $75 OO OO O O S 1.0 mole $165 O SO3H O O 1.0 mmole $175 $125 O 15 mmole $600 O 15 mmole $550 $700 Oligonucleotides

O QSY-7®* Biotin-dT NH NH

O O SO+ H SO3H N 3 O N H N NH N S 0.2 mmole $175 Abs. max: 560 nm 5' or Internal H O m O O N 1.0 mole $200 ε at Abs. max: 0.2 mmole $175 O m O 15 mole $700 H H 90,000 1.0 mmole $225 3' 3' OligonucOligonuleotidcleotiede 5'5' P N N N OO O S O SO3H O O 15 mmole $900 O O O O

SO3H SO3H QSY-9®* Biotin BB SO+ 3H SO3H N O N O O O H SO H SO H S P 3'3' OligonuOligonucclleotideotidee 5'5' P N 3 3 Abs. max: 562 nm 3' Terminus 5' or Internal O O N O O O O SO3H 0.2 mmole $175 H O O O ε at Abs. max: 88,000 0.2 mmole $75 $100 OH O H H m PP N N 1.0 mole $200 3' 3' OligonucOligonuleotidcleoteide 5'5' N HNN H O OO O S SO H m O 3 1.0 mole $125 $125 15 mmole $700 O O O O O m 15 mole $700 $425 O 3' Biotin BB shown

O QSY-21®* Dual Biotin SO+ 3H SO3H NHHN N O N OH H Abs. max: 661 nm N m 5' Terminus S 0.2 mole $175 O ε at Abs. max: 90,000 O O m O O 1.0 mmole $200 H H 0.2 mole $250 P N N N P 3' 3' OligonucOligonucleotidleotide e 5'5' SO H NHHN SO H OO OO O S SO H m 3 3 15 mmole $700 O 3 1.0 mole $350 -O O O O O O O H 15 mmole $900 N S O

SO3H SO3H O O O P H 3' 3'OligonucleotideOligonucleotide 5'5' P N QSY-35®* OO O-O O SO3H O O N N O O H H Abs. max: 475 nm P 3' 3' OligonucOligonucleotidleotidee 5' N N N NO2 0.2 mmole $175 O O H O SO3H SO3H SO3H O ε at Abs. max: 23,000 1.0 mmole $200 O O DNP-X* 15 mmole $700

O NO2 5', 3' or Internal O H H P SO3H SO3H 3' 3' OligonucOligonucleotidleotide e 5'5' P N N O OO N m O SO3H 0.2 mole $150 OO H O NO Black Hole Quencher® 1 or 2 1.0 mmole $175 O 2 m O O 15 mole $550 H H P N N N 3' OligonucOligonucleotidleotide e 5' 5' P N O O O O Abs. max: 480-580 nm O SO3H 5' or Internal 3' Terminus N N O O O NO2 ε at Abs. max: 34,000 0.2 mmole $275 $75 O 1.0 mmole $325 $150 15 mmole $850 $1200 3' BHQ1 shown

ε is noted in L mmole-1 cm-1. *These compounds require an extra purification step. See page 20 for details. *These compounds require an extra purification step. See page 20 for details.

30 www.trilinkbiotech.com [email protected] (800) 863-6801 3131 Oligonucleotide Modifications: Non-Fluorescent Conjugates Oligonucleotide Modifications: Inverted Oligonucleotides & Terminal Phosphates

Non-Fluorescent Conjugates Inverted Oligonucleotides

SO H SO3H DNP TEG 3 There are many applications for reverse linked nucleotide modifications of oligonucleotides. They impart nuclease resistance and allow the study of strand orientation effects on molecular biological function. When 5' or Internal O OH O NO2 H H placed internally the remaining portion of the oligo must be synthesized with reversed polarity. This and other 3' 3' OligonucOligonucleotidleotide e 5'5' PP O N O N 0.2 mmole $200 O O O O O O SO3H OO such complicated constructs, must be quoted on a case-by-case basis. Please see Internal modification prices 1.0 mmole $225 O NO 2 15 mmole $700 below to determine the cost of the reversed polarity portion of your oligonucleotide, if applicable.

SO3H SO3H Oligonucleotides Cholesteryl TEG OH OO H H PP O O N O 3'-3' Modifications 3' 3' OligonucOligonucleotidleotide e 5'5' N OO O O O O SO3H OO O O 5' Terminus 3' Terminus O O P 3' Oligonucleotide 5' Base O O 3' Terminus Internal 5' Oligonucleotide3' 0.2 mmole $150 $75 P O O O O O O 0.2 mmole $30 0.2 mmole $70 O P Base 1.0 mmole $175 $125 O O m m O 15 mmole $550 $700 1.0 mole $50 1.0 mole $75 15 mmole $400 15 mmole $300 Internal 3'-3' linkage shown SO H SO H Psoralen C6 3 3 Crosslinks to thymine or uracil when irradiated with longwave UV. 5'-5' Modifications OO O 5' or Internal O Base P O H 3' 3'OligonucleotideOligonucleotide 5'5' P N O O O 5' Terminus Internal P O O 0.2 mmole $125 O- O SO3H O O O O O O P 5' Oligonucleotide3' O 0.2 mmole $70 0.2 mmole $70 O O O 1.0 mmole $150 3' Oligonucleotide 5' P Base O O m m O O 15 mmole $475 1.0 mole $75 1.0 mole $75 O Oligonucleotides 15 mmole $300 15 mmole $300 Internal 5'-5' linkage shown

SO H SO H Psoralen C2 3 3 Crosslinks to thymine or uracil when irradiated with longwave UV. 5' or Internal OO O 0.2 mmole $125 P H 3' 3'OligonucleotideOligonucleotide 5'5' P O N OO O O O- O SO3H 1.0 mmole $150 O O 15 mmole $475 O O Terminal Phosphates

Acridine SO H SO H 3 3 Used as an effective intercalating agent. Cl N Phosphates can be placed on the 3' or 5' termini of oligonucleotides. A 3' terminal phosphate can be used to block elongation although other modifications, such as 3' Propyl or a 2',3'-Dideoxy modified base, are more 3' Terminus 5' or Internal OO O P H m 3' 3'OligonucleotideOligonucleotide 5'5' P NH N 0.2 mole $50 $225 O O O effective terminators. A 5' terminal phosphate is needed for enzymatic ligation of two oligonucleotides to form O- O SO3H m O 1.0 mole $100 $250 HO O longer oligonucleotides. 15 mmole $725 $750

SO3H SO3H Abasic 5' Phosphate Used to create highly base-labile abasic site. O OH O with PAGE purification with HPLC purification O H 3' 3' OligonucleOligonucotide leotide 5'5' PP N 5' or Internal 0.2 mmole $50 0.2 mmole $100 O O O O O SO3H O O OH O 0.2 mmole Inquire 1.0 mmole $75 1.0 mmole $100 O 1.0 mmole Inquire OH 15 mmole $275 15 mmole $375 m 15 mole Inquire 5' Phosphorylation requires additional processing in order to ensure high quality and yield.

SO3H SO3H 3' Phosphate O Did You Know? O 0.2 mmole $30 P H 3'3' OligonucOligleotidoneucleotide 5' P 5' N O O O O O SO3H 1.0 mmole $50 O O TriLink DNA Synthesis Reagents are now sold through Glen Research. To purchase any of the following 15 mmole $400 O products, please contact Glen Research via the phone, email or web address below. • MMT- and TFA-C6-Amino Linker • C6 and C3 Amino Modifier • Spacer 18, Spacer 9, C-3 Spacer • DADE, Carboxy Modifier • Propyl- and Dabcyl-CPG • CleanAmpTM Amidites

Phone: 703-437-6191 Email: [email protected] Website: www.glenres.com

32 www.trilinkbiotech.com [email protected] (800) 863-6801 3333 Oligonucleotide Modifications: Linkers Oligonucleotide Modifications: Linkers

Linkers Linkers

TriLink offers a wide range of linkers, allowing flexibility in oligonucleotide design. Linkers can be placed at Selective Placement Amino Linkers the 3' terminus, 5' terminus or internally. The most common linkers are listed below. If TriLink is synthesizing a The nucleoside linkers are attached to the pyrimidine base at the 5 position and the purine at the 8 position, conjugated oligonucleotide for you, the 5' or 3' C6-Amino linker is included for no additional charge at 0.2 which has a very moderate effect on hybridization with the corresponding target. Therefore you can place a and 1.0 µmole scales. modification such as a fluorescent dye where you want it. Oligonucleotides 5' and 3' Amino Linkers Thymidine-5-C2 and C6 Amino Linker O O O O These amino linkers are used to prepare oligonucleotides for most conjugations. The most common coupling partner is a succinimidyl NH2 NH2 NH N NH N ester (SE or NHS ester) to a primary amine, forming a covalent amide bond. H H 5' or Internal 3' Terminus (C-6 only) O O N O O N O O SO H SO H 3 3 0.2 mmole $125 $75 1.0 mmole $150 $100 5' C6 Amino Linker O O 15 mmole $525 $650 OO H 3' 3' OligonucleOligonucotide leotide 5'5' PP NNH2 0.2 mmole $45 O O O O O SO3H OO NH2 1.0 mmole $50 O 2'-Deoxyadenosine-8-C6 Amino Linker N N NH2 m N 15 mole $250 H 5' or Internal N O N O

SO3H SO3H 0.2 mmole $150 1.0 mmole $175 5' C12 Amino Linker O Oligonucleotides 15 mmole $575 OO 0.2 mmole $125 H 3' 3' OligonucleotOligonucide leotide 5'5' PP N NH2 O O O O O SO3H m OO 1.0 mole $150 NH O O 2'-Deoxycytidine-5-C6 Amino Linker 2 15 mmole $400 NH2 N N H

Additional 0.2 or 1.0 mmole scale couplings are only $35. 5' or Internal 3' Terminus O O N O SO H SO H 3 3 0.2 mmole $150 $100 m 3' C3 Amino Linker 1.0 mole $175 $125 O 15 mmole $625 $700 O O H P 0.2 mmole $30 P 3'3' OligonucOligleotidoneucleotide 5' 5' N NH O O O O 2 O SO3H NH O O 2 1.0 mmole $50 O 2'-Deoxyguanosine-8-C6 Amino Linker N N NH2 15 mmole $400 N H 5' or Internal NH N N SO H SO H 2 3 3 0.2 mmole $275 O O 3' C6 Amino Linker 1.0 mmole $300 m O O O 15 mole $1150 H NH P P 5' 0.2 mmole $20 2 3'3' OligonucOlleotidigoneucleotide 5' N O O O O O SO3H O O 1.0 mmole $40 O m C7 Internal Amino Linker 15 mole $325 O

SO H SO H 3 3 0.2 mmole $125 NH 3' C7 Amino Linker 1.0 mmole $150 2 15 mmole $400 O O O NH P H 0.2 mmole $30 2 3'3' OligonucOlleotidigoneucleotide 5' P 5' N O O O O O SO3H 1.0 mmole $50 O O O 15 mmole $400 OH

Did You Know?

TriLink is the only licensee of the patented Phthalimidyl-3'-C6-Amino-CPG and Phthalimidyl-3'-C6-Amino- CPG reagents. We offer this product as an oligonucleotide modification; see product details on page 34. We also sell the support, exclusively through Glen Research.

The phthalimidyl protected 3' amino linkers are the best available on the market. Because the amine is buried within the linker, it is not subject to the loss of the protecting group and capping during the subsequent oligonucleotide synthesis cycles that occur with the alternatives. This capping side reaction can substantially lower the yield of amine labeled oligonucleotide available for conjugation. We use this reagent routinely for the manufacture of the more unusual oligonucleotides with multiple modifications ordered by our customers.

34 www.trilinkbiotech.com [email protected] (800) 863-6801 3535 Oligonucleotide Modifications: Linkers Oligonucleotide Modifications: Spacers

Linkers Spacers

5' and 3' Thiol Linkers Spacers impart a number of desirable characteristics, such as stability to enzymatic degradation. Multiple spacers can be placed end on end to create extremely long spacers. They can also be placed between other Thiol linkers can be used to form either reversible disulfide bonds or stable thiol ether linkages with oligonucleotide modifications. Even though these spacers are shown on the 5' terminus of the oligonucleotide, maleimides. Care must be used when working with mercaptan labeled oligos. They will dimerize unless they they can be placed internally as well. The C3 (Propyl) Spacer can also be placed at the 3' terminus and is an are stored in a non-oxidizing environment. To avoid dimerization issues we recommend that you take delivery effective terminator. of the protected thiol (disulfide) form shown below. The reduction of the disulfide with TCEP can be done at the SO3H SO3H

same time the maleimide is conjugated. C3 (Propyl) Spacer Oligonucleotides

3' Terminus 5' or Internal OO The purified yields of thiol modified oligonucleotides are often lower than the analogous amino modified m H 0.2 mole $30 $75 3' 3' OligonucleotOligonucide leotide 5'5' PP N O O O O OH O SO3H oligonucleotide. 1.0 mmole $50 $100 OO O 15 mmole $300 $250 5' version shown Additional 5' or internal 0.2 or 1.0 mmole scale couplings only $35. SO H SO H 5' C6 Disulfide Linker 3 3 SO H SO H C6 Spacer 3 3 O 0.2 mmole $180 O P H 0.2 mmole $150 3' 3' OligonucOligonucleotideleotide 5'5' P N S S OO m O O 1.0 mole $180 O O O SO3H OH O m P H O 1.0 mole $150 3' Oligonucleotide3' Oligonucleotide 5'5' P OHN m O OO OO 15 mole $500 O- O SO3H 15 mmole $400 O Additional 0.2 or 1.0 mmole scale couplings only $35. O Oligonucleotides Additional 0.2 or 1.0 mmole scale couplings only $50.

3' C3 Disulfide Linker SO H SO H 3 3 SO3H SO3H Spacer 9 (Triethylene glycol; PEG-150) 0.2 mmole $75 O O H OO 1.0 mmole $120 P P 3'3' OligonucOligonucleotidleotide e 5' 5' N m H OH S S O O O O O SO H 0.2 mole $125 3 3' 3' OligonucleotOligonucide leotide 5'5' PP O N OH m O O OO OO O 15 mole $500 O SO3H O 1.0 mmole $125 OO 15 mmole $300 O

Additional 0.2 or 1.0 mmole scale couplings only $35.

SO H SO H Carboxyl Linkers 3 3 C12 Spacer Using our patented DADE Linker, we can readily prepare 5' carboxyl linkers. This linker can be used to OO conjugate to amines using conventional carbodiimide chemistry. Though less convenient than the solid phase 0.2 mmole $140 H 3' 3' OligonucleotOligonucide leotide 5'5' PP N OH OO OO O SO3H method described in the Technical Information section, solution phase methods may be necessary at times. 1.0 mmole $140 OO O The DADE linker is also useful for conjugation to amine bearing supports for microchip assembly. 15 mmole $350 Additional 0.2 or 1.0 mmole scale couplings only $35. SO3H SO3H DADE Linker (5' Carboxyl Linker) SO H SO H Spacer 18 (Hexaethylene glycol; PEG-282) 3 3 OO 0.2 mmole $125 H 3' 3' OligonucleOligonucotide leotide 5'5' PP N OH OO OO O SO H m 3 1.0 mole $150 OO m OO O O 0.2 mole $140 H P 15 mmole $400 3' 3' OligonucleOligonucotide leotide 5'5' P O N O O OO OO O O OH 1.0 mmole $140 O SO3H OO 15 mmole $350 O

Carboxy-dT Linker O O Additional 0.2 or 1.0 mmole scale couplings only $35.

NH O

0.2 mmole $150 O O N O rSpacer 1.0 mmole $175 O m O 15 mole $575 O 0.2 mmole $225 1.0 mmole $250 15 mmole $650 O OH Additional 0.2 or 1.0 mmole scale couplings only $100. Did You Know? dSpacer TriLink's DADE (decanoic acid diester) linker is unique on the market. It offers a novel way of preparing O O conjugates more economically and with much more flexibility. It was originally designed to allow high- 0.2 mmole $135 throughput screening of a large number of conjugates for therapeutic application. However, it has several 1.0 mmole $135 m other inherent advantages that make it a powerful tool for the scale up of oligonucleotide conjugates as 15 mole $350 O well. We now sell our patented DADE Synthesis Reagent through Glen Research. Additional 0.2 or 1.0 mmole scale couplings only $35.

36 www.trilinkbiotech.com [email protected] (800) 863-6801 3737 Oligonucleotide Modifications: Modified Bases Oligonucleotide Modifications: Modified Bases

Modified Bases for Oligonucleotides 2' Deoxyribonucleoside Analogs - Purines

TriLink can incorporate many different modified bases into your oligonucleotide. Our "hands-on" approach to oligonucleotide synthesis is uniquely suited to this chemistry. The following pages contain structure and 2-Aminopurine-2'-deoxyriboside 8-Amino-2'-deoxyadenosine NH2 pricing information. Please inquire if the modification you desire is not listed. TriLink also offers custom Use: Structure/activity studies Use: Triplex formation N N N phosphoramidite synthesis, see page 51. N H2N N m HO N 0.2 mole $125 HO N N 0.2 mmole $200 O O NH2 1.0 mmole $150 1.0 mmole $250 15 mmole $375 15 mmole $700 HO HO Oligonucleotides

8-Amino-2'-deoxyguanosine 8-Bromo-2'-deoxyadenosine Available Modified Bases O Use: Triplex formation Use: Crystallography, protein cross-linking N NH2 NH studies H2N N N N N NH2 Br 0.2 mmole $200 HO 0.2 mmole $110 O N 1.0 mmole $250 1.0 mmole $125 HO O N 15 mmole $700 m HO 15 mole $325 2' Deoxyribonucleoside (DNA) Ribonucleoside (RNA) HO

Purines 2-Aminopurine 8-Bromo-2'-deoxyguanosine 7-Deaza-8-aza-2'-deoxyadenosine 2-Aminopurine-2'-deoxyriboside 5-Bromouridine NH2 Use: Crystallography, protein cross-linking Use: Duplex stabilization 8-Amino-2'-deoxyadenosine 2,6-Diaminopurine O N studies N Inosine N 8-Amino-2'-deoxyguanosine NH N Br N 5-Iodouridine 0.2 mmole $100 m HO O Oligonucleotides 8-Bromo-2'-deoxyadenosine 0.2 mole $200 N 5-Methylcytidine HO N NH 8-Bromo-2'-deoxyguanosine 1.0 mmole $110 O 2 1.0 mmole $250 5-Methyluridine 7-Deaza-8-aza-2'-deoxyadenosine 15 mmole $300 m HO Puromycin 15 mole $325 7-Deaza-2'-deoxyadenosine HO 7-Deaza-2'-deoxyguanosine Pyrrolocytidine 7-Deaza-2'-deoxyxanthosine 4-Thiouridine 2,6-Diaminopurine-2'-deoxyriboside 7-Deaza-2'-deoxyadenosine 7-Deaza-2'-deoxyguanosine Etheno-2'-deoxyadenosine 2'-O-Methyl Ribonucleosides (2' OMe RNA) Use: Structure/activity studies NH Use: Structure/activity studies O N6-Methyl-2'-deoxyadenosine 2 6 O -Methyl-2'-deoxyguanosine N NH 6 0.2 mmole $200 0.2 mmole $200 O -Phenyl-2'-deoxyinosine 2-Aminopurine-2'-O-methylriboside N HO N HO O N O N NH 8-Oxo-2'-deoxyadenosine 5-Bromo-2'-O-methyluridine 1.0 mmole $250 1.0 mmole $250 2 8-Oxo-2'-deoxyguanosine 3-Deaza-5-aza-2'-O-methylcytidine 15 mmole $700 15 mmole $700 6-Thio-2'-deoxyguanosine 2,6-Diaminopurine-2'-O-methylribose HO HO 5-Fluoro-2'-O-methyluridine Pyrimidines 5-Fluoro-4-O-TMP-2'-O-methyluridine 5'-Aminothymidine 2'-O-Methylinosine 7-Deaza-2'-deoxyxanthosine 2,6-Diaminopurine-2'-deoxyriboside 5-Bromo-2'-deoxycytidine 5-Methyl-2'-O-methylcytidine Use: Structure/activity studies O Use: Duplex stabilization NH 5-Bromo-2'-deoxyuridine 5-Methyl-2'-O-methylthymidine 2 N 5-(Carboxy)vinyl-2'-deoxyuridine NH N 0.2 mmole $200 0.2 mmole $100 2'-Deoxypseudouridine HO N N Sugar Modified O N O HO O N NH 2'-Deoxyuridine 1.0 mmole $250 H 1.0 mmole $110 2 2,4-Difluorotoluyl m m Aracytidine 15 mole $700 15 mole $325 5,6-Dihydrothymidine HO 3'-Deoxyadenosine HO 5,6-Dihydro-2'-deoxyuridine 3'-Deoxycytidine 5-(C2-EDTA)-2'-deoxyuridine 6 3'-Deoxyguanosine N -Methyl-2'-deoxyadenosine N4-Ethyl-2'-deoxycytidine Etheno-2'-deoxyadenosine 3'-Deoxythymidine 5-Fluoro-2'-deoxyuridine Use: Mutagenic effect studies 2',3'-Dideoxyadenosine Use: Fluorescent nucleoside for structure/ N HN 5-Hydroxymethyl-2'-deoxyuridine activity studies 2',3'-Dideoxycytidine N N 5-Hydroxy-2'-deoxycytidine N N 2',3'-Dideoxyguanosine 0.2 mmole $100 0.2 mmole $130 5-Hydroxy-2'-deoxyuridine HO N 2',3'-Dideoxythymidine HO N N m O N 5-Iodo-2'-deoxycytidine 1.0 mmole $115 O 1.0 mole $145 5'-Iodothymidine 5-Iodo-2'-deoxyuridine 15 mmole $325 15 mmole $425 5'-O-Methylthymidine 5-Methyl-2'-deoxycytidine HO HO O4-Methylthymidine 5-Propynyl-2'-deoxycytidine Wobble and Universal Bases 5-Propynyl-2'-deoxyuridine 6 6 Pyrrolo-2'-deoxycytidine 2'-Deoxyinosine O -Methyl-2'-deoxyguanosine O -Phenyl-2'-deoxyinosine 4-Thio-2'-deoxyuridine 2'-Deoxyisoguanosine Use: Mutagenic effect studies O Use: Convertible nucleoside for structure/ O 2-Thiothymidine 2'-Deoxynebularine activity studies N N 4-Thiothymidine K-2'-deoxyribose N N 0.2 mmole $100 0.2 mmole $125 Thymidine Glycol 5-Methyl-2'-Deoxyisocytidine N OH N NH HO N 6-O-TMP-5-F-2'-deoxyuridine P-2'-deoxyribose 1.0 mmole $115 O 2 1.0 mmole $150 O N C4-(1,2,4-Triazol-1-yl)-2'-deoxyuridine 15 mmole $325 15 mmole $375 OH HO

38 www.trilinkbiotech.com [email protected] (800) 863-6801 3939 Oligonucleotide Modifications: Modified Bases Oligonucleotide Modifications: Modified Bases

2' Deoxyribonucleoside Analogs - Purines continued 2' Deoxyribonucleoside Analogs - Pyrimidines

8-Oxo-2'-deoxyadenosine 8-Oxo-2'-deoxyguanosine 5'-Aminothymidine 5-Bromo-2'-deoxycytidine

Use: Structure/activity studies of damaged Use: Structure/activity studies of damaged Use: Ligation blocking, peptide fusion O Use: Crystallography, protein cross-link- NH NH2 O 2 bases H bases H ing studies N N Br N NH 5' Terminus NH N 0.2 mmole $125 O 0.2 mmole $200 O 0.2 mmole $90 HO N HO N 0.2 mmole $120 N O O N O N NH 2NH HO N O m 1.0 mmole $250 2 O m O 1.0 mole $150 1.0 mmole $135 1.0 mole $100 m 15 mmole $375 15 mole $700 m 15 mmole $250

15 mole $400 Oligonucleotides HO HO HO HO

6-Thio-2'-deoxyguanosine 5-Bromo-2'-deoxyuridine 5-(Carboxy)vinyl-2'-deoxyuridine (Carboxy Thymidine)

Use: Photo-crosslinking and photoaffinity S Use: Crystallography, protein cross- O Use: Conjugation to amines O O labeling studies N linking studies Br NH NH HO NH 0.2 mmole $225 m m HO N N 0.2 mole $90 0.2 mole $130 O NH2 HO N O N O 1.0 mmole $250 1.0 mmole $100 O 1.0 mmole $145 HO O 15 mmole $775 15 mmole $250 15 mmole $450 HO HO HO

2'-Deoxypseudouridine 2'-Deoxyuridine

Use: Structure/activity studies O Use: Duplex destabilization O

HN NH NH

Oligonucleotides 0.2 mmole $180 0.2 mmole $90 OH O HO N O 1.0 mmole $200 O 1.0 mmole $100 O 15 mmole $600 15 mmole $250 HO HO

2,4-Difluorotoluyl 5,6-Dihydrothymidine Use: Non-polar thymidine mimic F Use: Structure/activity studies of O damaged bases NH 0.2 mmole $250 0.2 mmole $160 F HO N O Did You Know? 1.0 mmole $275 HO O 1.0 mmole $175 O 15 mmole Inquire 15 mmole $450 Prices listed are per coupling. You will receive a 25% discount HO HO on the second coupling and a 50% discount on all additional 5,6-Dihydro-2'-deoxyuridine 5-(C2-EDTA)-2'-deoxyuridine couplings of a particular base within a single oligonucleotide. O Use: Structure/activity studies of O Use: Sequence specific cleavage of O OH O H damaged bases single & double-stranded DNA & RNA N N O NH HO N N H 0.2 mmole $160 0.2 mmole $350 HO O NH HO N O O O N O 1.0 mmole $175 1.0 mmole $375 HO O 15 mmole $450 15 mmole $1000 HO HO

N4-Ethyl-2'-deoxycytidine 5-Fluoro-2'-deoxyuridine

Use: Duplex destabilization NH Use: Crystallography, protein cross- O linking studies F N NH 0.2 mmole $165 0.2 mmole $115 N O N O 1.0 mmole $180 HO O 1.0 mmole $130 HO O 15 mmole $475 15 mmole $350 HO HO

5-Hydroxymethyl-2'-deoxyuridine 5-Hydroxy-2'-deoxycytidine

Use: Structure/activity studies of OH O Use: Structure/activity studies of NH2 damaged bases damaged bases HO NH N 0.2 mmole $165 0.2 mmole $200 HO N O N O 1.0 mmole $180 O 1.0 mmole $225 HO O 15 mmole $475 15 mmole $625 HO HO

40 www.trilinkbiotech.com [email protected] (800) 863-6801 4141 Oligonucleotide Modifications: Modified Bases Oligonucleotide Modifications: Modified Bases

2' Deoxyribonucleoside Analogs - Pyrimidines continued Ribonucleoside Analogs

5-Hydroxy-2'-deoxyuridine 5-Iodo-2'-deoxycytidine 2-Aminopurine 5-Bromouridine O NH O Use: Structure/activity studies of Use: Crystallography, protein cross- 2 Use: Structure/activity studies Use: Crystallography, protein cross- HO I Br damaged bases NH linking studies N NH N N linking studies 0.2 mmole $165 0.2 mmole $115 0.2 mmole $225 HO N O N O 0.2 mmole $150 HO N O O HO O HO N N NH O 1.0 mmole $180 1.0 mmole $130 1.0 mmole $250 O 2 1.0 mmole $175 15 mmole $475 15 mmole $350 15 mmole $800 15 mmole $450

HO HO HOO H HO OH Oligonucleotides

5-Iodo-2'-deoxyuridine 5-Methyl-2'-deoxycytidine 2,6-Diaminopurine Inosine Use: Crystallography, protein O Use: Duplex stabilization NH 2 Use: Duplex stabilization O cross-linking studies I NH2 Use: Degenerate site NH N N N 0.2 mmole $90 0.2 mmole $90 N NH HO N O HO N O 0.2 mmole $225 0.2 mmole $150 N 1.0 mmole $100 O 1.0 mmole $100 O HO N N HO N 1.0 mmole $250 O NH2 m O 15 mmole $250 15 mmole $300 1.0 mole $175 15 mmole $800 15 mmole $450 HO HO HO OH HO OH O4-Methylthymidine 5-Propynyl-2'-deoxycytidine

Use: Mutagenic effect studies O Use: Duplex stabilization NH2 5-Iodouridine 5-Methylcytidine NH N Use: Crystallography, protein cross- O Use: Duplex stabilization NH 0.2 mmole $115 0.2 mmole $100 I

Oligonucleotides linking studies HO N O OH N O NH N m O m O 1.0 mole $130 1.0 mole $150 0.2 mmole $150 0.2 mmole $150 m m HO N O HO N O 15 mole $350 15 mole $300 1.0 mmole $175 O 1.0 mmole $175 O HO HO 15 mmole $450 15 mmole $450 HO OH HO OH 5-Propynyl-2'-deoxyuridine Pyrrolo-2'-deoxycytidine

Use: Duplex stabilization O Use: Probing of DNA structure and dynamics NH 5-Methyluridine Puromycin* N NH N O N 0.2 mmole $100 0.2 mmole $170 Use: Structure/activity studies Use: Peptide-RNA fusion, blocks elongation N N O OH N O HO 1.0 mmole $150 O 1.0 mmole $200 O NH N HO O N 15 mmole $300 15 mmole $600 0.2 mmole $150 3' Terminus HO N O HO HO 1.0 mmole $175 O 0.2 mmole $75 1.0 mmole $100 HN OH 15 mmole $450 O HOO H 15 mmole $575 NH 4-Thio-2'-deoxyuridine 2-Thiothymidine 2

Use: Photocrosslinking and photoaffinity S Use: Protein-DNA interaction studies O labeling experiments NH NH 0.2 mmole $200 0.2 mmole $200 N O N S OMe 1.0 mmole $225 HO O 1.0 mmole $225 HO O 15 mmole $650 15 mmole $650 HO HO *Additional coupling discount does not apply.

4-Thiothymidine Thymidine Glycol O

S Use: Photocrosslinking and photoaffinity Use: DNA repair enzyme studies HO NH labeling experiments Pyrrolocytidine 4-Thiouridine NH HO N HO O 0.2 mmole $200 O Use: Photocrosslinking and photoaffinity 0.2 mmole $250 Use: Probing RNA structure, fluorescence NH S HO N O 1.0 mmole $225 O 1.0 mmole $275 is sensitive to environment labeling experiments N NH 15 mmole $650 15 mmole $850 HO 0.2 mmole $250 0.2 mmole $225 HO Deprotected Version Shown N O HO N O 1.0 mmole $275 HO O 1.0 mmole $250 O 15 mmole $850 15 mmole $800

6-O-(TMP)-5-F-2'-deoxyuridine C4-(1,2,4-Triazol-1-yl)-2'-deoxyuridine HO OH HO OH N (O4-Triazolyl-2'-deoxyuridine) Use: Convertible nucleoside for structure/ N activity studies (converts to 5-F-dC) O Use: Convertible nucleoside for structure/ N F activity studies N N 0.2 mmole $160 0.2 mmole $115 N O HO N O 1.0 mmole $175 HO O 1.0 mmole $130 O 15 mmole $450 15 mmole $350 HO HO

42 www.trilinkbiotech.com [email protected] (800) 863-6801 4343 Oligonucleotide Modifications: Modified Bases Oligonucleotide Modifications: Modified Bases

2'-O-Methyl Ribonucleoside Analogs Sugar Modified Analogs

2-Aminopurine-2'-O-methylriboside 5-Bromo-2'-O-methyluridine Aracytidine 3'-Deoxyadenosine NH Use: Structure/activity studies Use: Crystallography, protein cross- O Use: Structure/activity studies 2 Use: Chain termination NH2 Br N N linking studies N N N NH 3' Terminus 0.2 mmole $200 0.2 mmole $165 m N N N N O 0.2 mole $115 HO N O HO HO N NH HO O 0.2 mmole $75 O 1.0 mmole $225 O 2 1.0 mmole $180 O m 1.0 mole $130 OH 1.0 mmole $200 15 mmole $650 15 mmole $475 15 mmole $350

15 mmole Inquire OH Oligonucleotides HO OMe HO OMe HO

3-Deaza-5-aza-2'-O-methylcytidine 2,6-Diaminopurine-2'-O-methylriboside 3'-Deoxycytidine 3'-Deoxyguanosine Use: 5-azacytidine mimic NH2 Use: Duplex stabilization NH NH 2 Use: Chain termination 2 Use: Chain termination O N N N N N NH 0.2 mmole $200 0.2 mmole $200 3' Terminus 3' Terminus HO N O HO N N N N N NH O HO NH2 1.0 mmole $225 O 1.0 mmole $225 O 2 0.2 mmole $75 HO O 0.2 mmole $75 O 15 mmole $650 15 mmole $650 1.0 mmole $200 1.0 mmole $200 HO OMe HO OMe 15 mmole Inquire OH 15 mmole Inquire OH

3'-Deoxythymidine 5-Fluoro-2'-O-methyluridine 5-Fluoro-4-O-TMP-2'-O-methyluridine O 2',3'-Dideoxyadenosine NH2 Use: Structure/activity studies O Use: Convertible nucleoside structure/ O Use: Chain termination Use: Chain termination N NH N F activity studies F

Oligonucleotides NH N N 0.2 mmole $250 3' Terminus HO O N N 0.2 mmole $200 0.2 mmole $200 O m HO O O N O HO O 0.2 mmole $75 1.0 mole $250 1.0 mmole $225 H O 1.0 mmole $225 O 1.0 mmole $200 15 mmole Inquire 15 mmole $650 15 mmole $650 OH 15 mmole Inquire Additional 0.2 and 1.0 umol couplings on another oligo are $50. HO OMe HO OMe Requires PAGE purification.

2'-O-Methylinosine 5-Methyl-2'-O-methylcytidine 2',3'-Dideoxycytidine 2',3'-Dideoxyguanosine NH NH Use: Degenerate site O Use: Duplex stabilization 2 Use: Chain termination 2 Use: Chain termination O N N N NH NH N 0.2 mmole $250 m 0.2 mmole $120 N 0.2 mmole $120 N 0.2 mole $400 N N HO N HO N O HO O HO NH2 1.0 mmole $135 O 1.0 mmole $135 O 1.0 mmole $250 O 1.0 mmole $400 O 15 mmole $400 15 mmole $400 15 mmole Inquire 15 mmole Inquire HO OMe HO OMe Additional 0.2 and 1.0 umol couplings on another oligo are $50. Additional 0.2 and 1.0 umol couplings on another oligo are $50. Requires PAGE purification. Requires PAGE purification. 5-Methyl-2'-O-methylthymidine

Use: Structure/activity studies O 2',3'-Dideoxythymidine 5'-Iodothymidine O Use: Chain termination Use: Chemical ligation NH O 0.2 mmole $120 NH HO N O NH 5' Terminus 1.0 mmole $135 O I N O 0.2 mmole $250 0.2 mmole $115 O m N O 15 mole $400 1.0 mmole $250 HO O 1.0 mmole $130 HO OMe 15 mmole Inquire 15 mmole $350 HO Additional 0.2 and 1.0 umol couplings on another oligo are $50. Requires PAGE purification.

5'-O-Methylthymidine

Use: Ligation blocking O

NH 5' Terminus O N O 0.2 mmole $115 O 1.0 mmole $130 15 mmole $350 OH

44 www.trilinkbiotech.com [email protected] (800) 863-6801 4545 Oligonucleotide Modifications: Modified Bases Oligonucleotide Modifications: Dimers & Trimers

Wobble and Universal Bases Cis-Syn Thymidine Dimers

2'-Deoxyinosine 2'-Deoxyisoguanosine Cis-syn thymidine dimers are used in DNA damage and repair research. One of the major causes of DNA damage is UV from sunlight that results in dimerization of adjacent pyrimidine bases, forming cyclobutane Use: Degenerate site O Use: Duplex stability studies NH2 N N dimers (CPDs). The majority of CPDs are cis-syn cyclobutane thymidine dimers. If you are interested in NH N 0.2 µmole $90 0.2 µmole $130 HO N N incorporating a cis-syn thymidine dimer in an oligonucleotide please inquire for a custom quotation. O N HO O N O 1.0 µmole $100 1.0 µmole $145 H 15 µmole $250 15 µmole $400 HO HO Oligonucleotides O 2'-Deoxynebularine K-2'-deoxyriboside O NH Use: Degenerate site Use: Degenerate site N N N N 3' Terminus 5' or Internal NH O O N O O 0.2 µmole $90 N µ HO N 0.2 mole $50 $160 HO N N O O NH2 1.0 µmole $100 1.0 µmole $100 $175 NH 15 µmole $300 15 µmole Inquire $450 O HO HO O P O N O O O 5-Methyl-2'-Deoxyisocytidine 5-Nitroindole-2'-deoxyriboside O Use: Duplex stability studies O Use: Universal base NO N 2 Oligonucleotides HO N 0.2 µmole $115 N NH2 0.2 µmole $110 O HO O 1.0 µmole $125 1.0 µmole $125 15 µmole $275 15 µmole $300 HO HO Trimer Oligonucleotide Synthesis 3-Nitropyrrole-2'-deoxyriboside P-2'-deoxyriboside O N Use: Universal base NO2 Use: Degenerate site 3' Terminus 5' or Internal NH N TriLink now offers oligonucleotide synthesis using 2'-deoxynucleoside trimer phosphoramidites. This chemistry HO O 0.2 µmole $110 0.2 µmole $50 $160 N O HO O is the simplest approach to oligonucleotide-directed mutagenesis. Although there are 64 possible codon 1.0 µmole $100 $175 1.0 µmole $125 combinations, all 20 amino acids can be achieved with only 20 codons. Please inquire for a custom quotation. 15 µmole $300 HO 15 µmole Inquire $450 HO Learn more about trimer oligonucleotides at www.trilinkbiotech.com.

Trimer* Amino Acid Abbrev. MW

d(AAA) Lys K 1911.5 NH2

d(AAC) Asn N 1887.5 N d(ACT) Thr T 1774.5 N d(ATC) Ile I 1774.5 O N O O N d(ATG) Met M 1780.5 NH d(CAG) Gln Q 1869.5 O d(CAT) His H 1774.5 O P O N O NH O 2 d(CCG) Pro P 1845.5 O d(CGT) Arg R 1756.5 N O d(CTG) Leu L 1756.5 O P O N O d(GAA) Glu E 1893.5 O d(GAC) Asp D 1869.5 O d(GCT) Ala A 1756.5 d(GGT) Gly G 1762.5 O d(GTT) Val V 1667.5 d(TAC) Tyr Y 1774.5 d(TCT) Ser S 1661.4 d(TGC) Cys C 1756.5 d(TGG) Trp W 1762.5 d(TTC) Phe F 1661.4

*Visit www.glenres.com for further information on trimer coding and physical parameters.

46 www.trilinkbiotech.com [email protected] (800) 863-6801 4747 Stocked Randomer Oligonucleotide Products

Stocked Randomer Oligonucleotide Products

Random Nonamers

5' Hydroxyl Random Nonamers (9mer "Wobble")

1 OD260 units O-30103-01 $30

2 OD260 units O-30103-02 $40

5' Amine Random Nonamers (9mer "Wobble")

1 OD260 units O-30104-01 $30

2 OD260 units O-30104-02 $40

6-FAM (Fluorescein) Antisense Oligo Controls

5' 6-FAM 20mer Phosphorothioate Oligonucleotides

2 OD260 units O-30020-02 $35 5 OD260 units O-30020-05 $50

Oligonucleotides S-18 Randomers

S-18 Randomer (Phosphorothioate 18mer Oligonucleotide)

50 nmoles O-30030-50 $75

S-18 Randomer Control (Phosphodiester 18mer Oligonucleotide)

50 nmoles O-30040-50 $75

48 www.trilinkbiotech.com 49 Nucleotides 51 H NMR, MS and 1 2 NR P O X X X P NMR, O O 31 O X X X O 2' Phosphoramidite O P N O O (800) 863-6801 O P 2 O NR Overview of Nucleotide Products Nucleotide of Overview P NMR and delivered as the lithium salt. Our P NMR and delivered as the lithium salt. O 2 31 PO P O OX NR X H NMR, MS and undergo a coupling test. O O 1 O P OX O XX O X P NMR, Amidites. If you would like a quotation for your custom 31 TM N x = your imagination x = your imagination

N X X 2 X X O NR H NMR and MS analysis along with a certificate of analysis. Please email or along with a certificate of analysis. H NMR and MS analysis 1 O P O Nucleoside Nucleoside Triphosphates X X O Nucleoside Triphosphate Synthesis Service Synthesis Triphosphate Nucleoside OH P NMR, 31 3' Phosphoramidite Custom Phosphoramidite Synthesis Service Service Synthesis Custom Phosphoramidite [email protected] N Custom

TriLink is unique in offering both custom phosphoramidite synthesis and incorporation into an oligonucleotide. is unique in offering both custom phosphoramidite synthesis TriLink quality phosphoramidites going As oligonucleotide chemists we understand how important it is to have high including amino linkers, into each synthesis. In fact, we manufacture many of our own phosphoramidites spacers and our patented CleanAmp disulfides, phosphoramidite email [email protected]. Please include the structure and the final yield required. phosphoramidite email [email protected]. Please include the structure All phosphoramidites are analyzed by RP-HPLC, service includes HPLC, TriLink has developed detailed protocols to convert nucleosides into triphosphates. In this custom service the protocols to convert nucleosides into triphosphates. In this custom service has developed detailed TriLink customer supplied or purchased if it is commercially available. Each starting nucleoside can be made in house, and greater than 90% by AX-HPLC to triphosphate is purified call for a quote on your specific compound. UV Spectroscopy. Your order will be accompanied by a certificate of analysis and the HPLC analysis. and certificate of analysis order will be accompanied by a Your UV Spectroscopy.

It is likely that among TriLink's wide selection of modified nucleoside triphosphates you will find a compound you will nucleoside triphosphates selection of modified wide that among TriLink's It is likely µmole aliquots in 1, 5 and 10 are sold individually nucleoside triphosphates application. Our to suit your inquire for larger most cases. Please mM solutions in as 100 5 mg respectively) 0.5, 2.5 and (approximately are analyzed by HPLC, concentrations. All of our nucleotides quantities or specific 5050 51 51 52 52 53 53 54 56 62 67 78 74 74 75 77 La Jolla, California © istockphoto.com/photo168 La

...... riphosphates www.trilinkbiotech.com ...... riphosphates riphosphates riphosphates riphosphates ...... riphosphates ...... ' Deoxynucleoside Triphosphates ...... Aminoallyl Nucleoside T CAP and mCAP Biotin Nucleoside T 2' Fluoro Nucleoside T eatured Products abeled Nucleoside Monophosphates Overview of Nucleotide Products Overview Custom Synthesis Services F NTP Quick Guide

Base Modified 2 Base Modified Ribonucleoside T Sugar Modified Nucleoside T Bisphosphate Nucleosides CAP and mCAP Alpha Phosphate Modified Nucleoside T L Nucleotide Kits

The purchase of these compounds does not convey any right or license to any patent rights and the use of aminoallyl The purchase of these compounds does not convey any right or license to any patent rights and the use of aminoallyl nucleotides in certain applications may be protected does not warrant that the use of these products will not infringe the claims of a US or foreign patent covering the compound or its use TriLink under third party patent rights. against any such claims. TriLink whether alone or in combination with other productsor in any process. Buyer agrees, as a condition of sale, to indemnify TriLink The purchase of a compound from under licenses that provide limited rights to end users for research purposes. TriLink Certain nucleoside triphosphates are sold by makes no representations or warranties that the use of any compound, alone or in combination with other TriLink does not convey rights for use in any particular application. or foreign patent. United States will not infringe the claims of a products, or in any process, See Licensing Information section for the disclaimer of license statement pertaining to PCR products. Nucleotides 50 52 52 Nucleotides Catalog # oligonucleotides. custom Biotin-labeled NTPsand TriLinkBiotin products. both competitors offers the limited that mixture 30% the to compared as 100%, as much as TriLink'sincorporated Biotin-dUTP study,recent a In incorporation. of level higher a equals quality higher incorporation; of level the to related directly is NTP labeled a of quality The RNA. labeled using detection target microarray as such detection, strong and hybridization sensitive require that assays in used frequently are acids nucleic labeled Biotin Catalog #Description Page synthesis. cDNA and reactions extension primer translation, nick PCR, in labeling DNA indirect for used NTPsare Aminoallyl molecule. DNA resultant the throughout conjugated be can protein, reactive amine an or hapten biotin, dye, fluorescent moiety,a reactive as such amine NTPs,an Aminoallyl of incorporation NTPs.Followingenzymatic pre-labeled of incorporation than DNA, of labeling density high for method efficient more a offer triphosphates nucleoside modified Aminoallyl Featured Products -04N Biotin-16-Aminoallylcytidine-5'-Triphosphate Biotin-16-Aminoallyl-2'-deoxycytidine-5'-Triphosphate N-5004 Biotin-16-Aminoallyl-2'-deoxyuridine-5'-Triphosphate N-5003 N-5002 N-5001 -065'-Biotin-dA-Monophosphate 5'-Biotin-dG-Monophosphate 5'-Biotin-A-Monophosphate N-6006 5'-Biotin-G-Monophosphate N-6005 58 Biotin-16-7-Deaza-7-Aminoallyl-2'-deoxyguanosine-5'-Triphosphate N-6004 Biotin-16-Aminoallyluridine-5'-Triphosphate N-6003 N-5006 N-5005 -025-Aminoallyluridine-5'-Triphosphate 5-Aminoallylcytidine-5'-Triphosphate 5-Aminoallyl-2'-deoxyuridine-5'-Triphosphate N-1062 5-Aminoallyl-2'-deoxycytidine-5'-Triphosphate N-1065 N-2049 N-2048 4 -Biotin-OBEA-2'-deoxycytidine-5'-Triphosphate Aminoallyl Nucleoside TriphosphatesNucleoside Aminoallyl Description Page Biotin Nucleoside TriphosphatesNucleoside Biotin www.trilinkbiotech.com

63 56 57 77 77 77 77 63 63 62 62 56 56 ε Catalog # place. market the in mCAP and CAP of source expensive TriLinkleast ribosome. the the is to translation. for ready mRNA makes that process the is mRNA immature the of end 5’ the to linkage 5’-5’ unique a via mCAP of addition vivo(italics) In transport. and splicing in role a play also may CAP ribosome. the to mRNA of attachment proper and recognition mRNA of study the in reagents important are mCAP and CAP Catalog #Description Page polymerase. RNA T7 used widely the including polymerases, RNA and DNA both for substrates excellent NTPsare Fluoro 2' response. immune reducing while resistance nuclease and affinity target increased impart they because siRNA, and antagomirs aptamers, of synthesis and design the in used are They RNA. and DNA both in stability vivo)italic (in in improvement for incorporated enzymatically monly com- most However,NTPsare kinases. Fluoro applicable 2' pharamceutically of investigation the in enzymes ATP-requiringscreening for tool universal a be to confirmed ATPwas Fluoro 2' Recently development. drug new and research in applications of number increasing an in utilized being are triphosphates nucleoside Fluoro 2' -01N Guanosine-5'-Triphosphate-5'-Guanosine N-7001 N-7002 -052'-Fluorothymidine-5'-Triphosphate 2'-Fluoro-2'-deoxyuridine-5'-Triphosphate 2'-Fluoro-2'-deoxyguanosine-5'-Triphosphate N-1055 2'-Fluoro-2'-deoxycytidine-5'-Triphosphate N-1010 2'-Fluoro-2'-deoxyadenosine-5'-Triphosphate N-1009 N-1008 N-1007

is notedinL mmole CAP also provides significant resistance to 5’ exonucleases, allowing mRNA the time needed to get to needed time the mRNA allowing exonucleases, 5’ to resistance significant provides also CAP -1 cm -1 [email protected] . 7 -Me-Guanosine-5'-Triphosphate-5'-Guanosine 2' Fluoro Nucleoside TriphosphatesNucleoside Fluoro 2' Description Page CAP and mCAP and CAP

(800) 863-6801 (800) Featured Products

74 74 71 72 71 71 71 53 Nucleotides 54 Nucleotides a. Name Cat.# N-1039 N-1038 N-1037 N-1036 N-1035 N-1034 N-1033 N-1032 N-1043 N-1040 N-1014 N-1013 N-1012 N-1011 N-1010 N-1009 N-1008 N-1007 N-1006 N-1004 N-1002 N-1001 N-1044 N-1041 N-1015 N-1042 N-1016 N-1017 N-1018 N-1019 N-1020 N-1021 N-1022 N-1023 N-1024 N-1025 N-1026 N-1027 N-1028 N-1029 N-1031 NTP Quick Guide Quick NTP -z-T 7.0 350 $2.0 62 $625.00 62 $365.00 $625.00 66 $77.50 $365.00 $625.00 $77.50 $365.00 N 64 $77.50 6-Aza-UTP $850.00 6-Aza-CTP $465.00 $97.50 2-Thio-CTP 5,6-Dihydro-UTP Ara-UTP -hoUP$75 $6.0$2.066 $625.00 $365.00 $77.50 Ara-CTP 2-Thio-UTP methyl-UTP 2'-O-Methyl-5- Amino-ATP 2'-O-Methyl-2- 64 $625.00 $365.00 $77.50 5-Methyl-CTP -ooUP$75 $6.0 650 64 $625.00 64 $365.00 $625.00 $77.50 $365.00 72 $77.50 N $415.00 71 5-Iodo-UTP $255.00 $850.00 71 5-Iodo-CTP $52.50 $465.00 $415.00 71 2'-Fluoro-2'-dUTP $625.00 $97.50 $255.00 2'-Fluoro-2'-dGTP $365.00 $52.50 62 2'-Fluoro-2'-dCTP $77.50 $2,250.00 $1,150.00 2'-Fluoro-2'-dATP $250.00 boside-TP 62 6-Chloropurineri- $625.00 8-Aza-ATP $365.00 TP $77.50 ropurineriboside- 2-Amino-6-Chlo- 2-Amino-ATP -ez-T 9.0 450 $5.0 64 $850.00 $465.00 $97.50 7-Deaza-GTP do-UTP 2'-O-Methylpseu- 72 $205.00 $115.00 $27.50 2'-O-Methyl-ATP N 'OMty-T 2.0 150 $0.0 72 $205.00 $115.00 $27.50 2'-O-Methyl-CTP 'OMty-T 2.0 150 $0.0 72 $205.00 $115.00 $27.50 2'-O-Methyl-GTP 'OMty-T 2.0 150 $0.0 73 $205.00 $115.00 $27.50 2'-O-Methyl-UTP suoUP$75 $6.0 650 65 $625.00 $365.00 $77.50 Pseudo-UTP ITP 'OMty-T 7.0 350 $2.0 72 $625.00 $365.00 $77.50 2'-O-Methyl-ITP uoyi-P$75 $6.0 800 66 $850.00 $465.00 $97.50 Puromycin-TP atoieT 5.0 250 $1.0 66 $415.00 $255.00 $52.50 Xanthosine-TP -ehlUP$75 $6.0$2.0 65 $625.00 $365.00 $77.50 5-Methyl-UTP -hoUP$75 $6.0 800 66 $850.00 $465.00 $97.50 4-Thio-UTP 'Aio2-CP$75 $6.0 650 67 $625.00 $365.00 $77.50 2'-Amino-2'-dCTP 'Aio2-UP$75 $6.0 650 67 $625.00 $365.00 $77.50 2'-Amino-2'-dUTP 'Aio2-CP$75 $6.0 650 68 $625.00 $365.00 $77.50 2'-Azido-2'-dCTP 'Aio2-UP$75 $6.0 650 68 $625.00 $365.00 $77.50 2'-Azido-2'-dUTP O 1 1 6 6 Mty-T 7.0 350 $2.0 65 $625.00 $365.00 $77.50 -Methyl-ATP 65 $625.00 $365.00 $77.50 -Methyl-GTP 65 $415.00 $255.00 $52.50 -Methyl-ATP Mty-T 150 $9.0 1250 65 $1,235.00 $695.00 $155.00 -Methyl-GTP 9.0 450 $5.0 68 $850.00 68 $465.00 $415.00 $97.50 $255.00 $52.50 5.0 250 $1.0 72 $415.00 $255.00 $52.50 72 $625.00 $365.00 $77.50 7.0 350 $2.0 64 $625.00 $365.00 $77.50 62 $850.00 $465.00 $97.50 5.0 250 $1.0 73 $415.00 $255.00 $52.50 5.0 250 450 64 $415.00 $255.00 $52.50 ml5µo 0µo Pg 10µmol 5µmol 1 µmol NTP QuickGuide www.trilinkbiotech.com www.trilinkbiotech.com N-2028 a. Name Cat.# N-2026 N-1045 N-2027 N-1046 N-1047 N-1048 N-1052 N-1054 N-1053 N-1055 N-1056 N-1057 N-1058 N-1059 N-1061 N-1062 N-1063 N-1064 N-1065 N-1066 N-1067 N-2002 N-2003 N-2004 N-2006 N-2008 N-2009 N-2010 N-2012 N-2011 N-2016 N-2017 N-2019 N-2020 N-2022 N-2023 N-2024 N-2025 -ehl2-CP$25 $5.0 450 60 $415.00 $255.00 $52.50 5-Methyl-2'-dCTP 'Aio2-AP$75 $6.0 650 68 $625.00 $365.00 $77.50 2'-Azido-2'-dATP O 'Aio2-AP$25 $5.0 450 67 $415.00 $255.00 $52.50 2'-Amino-2'-dATP riboside-TP Benzimidazole- Ara-ATP N dGTP -zd-T 5.0 250 $1.0 63 $415.00 $255.00 $52.50 8-Azido-ATP -rm-T 5.0 250 $1.0 63 $415.00 $255.00 63 $1,235.00 $52.50 $695.00 $155.00 5-Bromo-UTP 5-Bromo-CTP 'Fur-TP$75 $6.0 650 71 $625.00 $365.00 $77.50 2'-Fluoro-dTTP 'OMty-T 7.0 350 $2.0 73 $625.00 $365.00 $77.50 3'-O-Methyl-ATP 'OMty-T 7.0 350 $2.0 73 $625.00 $365.00 $77.50 3'-O-Methyl-CTP 'OMty-T 7.0 350 $2.0 73 $625.00 $365.00 $77.50 3'-O-Methyl-GTP 'OMty-T 7.0 350 $2.0 73 $625.00 $365.00 $77.50 3'-O-Methyl-UTP -ez-T 7.0 350 $2.0 64 $625.00 $365.00 $77.50 7-Deaza-ATP -mnallUP$25 $5.0 450 62 $415.00 $255.00 $52.50 5-Aminoallyl-UTP 'Aio2-GP$75 $6.0 650 68 $625.00 $365.00 $77.50 2'-Azido-2'-dGTP 'Aio2-GP$75 $6.0 650 67 $625.00 $365.00 $77.50 2'-Amino-2'-dGTP -mnallCP$75 $6.0 650 62 $625.00 $365.00 $77.50 5-Aminoallyl-CTP -x-T 5.0 250 $1.0 65 $415.00 $255.00 $52.50 8-Oxo-GTP riboside-TP 2-Aminopurine- deoxyriboside-TP chloropurine-2'- 2-Amino-6- -mn-'dT 7.0 350 $2.0 56 $625.00 $365.00 $77.50 2-Amino-2'-dATP 2'-deoxyribose-TP 2-Aminopurine- -rm-'dT 7.0 350 $2.0 57 $625.00 $365.00 $77.50 5-Bromo-2'-dCTP -rm-'dT 2.0 150 $0.0 57 $205.00 $115.00 $27.50 5-Bromo-2'-dUTP 2'-deoxyriboside-TP 6-Chloropurine- -ez-'dT 5.0 250 $1.0 58 $415.00 $255.00 $52.50 7-Deaza-2'-dATP -ez-'dT 150 $9.0 1250 58 $1,235.00 $695.00 2'-dITP $155.00 7-Deaza-2'-dGTP dCTP 5-Propynyl-2'- dUTP 5-Propynyl-2'- suo2-UP$75 $6.0 650 59 $625.00 $365.00 $77.50 Pseudo-2'-dUTP 2'-dUTP -loo2-UP$25 $5.0 450 59 $415.00 $255.00 $52.50 5-Fluoro-2'-dUTP -oo2-CP$75 $6.0 800 59 $850.00 $465.00 $97.50 5-Iodo-2'-dCTP -oo2-UP$25 $5.0 450 59 $415.00 $255.00 $52.50 5-Iodo-2'-dUTP N 6 2 6 Mty-'dT 9.0 450 $5.0 60 $850.00 $465.00 $97.50 -Methyl-2'-dATP -Methyl-2'- Mty-'dT 150 $9.0 1250 60 $1,235.00 $695.00 $155.00 -Methyl-2'-dGTP $250.00 9.0 450 $5.0 63 $850.00 $465.00 $97.50 7.0 350 $2.0 68 $625.00 $365.00 $77.50 9.0 450 $5.0 62 $850.00 $465.00 $97.50 9.0 450 $5.0 56 $850.00 $465.00 $97.50 9.0 450 $5.0 56 $850.00 $465.00 $97.50 7.0 350 $2.0 57 $625.00 $365.00 $77.50 7.0 350 $2.0 58 $625.00 $365.00 $77.50 7.0 350 $2.0 61 $625.00 $365.00 $77.50 7.0 350 $2.0 61 $625.00 $365.00 $77.50 2.0 150 $0.0 59 $205.00 $115.00 $27.50 ml5µo 0µo Pg 10µmol 5µmol 1 µmol 1100 $,5.0 60 $2,250.00 $1,150.00 a. Name Cat.# N-2057 N-2056 N-2055 N-2054 N-2053 N-2049 N-2048 N-2046 N-2045 N-2043 N-2042 N-2041 N-2039 N-2038 N-2037 N-2035 N-2034 N-2033 N-2031 N-3001 N-2058 N-3002 N-3003 N-3004 N-3005 N-4001 N-4002 N-4003 N-4004 N-4005 N-4007 N-4008 N-4009 N-4013 N-4012 N-4011 N-4010 N-4014

dCTP 'DoyLTP$75 $6.0 800 70 $850.00 $465.00 69 69 $97.50 $1,450.00 $850.00 69 N $775.00 $850.00 56 $465.00 $175.00 2'-Deoxy-L-TTP $415.00 $465.00 $97.50 56 2'-Deoxy-L-GTP $255.00 $625.00 $97.50 2'-Deoxy-L-CTP $52.50 $365.00 57 2'-Deoxy-L-ATP $625.00 $77.50 56 5-AA-2'-dUTP $365.00 61 $625.00 61 61 5-AA-2'-dCTP $2,250.00 $77.50 $365.00 $1,150.00 $850.00 $625.00 $250.00 8-Chloro-2'-dATP $77.50 $465.00 $365.00 6-Thio-2'-dGTP $97.50 $77.50 6-Aza-2'-dUTP 2-Thio-2'-dCTP 4-Thio-TTP 61 $850.00 dUTP 5-Hydroxy-2'- $465.00 60 dCTP $97.50 $625.00 60 5-Hydroxy-2'- $625.00 $365.00 2'-dPTP $365.00 $77.50 2-Thio-TTP $77.50 8-Oxo-2'-dGTP 8-Oxo-2'-dATP 2'-Deoxyribose-TP 5-Nitro-1-Indolyl- 3'-dATP ine-TP 2'-Deoxyzebular- 3'-dGTP 3'-dCTP -ehl3-UP$75 $6.0 650 70 $625.00 $365.00 $77.50 5-Methyl-3'-dUTP 3'-dUTP '3-dT 5.0 250 $1.0 70 $415.00 $255.00 $52.50 2',3'-ddATP '3-dT 5.0 250 $1.0 71 $415.00 $255.00 $52.50 2',3'-ddGTP '3-dT 2.0 150 $0.0 71 $205.00 $115.00 $27.50 2',3'-ddUTP '3-dT 2.0 150 $0.0 71 $205.00 $115.00 $27.50 2',3'-ddTTP '3-dT 2.0 150 $0.0 70 $205.00 $115.00 $27.50 2',3'-ddCTP ddATP 3'-Azido-2',3'- ddGTP 3'-Azido-2',3'- 'Aio3-TP$75 $6.0 800 69 $850.00 $465.00 $97.50 3'-Azido-3'-dTTP 'Aio3-TP$75 $6.0 800 67 $850.00 $465.00 $97.50 3'-Amino-3'-dTTP ddGTP 3'-Amino-2',3'- ddCTP 3'-Amino-2',3'- ddATP 3'-Amino-2',3'- ddCTP 3'-Azido-2',3'- 4 -Methyl-2'-

in [email protected] [email protected] 200 $,5.0 2200 60 $2,250.00 $1,150.00 $250.00 200 $,5.0 2200 59 $2,250.00 $1,150.00 $250.00 150 $7.0 1400 67 $1,450.00 $775.00 67 $175.00 $1,450.00 $775.00 67 $175.00 $1,450.00 $775.00 $175.00 7.0 350 $2.0 60 $625.00 $365.00 $77.50 9.0 450 $5.0 59 $850.00 59 $465.00 $625.00 $97.50 58 $365.00 $850.00 $77.50 $465.00 $97.50 7.0 350 $2.0 70 $625.00 $365.00 $77.50 7.0 350 $2.0 70 $625.00 $365.00 $77.50 7.0 350 $2.0 70 $625.00 $365.00 $77.50 7.0 350 $2.0 70 $625.00 $365.00 $77.50 9.0 450 $5.0 68 $850.00 $465.00 $97.50 9.0 450 $5.0 69 $850.00 $465.00 $97.50 9.0 450 $5.0 69 $850.00 $465.00 $97.50 ml5µo 0µo Pg 10µmol 5µmol 1 µmol NTP QuickGuide

a. Name Cat.# N-9001 N-8051 N-8015 N-8013 N-8012 N-8011 N-8010 N-8009 N-8008 N-8007 N-8006 N-8005 N-8004 N-8003 N-8002 N-8001 N-7002 N-7001 N-6010 N-6009 N-6006 N-6005 N-6004 N-6003 N-6002 N-6001 N-5007 N-5006 N-5005 N-5004 N-5003 N-5002 N-5001 N-4017 N-4016 N-4015 accoi-P$75 $6.0 650 72 $625.00 $365.00 $77.50 Ganciclovir-TP dCTP (1-Borano)-2'- ddUTP 76 (1-Thio)-2',3'- $415.00 2',3'-ddTTP $255.00 3'-Azido-(1-Thio)- $52.50 (1-Thio)-2',3'-ddTTP ddGTP 76 (1-Thio)-2',3'- $205.00 76 ddCTP $115.00 $205.00 (1-Thio)-2',3'- 75 $27.50 $115.00 $625.00 ddATP 75 (1-Thio)-2',3'- $205.00 $27.50 $365.00 $115.00 $77.50 (1-Thio)-UTP 75 $205.00 $27.50 (1-Thio)-GTP 75 $115.00 (1-Thio)-CTP $415.00 75 $27.50 $255.00 $205.00 (1-Thio)-ATP 75 $205.00 $52.50 $115.00 (1-Thio)-2'-dTTP $115.00 $27.50 (1-Thio)-2'-dGTP 77 $27.50 (1-Thio)-2'-dCTP $415.00 77 (1-Thio)-2'-dATP $255.00 $415.00 77 CAP $52.50 $255.00 $415.00 mCAP 77 $52.50 $255.00 77 $415.00 5'-Amino-AMP $415.00 $52.50 $255.00 77 5'-Amino-GMP $255.00 $415.00 $52.50 5'-Biotin-dAMP $52.50 $255.00 5'-Biotin-dGMP $52.50 5'-Biotin-AMP 5'-Biotin-GMP pGp 63 ppGpp N/A 2'-dUTP 5-(Dabcyl-3-AA)- N/A dGTP (Biotin-16)-AA-2'- 7-Deaza-7- $525.00 Biotin-16-AA-UTP 2'-dCTP itn1-ACP$2.0 / NA63 N/A N/A N $525.00 71 Biotin-16-AA-CTP $1,235.00 dCTP $695.00 Biotin-16-AA-2'- $155.00 dUTP Biotin-16-AA-2'- 2',3'-ddITP ddUTP 5-Bromo-2',3'- ddUTP 3'-Azido-2',3'- 4 Biotin-OBEA- (800) 863-6801 (800) 150 $7.0 1400 75 $1,450.00 $775.00 $175.00 74 $1,235.00 74 $695.00 $1,450.00 58 $155.00 $775.00 N/A $175.00 57 N/A N/A $725.00 N/A $525.00 550 NANA56 N/A 57 N/A N/A N/A $525.00 $525.00 5.0 250 $1.0 76 $415.00 75 $255.00 $850.00 $52.50 $465.00 $97.50 76 $415.00 76 $255.00 $625.00 $52.50 76 $365.00 $625.00 $77.50 $365.00 $77.50 74 $625.00 74 $365.00 $625.00 $77.50 $365.00 $77.50 9.0 450 $5.0 69 $850.00 69 $465.00 $850.00 $97.50 $465.00 $97.50 ml5µo 0µo Pg 10µmol 5µmol 1 µmol

/ / / 58 N/A N/A N/A NTP Quick Guide Quick NTP 55 Nucleotides 56 Nucleotides 6-Aza-2'-deoxyuridine-5'-Triphosphate 5-Aminoallyl-2'-deoxyuridine-5-Triphosphate 5-Aminoallyl-2'-deoxycytidine-5-Triphosphate 2-Amino-6-chloropurine-2'-deoxyriboside-5'-Triphosphate (Diaminopurine deoxyribose) 2-Amino-2'-deoxyadenosine-5'-Triphosphate Base Modified2'Deoxynucleoside Triphosphates ε 2-Aminopurine-2'-deoxyriboside-5'-Triphosphate Biotin-16-Aminoallyl-2'-deoxycytidine-5'-Triphosphate

is notedinL mmole N-2043-10 10 N-2043-5 5 N-2043-1 1 N-2049-10 10 N-2048-10 10 N-2003-10 10 N-2002-10 10 N-2049-5 5 N-2049-1 1 N-2048-5 5 N-2048-1 1 N-2003-5 5 N-2003-1 1 N-2002-5 5 N-2002-1 1 N-2004-10 10 N-2004-5 5 N-2004-1 1 N-5002-1 1 N-5002-050 Base Modified2'DeoxynucleosideTriphosphates -1 cm -1 . 50 nmoles$197.50 mmoles $625.00 mmoles $365.00 mmole $77.50 mmoles $415.00 mmoles $625.00 mmoles $625.00 mmoles $850.00 mmoles $255.00 mmole $52.50 mmoles $365.00 mmole $77.50 mmoles $365.00 mmole $77.50 mmoles $465.00 mmole $97.50 mmoles $850.00 mmoles $465.00 mmole $97.50 mmole $525.00 NH www.trilinkbiotech.com O S NH O 4 4 4 4 4 Li 4 Li Li Li Li + Li + + + + H N O O + O O O O O O O O O O PO PO PO PO PO PO O O O O O O O O O O O O P P P P P P O O O O O 2 2 NH O NH O O O O O O O O O O O O PO PO

PO PO PO PO O O O O O O O OH OH OH OH OH OH N H N O 4 O O O O Li O + O N N NH O N O O N N N N N N 2 PO NH N NH O O O O O N N Cl O NH P N O 2 N N H N O O N PO NH NH NH O 2 2 2 OH O NH N 2 N MW= 469.13 (freeacid) MW= 523.22 (freeacid) MW= 506.20 (freeacid) MW= 525.63 (freeacid) MW= 491.18 (freeacid) O MW= 947.80 (freeacid) MW= 522.0 (freeacid) ε ε ε ε ε ε ε = 6254@260nm = 8743@279nm = 7100@290nm = 5041@290nm = 6590@306nm = 6422@303nm = 7100@290nm C C 10 C C C C C 32 H 12 12 10 10 >90% purity 8 >90% purity >90% purity >90% purity >90% purity H >90% purity Lithium salt 15 Lithium salt Lithium salt Lithium salt Lithium salt >90% purity H Lithium salt H H H H Lithium salt 53 N 14 17 20 21 16 N 5 N N N N N O 8 3 O 3 4 6 5 12 O O O O O 17 P 14 14 13 12 12 P 3 P P P P Cl P 3 3 S 3 3 3 3 56 ε 6-Chloropurine-2'-deoxyriboside-5'-Triphosphate 8-Chloro-2'-deoxyadenosine-5'-Triphosphate 5-Bromo-2'-deoxyuridine-5'-Triphosphate 5-Bromo-2'-deoxycytidine-5'-Triphosphate N Biotin-16-Aminoallyl-2'-deoxyuridine-5'-Triphosphate

is notedinL mmole 4 -Biotin-OBEA-2'-deoxycytidine-5'-Triphosphate Base Modified2'DeoxynucleosideTriphosphates continued N-2009-10 10 N-2046-10 10 N-2006-10 10 N-5004-1 1 N-2009-5 5 N-2009-1 1 N-2046-5 5 N-2046-1 1 N-2008-10 10 N-2008-5 5 N-2008-1 1 N-2006-5 5 N-2006-1 1 N-5004-050 N-5001-1 1 N-5001-050 -1 cm -1 [email protected] . 50 nmoles$197.50 50 nmoles$197.50 mmoles $625.00 mmoles $625.00 mmoles $625.00 mmole $525.00 mmoles $365.00 mmole $77.50 mmoles $365.00 mmole $77.50 mmoles $205.00 mmoles $115.00 mmole $27.50 mmoles $365.00 mmole $77.50 mmole $525.00 Base Modified2'Deoxynucleoside Triphosphates

NH O S NH NH O S NH O 4 4 4 4 Li Li Li Li H N + + + + O O O O O O O O PO PO PO PO O O O O O O O H N O O P P P P O O O O O O O O O O O O PO PO PO PO O O 4 O O TE O (800) 863-6801 (800) A Br Br OH + OH OH OH Cl N H O O 4 Li PO O O + O O O O O O N NH O N O N N N N PO N H 2 P N O NH O O O O O O O P NH N N Cl PO O O 2 H N N N O O O PO O OH O OH NH N O N O N O NH O MW= 510.61 (freeacid) MW= 525.63 (freeacid) MW= 547.04 (freeacid) MW= 546.05 (freeacid) MW= 893.70 (freeacid) MW= 946.70 (freeacid) Triethylammonium salt ε ε ε ε ε = 14900@262nm ε = 6600@264nm = 9220@279nm = 6825@288nm = 9100@272nm = 5040@290nm C C C C C C 10 10 29 9 9 H H H H 32 H >90% purity >90% purity >90% purity >90% purity >90% purity 14 15 H 14 15 Lithium salt Lithium salt Lithium salt Lithium salt >90% purity 50 Lithium salt N N N N 52 N 2 3 N 4 5 7 O O O O O 7 O 14 13 12 12 17 P P P P 18 P 3 3 3 3 P

3 Br Br Cl Cl

S 3 S 57 Nucleotides 58 Nucleotides 7-Deaza-7-(Biotin-16)aminoallyl-2'-deoxyguanosine-5'-Triphosphate 5-(Dabcyl-3-Aminoallyl)-2'-deoxyuridine-5'-Triphosphate ε 2'-Deoxy-P-nucleoside-5-Triphosphate 2'-Deoxyinosine-5'-Triphosphate 7-Deaza-2'-deoxyguanosine-5'-Triphosphate 7-Deaza-2'-deoxyadenosine-5'-Triphosphate Base Modified2'Deoxynucleoside Triphosphates

is notedinL mmole Base Modified2'DeoxynucleosideTriphosphates continued N-5007-0100 N-5006-1 1 N-5006-1 N-5006-050 N-2011-10 10 N-2037-10 10 N-2012-10 10 N-2011-5 5 N-2010-10 10 N-2037-5 5 N-2037-1 1 N-2012-5 5 N-2012-1 1 N-2011-1 1 N-2010-5 5 N-2010-1 1 -1 cm 100nmole$197.50 -1 50 nmoles$215.00 . mmole $725.00 mmoles $1235.00 mmoles $850.00 mmoles $625.00 mmoles $695.00 mmoles $415.00 mmoles $465.00 mmole $97.50 mmoles $365.00 mmole $77.50 mmole $155.00 mmoles $255.00 mmole $52.50 www.trilinkbiotech.com O S NH HN N N 4 O Li 4 4 4 + N Li Li Li H N + + O + O O O O O O O PO N PO PO PO O O O O 4 O Li O O O P + P O P P O O O O O O O PO O O PO O O O O O O

PO PO P O O O O O P 4 O OH Li O O + H N OH OH OH O O O O O N H PO PO O O O O O N O O N N N N N P OH NH N O O O O O O NH N N O NH PO O O N O NH 2 NH N H N 2 NH OH O O N N O MW= 774.51 (freeacid) MW= 985.80 (freeacid) NH MW= 509.20 (freeacid) MW= 492.17 (freeacid) MW= 506.00 (freeacid) MW= 490.20 (freeacid) ε ε ε = [email protected] NH ε = 32000@467nm ε ε = 10100@298nm = 10069@259nm 2 = 9500@271nm = 6473@293nm C C 34 C C C C 27 >90% purity H Lithium salt 10 11 11 11 >90% purity H >90% purity >90% purity >90% purity >90% purity Lithium salt Lithium salt Lithium salt Lithium salt Lithium salt 54 H H H H 33 N 17 17 18 15 N 9 N N N N 8 O O 3 4 4 4 17 O O O O 15 P 14 13 13 12 P 3 P P P P 3 S 3 3 3 3 ε 5-Iodo-2'-deoxyuridine-5'-Triphosphate 5-Iodo-2'-deoxycytidine-5'-Triphosphate 5-Hydroxy-2'-deoxyuridine-5'-Triphosphate 5-Hydroxy-2'-deoxycytidine-5'-Triphosphate 2'-Deoxyzebularine-5'-Triphosphate 2'-Deoxyuridine-5'-Triphosphate 2'-Deoxypseudouridine-5'-Triphosphate 5-Fluoro-2'-deoxyuridine-5'-Triphosphate

is notedinL mmole Base Modified2'DeoxynucleosideTriphosphates continued N-2024-10 10 N-2023-10 10 N-2039-10 10 N-2038-10 10 N-2058-10 10 N-2058-5 5 N-2019-10 10 N-2024-5 5 N-2024-1 1 N-2023-5 5 N-2023-1 1 N-2039-5 5 N-2039-1 1 N-2038-5 5 N-2038-1 1 N-2058-1 1 N-2020-10 10 N-2020-5 5 N-2020-1 1 N-2019-5 5 N-2019-1 1 N-2022-10 10 N-2022-5 5 N-2022-1 1 -1 cm -1 [email protected] . mmoles $415.00 mmoles $850.00 mmoles $850.00 mmoles $625.00 mmoles $2250.00 mmoles $1150.00 mmoles $625.00 mmoles $255.00 mmole $52.50 mmoles $465.00 mmole $97.50 mmoles $465.00 mmole $97.50 mmoles $365.00 mmole $77.50 mmole $250.00 mmoles $205.00 mmoles $115.00 mmole $27.50 mmoles $365.00 mmole $77.50 mmoles $415.00 mmoles $255.00 mmole $52.50 Base Modified2'Deoxynucleoside Triphosphates

4 4 4 4 4 4 Li 4 4 Li Li Li Li Li + Li Li + + + + + O O + + O O O O O O O O O O O O PO O O PO PO PO PO PO O PO PO O O O O O O O O O O O O O P O O P P O P P P P P O O O O O O O O O O O O O PO O O O O O O O O O O O PO PO PO PO PO PO PO O O O O O O O OH (800) 863-6801 (800) OH OH OH OH OH OH OH OH I OH I O F O O NH N O O O O O O N NH N N O O N N NH O N O N 2 2 O N NH NH N NH NH NH O O O O O O O MW= 484.14 (freeacid) MW= 483.16 (freeacid) MW= 458.19 (freeacid) MW= 468.14 (freeacid) MW= 468.14 (freeacid) MW= 594.04 (freeacid) MW= 593.05 (freeacid) MW= 486.13 (freeacid) ε ε ε ε ε ε ε ε = 10100@262nm = 8300@287nm = 5300@294nm = 8200@280nm = 6500@293nm = 5800@302nm = 7385@260nm = 7350@268nm C C C C C C C 9 9 9 H >90% purity >90% purity >90% purity >90% purity >90% purity >90% purity >90% purity 9 9 9 9 >90% purity H H Lithium salt Lithium salt Lithium salt Lithium salt Lithium salt Lithium salt Lithium salt Lithium salt H H H H 14 C 14 15 15 15 15 16 N 9 N N H N N N N 2 2 3 O 12 2 2 2 3 O O O O O O N 14 14 13 14 14 15 14 P 2 P P

O P P P P 3

3 3 F 4 3 3 3 3 I I 59 Nucleotides 60 Nucleotides N ε (8-Hydroxy-2'-deoxyguanosine) 8-Oxo-2'-deoxyguanosine-5'-Triphosphate (8-Hydroxy-2'-deoxyadenosine) 8-Oxo-2'-deoxyadenosine-5'-Triphosphate 5-Nitro-1-indolyl-2'-deoxyribose-5-Triphosphate O N N 5-Methyl-2'-deoxycytidine-5'-Triphosphate Base Modified2'Deoxynucleoside Triphosphates

6 is notedinL mmole 4 2 6 -Methyl-2'-deoxyadenosine-5'-Triphosphate -Methyl-2'-deoxycytidine-5'-Triphosphate -Methyl-2'-deoxyguanosine-5'-Triphosphate -Methyl-2'-deoxyguanosine-5'-Triphosphate Base Modified2'DeoxynucleosideTriphosphates continued N-2025-10 10 N-2025-5 5 N-2025-1 1 N-2027-10 10 N-2034-10 10 N-2033-10 10 N-2031-10 10 N-2031-5 5 N-2027-5 5 N-2057-10 10 N-2028-10 10 N-2028-5 5 N-2026-10 10 N-2034-5 5 N-2034-1 1 N-2033-5 5 N-2033-1 1 N-2031-1 1 N-2027-1 1 N-2057-5 5 N-2057-1 1 N-2028-1 1 N-2026-5 5 N-2026-1 1 -1 cm -1 . mmoles $850.00 mmoles $465.00 mmole $97.50 mmoles $1235.00 mmoles $625.00 mmoles $625.00 mmoles $2250.00 mmoles $1150.00 mmoles $695.00 mmoles $625.00 mmoles $2250.00 mmoles $1150.00 mmoles $415.00 mmoles $365.00 mmole $77.50 mmoles $365.00 mmole $77.50 mmole $250.00 mmole $155.00 mmoles $365.00 mmole $77.50 mmole $250.00 mmoles $255.00 mmole $52.50 www.trilinkbiotech.com 4 4 4 4 4 4 4 Li Li 4 Li Li Li Li Li Li + + + + + + + O O + O O O O O O O O O O O O O O PO PO PO PO PO PO PO O PO O O O O O O O O O O O O O O P O P P P P O P P P O O O O O O O O O O O O O O O O O PO O O O O O O

PO PO O PO PO PO PO PO O O O O O O O OH 3 OH OH OH CH OH OH O O O OH OH O O O O O N NH O O N NH N H N N N N N H N N N 2 N N N N O O NH O N N O N 2 O NH N N N N NH NH NO N NH NH 2 2 2 H N MW= 505.21 (freeacid) MW= 523.18 (freeacid) MW= 507.18 (freeacid) MW= 518.20 (freeacid) MW= 521.21 (freeacid) MW= 481.18 (freeacid) MW= 521.21 (freeacid) MW= 481.18 (freeacid) ε ε ε ε ε ε ε ε = 15400@269nm = 14800@254nm = 13600@267nm = 21735@265nm = 11700 @270nm = 8900@278nm = 7868@292nm = 7100@279nm C C C C C C C C 11 10 10 13 10 10 11 11 >90% purity >90% purity >90% purity >90% purity >90% purity >90% purity >90% purity >90% purity Lithium salt Lithium salt Lithium salt Lithium salt Lithium salt Lithium salt Lithium salt Lithium salt H H H H H H H H 18 18 18 18 16 16 17 18 N N N N N N N N 5 5 5 2 5 3 5 3 O O O O O O O O 12 14 13 14 13 13 13 13 P P P P P P P P 3 3 3 3 3 3 3 3 60 61 ε 2-Thio-2'-deoxycytidine-5'-Triphosphate 5-Propynyl-2'-deoxyuridine-5'-Triphosphate 5-Propynyl-2'-deoxycytidine-5'-Triphosphate 6-Thio-2'-deoxyguanosine-5'-Triphosphate 4-Thiothymidine-5'-Triphosphate 2-Thiothymidine-5'-Triphosphate

is notedinL mmole Base Modified2'DeoxynucleosideTriphosphates continued N-2042-10 10 N-2017-10 10 N-2016-10 10 N-2042-5 5 N-2042-1 1 N-2017-5 5 N-2017-1 1 N-2016-5 5 N-2016-1 1 N-2045-10 10 N-2045-5 5 N-2045-1 1 N-2035-10 10 N-2035-5 5 N-2035-1 1 N-2041-10 10 N-2041-5 5 N-2041-1 1 -1 cm -1 [email protected] . mmoles $850.00 mmoles $625.00 mmoles $625.00 mmoles $465.00 mmole $97.50 mmoles $365.00 mmole $77.50 mmoles $365.00 mmole $77.50 mmoles $2,250.00 mmoles $1,150.00 mmole $250.00 mmoles $850.00 mmoles $465.00 mmole $97.50 mmoles $625.00 mmoles $365.00 mmole $77.50 Base Modified2'Deoxynucleoside Triphosphates

4 4 4 4 Li Li Li Li 4 4 + + Li Li + + O O O O + + O O O O O O O O PO PO PO PO PO O PO O O O O O O O O O O O P P P P O P P O O O O O O O O O O O O O O O PO O O PO PO PO O PO PO O O O O O (800) 863-6801 (800) OH OH OH OH 3 3 OH OH CH CH O O O O O O N N N NH O N N NH O N N S 2 2 N NH N NH NH S N S O O S O NH NH 2 MW= 483.22 (freeacid) MW= 506.19 (freeacid) MW= 505.21 (freeacid) MW= 523.24 (freeacid) MW= 498.23 (freeacid) MW= 498.23 (freeacid) ε ε ε ε ε ε = 11050 @291nm = 24800@341nm = 16300@335nm = 13300@277nm = 6500@293nm = 9500@295nm C C C C C C 10 9 10 10 12 12 H H >90% purity >90% purity >90% purity >90% purity H H Lithium salt Lithium salt Lithium salt >90% purity >90% purity Lithium salt H H 16 Lithium salt Lithium salt 16 17 17 17 18 N N N N N N 3 5 O 2 2 O 2 3 O O O O 12 12 13 13 14 13 P P P P

3 P P 3

S 3 3 S 3 3 S S 61 Nucleotides 62 Nucleotides 6-Azauridine-5'-Triphosphate 6-Azacytidine-5'-Triphosphate 2-Amino-6-chloropurineriboside-5'-Triphosphate ε 8-Azaadenosine-5'-Triphosphate 2-Aminopurine-riboside-5'-Triphosphate 5-Aminoallyluridine-5'-Triphosphate 5-Aminoallylcytidine-5 (Diaminopurine) 2-Aminoadenosine-5'-Triphosphate Base ModifiedRibonucleosideTriphosphates

is notedinL mmole N-1038-10 10 N-1037-10 10 N-1002-10 10 N-1038-5 5 N-1038-1 1 N-1037-5 5 N-1037-1 1 N-1002-5 5 N-1002-1 1 N-1004-10 10 N-1004-5 5 N-1067-10 10 N-1062-10 10 N-1065-10 10 N-1001-10 10 N-1004-1 1 N-1067-5 5 N-1067-1 1 N-1062-5 5 N-1062-1 1 N-1065-5 5 N-1065-1 1 N-1001-5 5 N-1001-1 1 -1 cm Base ModifiedRibonucleosideTriphosphates -1 . mmoles $625.00 mmoles $625.00 mmoles $850.00 mmoles $365.00 mmole $77.50 mmoles $365.00 mmole $77.50 mmoles $465.00 mmole $97.50 mmoles $2250.00 mmoles $1150.00 mmoles $850.00 mmoles $415.00 mmoles $625.00 mmoles $625.00 mmole $250.00 mmoles $465.00 mmole $97.50 mmoles $255.00 mmole $52.50 mmoles $365.00 mmole $77.50 mmoles $365.00 mmole $77.50 '-Triphosphate www.trilinkbiotech.com 4 4 4 4 4 4 Li 4 4 Li Li Li Li Li Li Li + + + + + + + + O O O O O O O O O O O O O O O O PO PO PO PO PO PO PO PO O O O O O O O O O O O O O O O O P P P P P P P P O O O O O O O O 2 2 NH O O NH O O O O O O O O O O O O O O P P P P

PO P P P O O O O O O O O O O O O O O O OH OH OH OH OH OH OH OH N N O O O O O O O O N O N N NH N NH N N OH OH N N O OH N N N OH N OH N OH OH OH 2 2 NH N N NH O O O O N N N Cl N NH NH 2 2 N N N N NH NH NH 2 2 2 MW= 541.63 (freeacid) MW= 485.13 (freeacid) MW= 484.14 (freeacid) MW= 508.17 (freeacid) MW= 507.20 (freeacid) MW= 539.22 (freeacid) MW= 538.23 (freeacid) MW= 522.20 (freeacid) ε ε ε ε ε ε ε ε = 9894@279nm = 17830@277nm = 6254@260nm = 7473@262nm = 6709@307nm = 7100@290nm = 5041@290nm = 6422@303nm C 10 C C C C C C H C 10 12 12 10 >90% purity >90% purity >90% purity 8 8 >90% purity >90% purity >90% purity >90% purity >90% purity 15 Lithium salt Lithium salt Lithium salt 9 Lithium salt Lithium salt Lithium salt Lithium salt Lithium salt H H H H H H H N 14 15 20 21 17 15 16 5 N N O N N N N N 3 4 6 5 3 4 6 13 O O O O O O O P 15 14 13 13 15 14 13 3 P P Cl P P P P P 3 3 3 3 3 3 3 8-Azidoadenosine-5'-Triphosphate Biotin-16-Aminoallyluridine-5'-Triphosphate ε 5-Bromouridine-5'-Triphosphate 5-Bromocytidine-5'-Triphosphate Benzimidazole-riboside-5'-Triphosphate Biotin-16-Aminoallylcytidine-5'-Triphosphate

is notedinL mmole N-1052-10 10 N-1052-5 5 N-1052-1 1 N-5005-2 2 N-5005-1 1 N-5005-0250 N-1053-10 10 N-1054-10 10 N-1053-5 5 N-1054-5 5 N-1054-1 1 N-1053-1 1 N-1047-10 10 N-5003-2 2 N-1047-5 5 N-1047-1 1 N-5003-1 1 N-5003-0250 Base ModifiedRibonucleosideTriphosphates continued -1 cm 250 nmoles$150.00 -1 250 nmoles$197.50 [email protected] . mmoles $415.00 mmoles $255.00 mmole $52.50 mmoles $750.00 mmole $525.00 mmoles $1235.00 mmoles $415.00 mmoles $695.00 mmoles $255.00 mmole $52.50 mmole $155.00 mmoles $850.00 mmoles $915.00 mmoles $465.00 mmole $97.50 mmole $525.00

HNH NH HNH NH O S O S Base ModifiedRibonucleosideTriphosphates 4 O O 4 4 Li 4 Li Li Li + H N + + + O O H N O O O O O O PO PO PO PO O O O O O O O O P P P P O O O O O O O O O O O O P P P P O O O O O O O O (800) 863-6801 (800) O O Br Br OH N OH OH OH 3 N H N H O O O 4 O 4 Li Li + N O N N N NH OH + OH OH N O O N OH O O 2 PO NH N PO O O O O N O NH O O P O 2 P N O O H N O H N O O O P P O O O O OH OH O O N NH OH N O OH 2 N NH MW= 548.20 (freeacid) MW= 963.80 (freeacid) MW= 563.04 (freeacid) MW= 562.05 (freeacid) MW= 490.19 (freeacid) MW= 961.70 (freeacid) O O ε ε ε ε ε = 14500@281nm ε = 19800@251nm = 7100@290nm = 9220@279nm = 6825@288nm = 5040@290nm C C C C C 32 9 9 C 32 H H 10 H >90% purity >90% purity 12 H >90% purity >90% purity Lithium salt Lithium salt >90% purity >90% purity 14 15 Lithium salt Lithium salt H 52 Lithium salt Lithium salt H 52 N N 15 N 17 N 2 3 N 7 O O N 8 O O 8 15 14 2 O 19 O 18 P P 13 P 13 P 3 3 P 3

Br Br P 3 S

S 3 3 63 Nucleotides 64 Nucleotides 5-Methylcytidine-5'-Triphosphate 5-Iodouridine-5'-Triphosphate 5-Iodocytidine-5'-Triphosphate Inosine-5'-Triphosphate Base ModifiedRibonucleosideTriphosphates ε 5,6-Dihydrouridine-5'-Triphosphate 7-Deazaguanosine-5'-Triphosphate (Tubercidin Triphosphate) 7-Deazaadenosine-5'-Triphosphate 6-Chloropurineriboside-5'-Triphosphate

is notedinL mmole N-1014-10 10 N-1012-10 10 N-1011-10 10 N-1014-5 5 N-1014-1 1 N-1012-5 5 N-1012-1 1 N-1011-5 5 N-1011-1 1 N-1020-10 10 N-1020-5 5 N-1020-1 1 N-1035-10 10 N-1044-10 10 N-1061-10 10 N-1006-10 10 N-1035-5 5 N-1035-1 1 N-1044-5 5 N-1044-1 1 N-1061-5 5 N-1061-1 1 N-1006-5 5 N-1006-1 1 Base ModifiedRibonucleosideTriphosphates continued -1 cm -1 . mmoles $625.00 mmoles $625.00 mmoles $625.00 mmoles $365.00 mmole $77.50 mmoles $365.00 mmole $77.50 mmoles $365.00 mmole $77.50 mmoles $415.00 mmoles $255.00 mmole $52.50 mmoles $850.00 mmoles $850.00 mmoles $625.00 mmoles $625.00 mmoles $465.00 mmole $97.50 mmoles $465.00 mmole $97.50 mmoles $365.00 mmole $77.50 mmoles $365.00 mmole $77.50 www.trilinkbiotech.com 4 4 4 4 4 4 Li Li Li 4 Li Li 4 Li + + Li + + Li + + O O O O + O O O O + O O O O O O PO PO O O PO PO PO PO O PO O O PO O O O O O O O O O O O P O P O P P P O P O P O P O O O O O O O O O O O O O O O P O O P O O O O P P O P

P O P O P O O O O O O O O O O O OH O O OH OH OH OH I I OH OH OH O O O O O O N O O OH O N N OH N NH O N N NH OH OH NH OH N N N N OH 2 OH OH 2 N NH N O N O O O O N N N O NH Cl NH 2 NH N N NH 2 MW= 497.18 (freeacid) MW= 610.04 (freeacid) MW= 609.05 (freeacid) MW= 508.17 (freeacid) MW= 526.61 (freeacid) MW= 486.16 (freeacid) MW= 522.19 (freeacid) MW= 506.19 (freeacid) ε ε ε ε ε ε ε = 12700@248nm = 7808@279nm = 6533@287nm = 8275@294nm = 9145@259nm = 9500@271nm = 9754@262nm C C C C 10 C C C C 10 H 9 9 10 >90% purity >90% purity >90% purity 11 11 >90% purity H H Lithium salt Lithium salt Lithium salt >90% purity >90% purity >90% purity >90% purity Lithium salt 9 H 14 Lithium salt Lithium salt Lithium salt Lithium salt H H H H 14 15 18 N 15 17 17 17 N N N 4 N ε N N N O 2 3 3 O O 4 = N/A 2 4 4 O 13 O O O O 15 14 14 P 14 15 14 13 P P P 3 P P P P Cl 3 3 3 I I 3 3 3 3 64 O ε 8-Oxoguanosine-5'-Triphosphate N (Thymidine RNA analog) 5-Methyluridine-5'-Triphosphate Pseudouridine-5'-Triphosphate N N

is notedinL mmole 1 6 6 1 -Methyladenosine-5'-Triphosphate -Methylguanosine-5'-Triphosphate -Methyladenosine-5'-Triphosphate -Methylguanosine-5'-Triphosphate N-1031-10 10 N-1031-5 5 N-1031-1 1 N-1066-10 10 N-1042-10 10 N-1024-10 10 N-1066-5 5 N-1066-1 1 N-1042-5 5 N-1042-1 1 N-1024-5 5 N-1024-1 1 N-1019-10 10 N-1019-5 5 N-1019-1 1 N-1013-10 10 N-1013-5 5 N-1013-1 1 N-1039-10 10 N-1039-5 5 N-1039-1 1 Base ModifiedRibonucleosideTriphosphates continued -1 cm -1 [email protected] . mmoles $1235.00 mmoles $695.00 mmole $155.00 mmoles $415.00 mmoles $625.00 mmoles $625.00 mmoles $255.00 mmole $52.50 mmoles $365.00 mmole $77.50 mmoles $365.00 mmole $77.50 mmoles $625.00 mmoles $365.00 mmole $77.50 mmoles $415.00 mmoles $255.00 mmole $52.50 mmoles $625.00 mmoles $365.00 mmole $77.50

Base ModifiedRibonucleosideTriphosphates 4 4 Li Li 4 4 4 4 + 4 + Li Li Li Li O O Li O O + + + + + O O PO O O O O O O PO O O O PO PO PO O PO PO O O O O O O O P O O O O P O O P P P O P O O P O O O O O O P O O O O O O O P O O O P O O P P O P O O O O P O O O O O O OH (800) 863-6801 (800) O O OH O OH OH OH O OH OH O O NH O N N H O OH O N N O N O OH O OH N OH N OH N N OH NH N NH N N OH O O N O NH NH N O N O N NH N NH NH CH N 2 3 N NH NH 2 2 MW= 537.21 (freeacid) MW= 539.18 (freeacid) MW= 523.23 (freeacid) MW= 498.17 (freeacid) MW= 484.14 (freeacid) MW= 521.21 (freeacid) MW= 537.21 (freeacid) ε ε ε ε ε ε ε = 14600@257nm = 15567@265nm = 12950@254nm = 5460@280nm = 7868@296nm = 9650@267nm = 7546@262nm C C C C C C C 10 10 11 11 11 11 >90% purity >90% purity >90% purity >90% purity >90% purity >90% purity >90% purity 9 Lithium salt Lithium salt Lithium salt Lithium salt Lithium salt Lithium salt Lithium salt H H H H H H H 18 17 16 20 15 18 18 N N N N N N N 5 2 5 5 2 5 5 O O O O O O O 14 15 15 13 15 13 14

P P P P P P P

3 3 3 3 3 3 3 65 Nucleotides 66 Nucleotides Xanthosine-5'-Triphosphate 4-Thiouridine-5'-Triphosphate 2-Thiouridine-5'-Triphosphate 2-Thiocytidine-5'-Triphosphate Base ModifiedRibonucleosideTriphosphates ε Puromycin-5'-Triphosphate

is notedinL mmole N-1023-10 10 N-1025-10 10 N-1032-10 10 N-1036-10 10 N-1023-5 5 N-1023-1 1 N-1025-5 5 N-1025-1 1 N-1032-5 5 N-1032-1 1 N-1036-5 5 N-1036-1 1 N-1022-10 10 N-1022-5 5 N-1022-1 1 Base ModifiedRibonucleosideTriphosphates continued -1 cm -1 . mmoles $415.00 mmoles $850.00 mmoles $625.00 mmoles $625.00 mmoles $255.00 mmole $52.50 mmoles $465.00 mmole $97.50 mmoles $365.00 mmole $77.50 mmoles $365.00 mmole $77.50 mmoles $850.00 mmoles $465.00 mmole $97.50 www.trilinkbiotech.com 4 Li + 4 4 4 4 O O Li Li Li Li + + PO + + O O O O O O O O O PO PO PO PO O O O O O P O O O O O P P P O P O O O O O P O O O 3 O O O O O O O CH P P O PO P

O O O O O NH O O O OH OH O OH NH OH 2 N N O OH O O O N NH N O S N OH OH OH N N OH 2 N N N NH NH N S O S H N O NH O MW= 500.20 (freeacid) MW= 500.20 (freeacid) MW= 499.22 (freeacid) MW= 524.17 (freeacid) MW= 711.45 (freeacid) ε ε ε ε ε = 16526@331nm = 13120@274nm = 18800@275nm = 11124 @247nm = 8900@278nm C C C C C 9 9 9 10 H H H 22 >90% purity >90% purity >90% purity >90% purity >90% purity Lithium salt Lithium salt Lithium salt Lithium salt Lithium salt H 15 15 16 H 15 N N N 32 N 2 2 3 N O O O 4 7 O 14 14 13 O 15 P P P 14 P 3 3 3 P S S S 3 3 66 67 3'-Amino-2',3'-dideoxyadenosine-5'-Triphosphate 2'-Amino-2'-deoxyuridine-5'-Triphosphate 2'-Amino-2'-deoxycytidine-5'-Triphosphate 2'-Amino-2'-deoxyadenosine-5'-Triphosphate ε 3'-Amino-2',3'-dideoxythymidine-5'-Triphosphate 3'-Amino-2',3'-dideoxyguanosine-5'-Triphosphate 3'-Amino-2',3'-dideoxycytidine-5'-Triphosphate 2'-Amino-2'-deoxyguanosine-5'-Triphosphate

is notedinL mmole N-4010-10 10 N-4010-5 5 N-4010-1 1 N-1027-10 10 N-1026-10 10 N-1046-10 10 N-1027-5 5 N-1027-1 1 N-1026-5 5 N-1026-1 1 N-1046-5 5 N-1046-1 1 N-4012-10 10 N-4011-10 10 N-4013-10 10 N-4012-5 5 N-4011-5 5 N-1064-10 10 N-4013-5 5 N-4013-1 1 N-4012-1 1 N-4011-1 1 N-1064-5 5 N-1064-1 1 Sugar ModifiedNucleosideTriphosphates continued -1 cm -1 [email protected] . mmoles $1450.00 mmoles $775.00 mmole $175.00 mmoles $625.00 mmoles $625.00 mmoles $415.00 mmoles $365.00 mmole $77.50 mmoles $365.00 mmole $77.50 mmoles $255.00 mmole $52.50 mmoles $1450.00 mmoles $1450.00 mmoles $850.00 mmoles $775.00 mmoles $775.00 mmoles $625.00 mmoles $465.00 mmole $97.50 mmole $175.00 mmole $175.00 mmoles $365.00 mmole $77.50

Sugar ModifiedNucleosideTriphosphates 4 4 Li 4 4 4 4 4 4 Li Li + Li Li Li Li Li + + O O + + + + + O O O O O O O O O O O O PO O O PO PO PO PO PO PO O PO O O O O O O O O O O O O O O P O P P P P O P P P O O O O O O O O O O O O O O O O O PO O O O O O O PO PO O PO PO PO PO PO O O O O O O O OH (800) 863-6801 (800) 2 2 2 OH OH OH NH NH NH 2 NH O O O O O O O N O N NH O N NH N N N O N N N NH N NH NH NH 2 N N 2 2 2 N 2 NH 2 NH N O O O O O N N NH NH NH N O 2 2 N N NH NH 2 NH 2 MW= 490.20 (freeacid) MW= 483.16 (freeacid) MW= 482.17 (freeacid) MW= 522.20 (freeacid) MW= 481.18 (freeacid) MW= 506.20 (freeacid) MW= 466.17 (freeacid) MW= 522.20 (freeacid) ε ε ε ε ε ε ε ε = 15400@258nm = 10100@262nm = 15400@258nm = 13600@252nm = 13600@252nm = 9100@271nm = 9650@267nm = 9100@271nm C C C C C C C C 10 10 10 10 10 >90% purity >90% purity >90% purity >90% purity 9 9 >90% purity >90% purity >90% purity >90% purity Lithium salt Lithium salt Lithium salt Lithium salt 9 Lithium salt Lithium salt Lithium salt Lithium salt H H H H H H H H 16 17 17 17 18 17 17 17 N N N N N N N N 3 4 6 6 3 6 4 6 O O O O O O O O 14 13 12 11 13 12 12 13

P P P P P P P P

3 3 3 3 3 3 3 3 67 Nucleotides 68 Nucleotides 2'-Azido-2'-deoxyuridine-5'-Triphosphate 2'-Azido-2'-deoxycytidine-5'-Triphosphate 2'-Azido-2'-deoxyadenosine-5'-Triphosphate Arauridine-5'-Triphosphate Aracytidine-5'-Triphosphate 2'-Azido-2'-deoxyguanosine-5'-Triphosphate Araadenosine-5'-Triphosphate ε 3'-Azido-2',3'-dideoxyadenosine-5'-Triphosphate Sugar ModifiedNucleosideTriphosphates

is notedinL mmole N-1029-10 10 N-1028-10 10 N-1045-10 10 N-1034-10 10 N-1033-10 10 N-1063-10 10 N-1048-10 10 N-1029-5 5 N-1029-1 1 N-1028-5 5 N-1028-1 1 N-1045-5 5 N-1045-1 1 N-1034-5 5 N-1034-1 1 N-1033-5 5 N-1033-1 1 N-1063-5 5 N-1063-1 1 N-1048-5 5 N-1048-1 1 N-4007-10 10 N-4007-5 5 N-4007-1 1 Sugar ModifiedNucleosideTriphosphates continued -1 cm -1 . mmoles $625.00 mmoles $625.00 mmoles $625.00 mmoles $850.00 mmoles $415.00 mmoles $625.00 mmoles $625.00 mmoles $365.00 mmole $77.50 mmoles $365.00 mmole $77.50 mmoles $365.00 mmole $77.50 mmoles $465.00 mmole $97.50 mmoles $255.00 mmole $52.50 mmoles $365.00 mmole $77.50 mmoles $365.00 mmole $77.50 mmoles $850.00 mmoles $465.00 mmole $97.50 www.trilinkbiotech.com 4 Li 4 4 4 4 4 4 4 + Li Li Li Li Li Li Li O O + + + + + + + O O PO O O O O O O O O O O O O O PO PO PO PO PO PO PO O O O O O O O O O P O O O O O O O P P P P P P P O O O O O O O O O PO O O O O O O O O O O O O O O O PO PO PO PO PO PO PO

O O O O O O O OH N OH OH OH OH OH OH 3 O O O O O O N O O N N 3 O N NH N O N N NH N N N N N N N N N OH OH 3 3 OH 2 2 3 N NH NH N N O O O O O NH N N N NH NH NH 2 NH 2 2 N N N 2 MW= 509.15 (freeacid) MW= 508.17 (freeacid) MW= 532.20 (freeacid) MW= 482.12 (freeacid) MW= 483.16 (freeacid) MW= 548.19 (freeacid) MW= 507.18 (freeacid) MW= 516.20 (freeacid) ε ε ε ε ε ε ε ε = 10100@262nm = 10100@262nm = 15400@258nm = 13600@252nm = 15400@258nm = 15400@258nm = 9100@271nm = 9100@271nm C C C C C C C C 10 10 10 10 >90% purity >90% purity >90% purity >90% purity >90% purity >90% purity >90% purity 9 9 9 9 >90% purity Lithium salt Lithium salt Lithium salt Lithium salt Lithium salt Lithium salt Lithium salt Lithium salt H H H H H H H H 15 16 14 15 15 16 15 15 N N N N N N N N 5 6 8 2 3 8 5 8 O O O O O O O O 14 13 12 15 14 13 13 11 P P P P P P P P 3 3 3 3 3 3 3 3 68 69 3'-Azido-2',3'-dideoxyuridine-5'-Triphosphate 3'-Azido-2',3'-dideoxythymidine-5'-Triphosphate 3'-Azido-2',3'-dideoxycytidine-5'-Triphosphate ε 5-Bromo-2',3'-dideoxyuridine-5'-Triphosphate 3'-Azido-2',3'-dideoxyguanosine-5'-Triphosphate 2'-Deoxy-L-cytidine-5-Triphosphate 2'-Deoxy-L-guanosine-5-Triphosphate 2'-Deoxy-L-adenosine-5-Triphosphate

is notedinL mmole N-4015-10 10 N-4009-10 10 N-4014-10 10 N-4015-5 5 N-4015-1 1 N-4009-5 5 N-4009-1 1 N-4014-5 5 N-4014-1 1 N-4016-10 10 N-4008-10 10 N-4016-5 5 N-4016-1 1 N-4008-5 5 N-4008-1 1 N-2055-10 10 N-2054-10 10 N-2055-5 5 N-2053-10 10 N-2054-5 5 N-2054-1 1 N-2055-1 1 N-2053-5 5 N-2053-1 1 Sugar ModifiedNucleosideTriphosphates continued -1 cm [email protected] -1 . mmoles $850.00 mmoles $850.00 mmoles $850.00 mmoles $465.00 mmole $97.50 mmoles $465.00 mmole $97.50 mmoles $465.00 mmole $97.50 mmoles $850.00 mmoles $850.00 mmoles $465.00 mmole $97.50 mmoles $465.00 mmole $97.50 mmoles $1450.00 mmoles $850.00 mmoles $775.00 mmoles $850.00 mmoles $465.00 mmole $97.50 mmole $175.00 mmoles $465.00 mmole $97.50

Sugar ModifiedNucleosideTriphosphates 4 4 4 4 4 Li Li 2 Li NH Li Li + + + + + O O O O O O O O O O N NH PO PO PO PO PO O O O NH O O N O N O O O O O O 2 P P P P N P O O O O O N O O O O N N O O N O N NH O O O PO PO PO PO PO 2 O O O O O O O O (800) 863-6801 (800) OH OH OH N N N Br N 3 3 3 3 O O O O O O O O O O O P P P O N N NH O N O N O O O O O O N N 2 O O O NH N NH NH P P P O O O O O O O N O O O O PO PO PO NH O O O O O O NH 4 4 4 2 Li Li Li + + + MW= 493.16 (freeacid) MW= 507.18 (freeacid) MW= 492.17 (freeacid) MW= 531.04 (freeacid) MW= 532.20 (freeacid) MW= 467.16 (freeacid) MW= 507.18 (freeacid) MW= 491.18 (freeacid) ε ε ε ε ε ε ε = 10100@262nm ε = 13600@252nm = 13600@252nm = 15400@258nm = 9650@267nm = 9100@271nm = 9200@279nm = 9100@271nm C C C 9 C C C C H C 10 10 10 10 >90% purity >90% purity >90% purity 9 9 >90% purity >90% purity >90% purity >90% purity >90% purity Lithium salt Lithium salt Lithium salt 14 9 Lithium salt Lithium salt H H H Lithium salt Lithium salt Lithium salt H H H H N 14 16 15 15 16 16 16 2 N N N N O N N N 5 5 6 8 3 5 5 13 O O O O O O O P 13 13 12 12 13 13 12 3

P P P P Br P P P

3 3 3 3 3 3 3 69 Nucleotides 70 Nucleotides ε 3'-Deoxy-5-methyluridine-5'-Triphosphate 3'-Deoxyuridine-5'-Triphosphate 3'-Deoxycytidine-5'-Triphosphate (Cordycepin) 3'-Deoxyadenosine-5'-Triphosphate Sugar ModifiedNucleosideTriphosphates 2',3'-Dideoxycytidine-5'-Triphosphate 2',3'-Dideoxyadenosine-5'-Triphosphate 3'-Deoxyguanosine-5'-Triphosphate 2'-Deoxy-L-thymidine-5-Triphosphate

is notedinL mmole N-3004-10 10 N-3004-5 5 N-3004-1 1 N-3005-10 10 N-3003-10 10 N-3001-10 10 N-4001-10 10 N-3005-5 5 N-3005-1 1 N-3003-5 5 N-3003-1 1 N-3001-5 5 N-3001-1 1 N-4005-10 10 N-4005-5 5 N-4005-1 1 N-4001-5 5 N-4001-1 1 N-3002-10 10 N-3002-5 5 N-3002-1 1 N-2056-10 10 N-2056-5 5 N-2056-1 1 Sugar ModifiedNucleosideTriphosphates continued -1 cm -1 . mmoles $625.00 mmoles $365.00 mmole $77.50 mmoles $625.00 mmoles $625.00 mmoles $625.00 mmoles $415.00 mmoles $365.00 mmole $77.50 mmoles $365.00 mmole $77.50 mmoles $365.00 mmole $77.50 mmoles $205.00 mmoles $115.00 mmole $27.50 mmoles $255.00 mmole $52.50 mmoles $625.00 mmoles $365.00 mmole $77.50 mmoles $850.00 mmoles $465.00 mmole $97.50 www.trilinkbiotech.com 4 4 4 4 4 Li 4 Li Li 4 Li Li Li + Li + + + + + + O O O O O O O O O O O O O O PO PO PO PO PO PO PO O O O O O O O O O O O O O O O P P P P P P P O NH O O O O O O O O O O O O O O O O O O O N O O PO

PO PO PO PO PO PO O O O O O O O O OH O O O O O O O O P O O O N NH OH N O N N O N N N N N NH N O OH OH OH OH 2 P N 2 NH NH N O O O O O O N N N NH PO NH O O O 2 2 N N NH 4 Li NH + 2 MW= 482.17 (freeacid) MW= 468.14 (freeacid) MW= 467.16 (freeacid) MW= 491.18 (freeacid) MW= 451.16 (freeacid) MW= 475.18 (freeacid) MW= 507.18 (freeacid) MW= 482.17 (freeacid) ε ε ε ε ε ε ε ε = 15400@258nm = 10100@262nm = 13600@252nm =15400 @258nm = 8700@260nm = 9100@271nm = 9100@271nm = 9650@267nm C C C C C C C C 10 10 10 10 10 >90% purity >90% purity >90% purity >90% purity >90% purity >90% purity 9 9 9 >90% purity >90% purity Lithium salt Lithium salt Lithium salt Lithium salt Lithium salt Lithium salt Lithium salt H Lithium salt H H H H H H H 17 16 15 16 16 16 16 17 N N N N N N N N 2 2 3 5 3 5 5 2 O O O O O O O O 14 14 13 12 12 11 13 14 P P P P P P P P 3 3 3 3 3 3 3 3 70 71 2',3'-Dideoxyuridine-5'-Triphosphate 2'-Fluoro-2'-deoxythymidine-5'-Triphosphate ε 2',3'-Dideoxythymidine-5'-Triphosphate 2',3'-Dideoxyguanosine-5'-Triphosphate 2'-Fluoro-2'-deoxyguanosine-5'-Triphosphate 2'-Fluoro-2'-deoxycytidine-5'-Triphosphate 2'-Fluoro-2'-deoxyadenosine-5'-Triphosphate 2',3-Dideoxyinosine-5-Triphosphate

is notedinL mmole N-1055-10 10 N-4003-10 10 N-4003-5 5 N-4003-1 1 N-1055-5 5 N-1055-1 1 N-4017-10 10 N-4002-10 10 N-1009-10 10 N-1008-10 10 N-1007-10 10 N-4017-5 5 N-4004-10 10 N-4004-5 5 N-4004-1 1 N-4002-5 5 N-4002-1 1 N-1009-5 5 N-1009-1 1 N-1008-5 5 N-1008-1 1 N-1007-5 5 N-1007-1 1 N-4017-1 1 Sugar ModifiedNucleosideTriphosphates continued -1 cm -1 [email protected] . mmoles $625.00 mmoles $205.00 mmoles $115.00 mmole $27.50 mmoles $365.00 mmole $77.50 mmoles $1235.00 mmoles $415.00 mmoles $850.00 mmoles $415.00 mmoles $625.00 mmoles $695.00 mmoles $205.00 mmoles $115.00 mmole $27.50 mmoles $255.00 mmole $52.50 mmoles $465.00 mmole $97.50 mmoles $255.00 mmole $52.50 mmoles $365.00 mmole $77.50 mmole $155.00

Sugar ModifiedNucleosideTriphosphates 4 4 4 4 4 Li Li 4 Li Li 4 Li Li 4 Li + + + + + Li + + O O O O O O O O O O + O O O O PO O O PO PO PO PO PO PO O O O PO O O O O O O O O O O O O O P P P P P P P P O O O O O O O O O O O O O O O O O O O O O O O O PO PO PO PO PO PO PO PO O O O O O O O O (800) 863-6801 (800) OH OH OH OH O O O O O O O O O N N NH N F F O N O N N N N N N F N N F 2 NH N NH NH O O O O N NH N N O N O O 2 N NH NH NH NH NH 2 2 MW= 452.14 (freeacid) MW= 500.16 (freeacid) MW= 466.17 (freeacid) MW= 491.18 (freeacid) MW= 525.17 (freeacid) MW= 476.17 (freeacid) MW= 485.15 (freeacid) MW= 509.17 (freeacid) ε ε ε ε ε ε ε ε = 10100@262nm = 13600@252nm = 13600@252nm = 15400@258nm = 12300@248nm = 9650@267nm = 9650@267nm = 9100@271nm C C C 10 C C C C 10 C 9 H 10 10 10 10 H H >90% purity >90% purity 9 >90% purity >90% purity >90% purity >90% purity >90% purity >90% purity Lithium salt Lithium salt Lithium salt Lithium salt Lithium salt Lithium salt Lithium salt Lithium salt H 16 H H H H 15 15 15 N 17 16 15 15 N N N 2 N N N N 3 5 O O O 2 2 5 5 4 O O O O O 14 13 12 13 P 13 12 13 12 P P

P 3 P P P P 3 3

F F F 3 3 3 3 3 71 Nucleotides 72 Nucleotides ε 2'-O-Methylcytidine-5'-Triphosphate Sugar ModifiedNucleosideTriphosphates 2'-Fluoro-2'-deoxyuridine-5'-Triphosphate 2'-O-Methylinosine-5'-Triphosphate 2'-O-Methylguanosine-5'-Triphosphate 2'-O-Methyladenosine-5'-Triphosphate 2'-O-Methyl-5-methyluridine-5'-Triphosphate (Diaminopurine) 2'-O-Methyl-2-aminoadenosine-5'-Triphosphate Ganciclovir-5'-Triphosphate (S-isomer)

is notedinL mmole N-1016-10 10 N-1016-5 5 N-1016-1 1 N-1010-10 10 N-1010-5 5 N-1010-1 1 N-1021-10 10 N-1043-10 10 N-1040-10 10 N-9001-10 10 N-1021-5 5 N-1021-1 1 N-1017-10 10 N-1017-5 5 N-1017-1 1 N-1015-10 10 N-1015-5 5 N-1015-1 1 N-1043-5 5 N-1043-1 1 N-1040-5 5 N-1040-1 1 N-9001-5 5 N-9001-1 1 Sugar ModifiedNucleosideTriphosphates continued -1 cm -1 . mmoles $205.00 mmoles $115.00 mmole $27.50 mmoles $415.00 mmoles $255.00 mmole $52.50 mmoles $625.00 mmoles $415.00 mmoles $625.00 mmoles $625.00 mmoles $365.00 mmole $77.50 mmoles $205.00 mmoles $115.00 mmole $27.50 mmoles $205.00 mmoles $115.00 mmole $27.50 mmoles $255.00 mmole $52.50 mmoles $365.00 mmole $77.50 mmoles $365.00 mmole $77.50 www.trilinkbiotech.com 4 4 Li 4 4 4 Li 4 4 4 Li + Li Li Li Li Li + + O O + + + + + O O O O O O O O O O O O O PO O PO PO PO PO O PO PO PO O O O O O O O O O O O O P O O O P P P O P P P P O O O O O O O O O O O O O O O O O PO O O O O O O PO PO

O PO PO PO PO O PO O O O O O O OH OH OH OH OH OH OH OH O O O O O O O N O N OCH N NH N OCH N O N N O N N OCH F N OCH N N 2 OCH N OCH N 3 NH 3 NH N O 3 3 O 3 N 3 O O O N NH NH N NH N O NH 2 N 2 N NH NH 2 NH NH 2 2 MW= 497.18 (freeacid) MW= 486.13 (freeacid) MW= 522.25 (freeacid) MW= 537.21 (freeacid) MW= 521.21 (freeacid) MW= 512.19 (freeacid) MW= 536.22 (freeacid) MW= 495.20 (freeacid) ε ε ε ε ε ε ε ε = 10100@262nm = 13600@252nm = 12200@248nm = 13600@252nm = 15400@258nm = 10360@265nm = 9100@271nm = 8675@280nm C C C C C C C C 9 10 >90% purity H 10 11 11 11 11 >90% purity Lithium salt >90% purity >90% purity >90% purity >90% purity >90% purity >90% purity 9 H Lithium salt Lithium salt Lithium salt Lithium salt Lithium salt Lithium salt Lithium salt H H H H H H 14 18 N 19 16 13 17 18 19 N N N N N N N 2 O 3 4 2 5 2 6 5 O O O O O O O 14 14 P 13 15 13 15 13 13 P 3 P P P P P P F 3 3 3 3 3 3 3 72 73 ε 3'-O-Methylguanosine-5-Triphosphate 3'-O-Methyladenosine-5'-Triphosphate 3'-O-Methyluridine-5 3'-O-Methylcytidine-5 2'-O-Methyluridine-5'-Triphosphate 2'-O-Methylpseudouridine-5'-Triphosphate

is notedinL mmole N-1058-10 10 N-1056-10 10 N-1058-5 5 N-1058-1 1 N-1056-5 5 N-1056-1 1 N-1059-10 10 N-1057-10 10 N-1041-10 10 N-1059-5 5 N-1059-1 1 N-1057-5 5 N-1057-1 1 N-1018-10 10 N-1018-5 5 N-1018-1 1 N-1041-5 5 N-1041-1 1 Sugar ModifiedNucleosideTriphosphates continued -1 cm -1 [email protected] . mmoles $625.00 mmoles $625.00 mmoles $365.00 mmole $77.50 mmoles $365.00 mmole $77.50 mmoles $625.00 mmoles $625.00 mmoles $415.00 mmoles $365.00 mmole $77.50 mmoles $365.00 mmole $77.50 mmoles $205.00 mmoles $115.00 mmole $27.50 mmoles $255.00 mmole $52.50 '-Triphosphate '-Triphosphate

Sugar ModifiedNucleosideTriphosphates 4 4 4 4 4 Li 4 Li Li Li Li Li + + + + + + O O O O O O O O O O O O PO PO PO PO PO PO O O O O O O O O O O O O P P P P P P O O O O O O O O O O O O O O O O O O PO PO PO PO PO PO O O O O O O H H H H (800) 863-6801 (800) 3 3 3 3 CO CO CO CO OH OH NH O O O O O O O N NH N O O N N N NH N OCH OH OH OCH OH OH 2 2 NH N NH N NH 3 3 O O O O O N NH 2 N MW= 537.21 (freeacid) MW= 521.21 (freeacid) MW= 498.17 (freeacid) MW= 497.18 (freeacid) MW= 498.17 (freeacid) MW= 498.17 (freeacid) ε ε ε ε ε ε = 13600@252nm = 15400@258nm = 10100@262nm = 10100@262nm = 9100@271nm = 6475@262nm C C C C C C 11 11 10 10 10 10 >90% purity >90% purity >90% purity >90% purity >90% purity >90% purity Lithium salt Lithium salt Lithium salt Lithium salt Lithium salt Lithium salt H H H H H H 18 18 17 18 17 17 N N N N N N 5 5 2 3 2 2 O O O O O O 14 13 15 14 15 15

P P P P P P

3 3 3 3 3 3 73 Nucleotides 74 Nucleotides Bisphosphate Nucleosides (ppGpp) Guanosine-3',5'-Bisdiphosphate (mCAP) N (pGp) Guanosine-3',5'-Bisphosphate (CAP) Guanosine-5'-Triphosphate-5'-Guanosine 7 -Me-Guanosine-5'-Triphosphate-5'-Guanosine N-6001-10 10 N-6001-5 5 N-6001-1 1 N-6002-10 10 N-6002-5 5 N-7001-10 10 N-6002-1 1 N-7001-5 5 N-7001-1 1 N-7002-10 10 N-7002-5 5 N-7002-1 1 mmoles $1450.00 mmoles $775.00 mmole $175.00 mmoles $1235.00 mmoles $695.00 mmoles $625.00 mmole $155.00 mmoles $365.00 mmole $77.50 mmoles $625.00 mmoles $365.00 mmole $77.50 Bisphosphate Nucleosides Bisphosphate CAP and mCAP and CAP www.trilinkbiotech.com 2 NH 2 NH NH NH O N O N 6 Li 4 OH N N OH + Li N N + + O O O O O O PO O O O OH PO OH PO O O O O O O O PO O O PO O O PO O O O OP O O O

P OP P O O O 3 O O 3 NH O O O Li N N PO OH + PO + 4 O N O N OH OH N OH O N O O NH O NH N N OH N N NH OH NH 2 2 N O O N NH NH NH NH 2 2 MW= 603.16 (freeacid) MW= 443.20 (freeacid) MW= 803.45 (freeacid) MW= 788.41 (freeacid) ε ε ε ε = 13600@252nm = 13600@252nm = 21000@254nm = 21600@260nm Ammonium salt C C C C 21 20 10 10 >90% purity >90% purity Lithium salt H >90% purity Lithium salt H H Lithium salt H 30 27 17 15 N N N N 10 10 5 5 O O O O 17 18 11 18 P P P P 4 2 3 3 Cytidine-5'-O-(1-Thiotriphosphate) (mixedisomers) 2'-Deoxyadenosine-5'-O-(1-Thiotriphosphate) (mixedisomers) ε isomers) 2'-Deoxycytidine-5'-O-(1-Boranotriphosphate) (mixed 2'-Deoxythymidine-5'-O-(1-Thiotriphosphate) (mixedisomers) 2'-Deoxyguanosine-5'-O-(1-Thiotriphosphate) (mixedisomers) isomers) 2'-Deoxycytidine-5'-O-(1-Thiotriphosphate) (mixed 3'-Azido-2',3'-dideoxythymidine-5'-O-(1-Thiotriphosphate) (mixed isomers) 3'-Azido-2',3'-dideoxythymidine-5'-O-(1-Thiotriphosphate) (mixed Adenosine-5'-O-(1-Thiotriphosphate) (mixedisomers)

is notedinL mmole N-8006-10 10 N-8006-5 5 N-8006-1 1 N-8001-10 10 N-8001-5 5 N-8001-1 1 N-8051-10 10 N-8051-5 5 N-8051-1 1 N-8003-10 10 N-8004-10 10 N-8004-5 5 N-8004-1 1 N-8003-5 5 N-8003-1 1 N-8002-10 10 N-8002-5 5 N-8002-1 1 N-8013-10 10 N-8013-5 5 N-8013-1 1 N-8005-10 10 N-8005-5 5 N-8005-1 1 Alpha PhosphateModifiedNucleosideTriphosphates -1 cm -1 [email protected] . mmoles $625.00 mmoles $365.00 mmole $77.50 mmoles $205.00 mmoles $115.00 mmole $27.50 mmoles $1450.00 mmoles $775.00 mmole $175.00 mmoles $415.00 mmoles $205.00 mmoles $115.00 mmole $27.50 mmoles $255.00 mmole $52.50 mmoles $205.00 mmoles $115.00 mmole $27.50 mmoles $850.00 mmoles $465.00 mmole $97.50 mmoles $205.00 mmoles $115.00 mmole $27.50 Alpha Phosphate ModifiedNucleosideTriphosphatesAlpha

4 4 4 4 4 Li 4 4 4 Li Li Li Li Li + Li Li + + + + + O O + + O O O O O O O O O O O O O O PO PO PO PO PO PO PO PO O O O O O O O O O O O O O O O O P P P P P P P P BH O O O O O O O O O S 3 O O O O S O S S S - O O S S PO PO PO PO PO P PO PO O O O O O O O O O (800) 863-6801 (800) OH N 3 OH OH OH OH 3 OH CH OH CH 3 O O O O O O O O N N N NH N NH O N N NH N O N OH N N N OH 2 2 2 N N NH N NH N O NH O O O O N N O NH 2 N 2 NH N NH 2 MW= 499.22 (freeacid) MW= 507.24 (freeacid) MW= 463.90 (freeacid) MW= 498.23 (freeacid) MW= 523.24 (freeacid) MW= 483.22 (freeacid) MW= 523.24 (freeacid) MW= 523.24 (freeacid) ε ε ε ε ε ε ε = 15400@258nm ε = 13600@252nm = 15400@258nm = 9100@271nm = 9300@271nm = 9650@267nm = 9100@271nm = 9650@267nm C C C C C C C C 10 10 10 10 10 9 9 9 H H H H H H >90% purity >90% purity >90% purity H H >90% purity >90% purity >90% purity Lithium salt Lithium salt >90% purity >90% purity Lithium salt Lithium salt Lithium salt Lithium salt 16 18 Lithium salt Lithium salt 16 17 16 16 16 16 N BN N N N N N N 3 5 2 5 3 5 5 O O O O O O O 3 O 13 11 13 12 12 12 12 P P 12 P P P P P

3 3 P 3 3 3

3 3 S S S S S S S 3 75 Nucleotides 76 76 Nucleotides 2',3'-Dideoxyguanosine-5'-O-(1-Thiotriphosphate) (mixedisomers) 2',3'-Dideoxythymidine-5'-O-(1-Thiotriphosphate) (mixedisomers) isomers) 2',3'-Dideoxycytidine-5'-O-(1-Thiotriphosphate) (mixed 2',3'-Dideoxyadenosine-5'-O-(1-Thiotriphosphate) (mixedisomers) Phosphate ModifiedNucleosideTriphosphatesAlpha ε Uridine-5'-O-(1-Thiotriphosphate) (mixedisomers) (mixed isomers) Guanosine-5'-O-(1-Thiotriphosphate) 2',3-Dideoxyuridine-5-O-(1-Thiotriphosphate) (mixedisomers) Alpha PhosphateModifiedNucleosideTriphosphates

is notedinL mmole N-8011-10 10 N-8011-5 5 N-8011-1 1 N-8012-10 10 N-8010-10 10 N-8009-10 10 N-8012-5 5 N-8012-1 1 N-8010-5 5 N-8010-1 1 N-8009-5 5 N-8009-1 1 N-8015-10 10 N-8008-10 10 N-8008-5 5 N-8008-1 1 N-8007-10 10 N-8007-5 5 N-8007-1 1 N-8015-5 5 N-8015-1 1 -1 cm -1 . mmoles $415.00 mmoles $255.00 mmole $52.50 mmoles $415.00 mmoles $625.00 mmoles $625.00 mmoles $255.00 mmole $52.50 mmoles $365.00 mmole $77.50 mmoles $365.00 mmole $77.50 mmoles $415.00 mmoles $205.00 mmoles $115.00 mmole $27.50 mmoles $205.00 mmoles $115.00 mmole $27.50 mmoles $255.00 mmole $52.50 www.trilinkbiotech.com 4 4 4 4 4 4 4 Li Li Li Li Li Li Li + + + + + + + O O O O O O O O O O O O O O PO PO PO PO PO PO PO O O O O O O O O O O O O O O P P P P P P P O O O O O O O O O O S S S O S O S O S O S PO PO PO

PO PO PO PO O O O O O O O HO 3 CH O OH O O O O O O O O N N NH N N O N N O N N N N OH 2 H N NH NH NH O O O N NH O N O N O 2 N NH NH NH NH 2 2 MW= 507.24 (freeacid) MW= 482.23 (freeacid) MW= 467.22 (freeacid) MW= 491.25 (freeacid) MW= 500.20 (freeacid) MW= 539.24 (freeacid) MW= 468.2 (freeacid) ε ε ε ε ε ε ε = 13600@252nm continued = 15400@258nm = 10100@262nm = 13600@252nm = 10100@262nm = 9100@271nm = 9650@267nm C C C C C C C 10 10 10 10 9 9 9 H H H H >90% purity H H H >90% purity >90% purity >90% purity Lithium salt >90% purity >90% purity >90% purity Lithium salt Lithium salt Lithium salt Lithium salt Lithium salt Lithium salt 16 17 16 16 15 16 15 N N N N N N N 5 2 5 3 2 5 2 O O O O O O O 11 12 10 11 14 13 12 P P P P P P P 3 3 3 3 3 3 3 S S S S S S S ε Adenosine-5'-[N-(6-Biotinylamidohexyl)]-Monophosphate 5'-Biotin-A-Monophosphate Guanosine-5'-[N-(6-Biotinylamidohexyl)]-Monophosphate 5'-Biotin-G-Monophosphate 2'-Deoxyguanosine-5'-[N-(6-Biotinylamidohexyl)]-Monophosphate 5'-Biotin-dG-Monophosphate 2'-Deoxyadenosine-5'-[N-(6-Biotinylamidohexyl)]-Monophosphate 5'-Biotin-dA-Monophosphate Guanosine-5'-(6-Aminohexyl)-Monophosphate 5'-Amino-G-Monophosphate Adenosine-5'-(6-Aminohexyl)-Monophosphate 5'-Amino-A-Monophosphate

is notedinL mmole N-6003-10 10 N-6006-10 10 N-6004-10 10 N-6005-10 10 N-6009-10 10 N-6003-5 5 N-6003-1 1 N-6006-5 5 N-6006-1 1 N-6004-5 5 N-6004-1 1 N-6005-5 5 N-6005-1 1 N-6009-5 5 N-6009-1 1 N-6010-10 10 N-6010-5 5 N-6010-1 1 -1 cm -1 [email protected] . mmoles $415.00 mmoles $415.00 mmoles $415.00 mmoles $415.00 mmoles $415.00 mmoles $255.00 mmole $52.50 mmoles $255.00 mmole $52.50 mmoles $255.00 mmole $52.50 mmoles $255.00 mmole $52.50 mmoles $255.00 mmole $52.50 mmoles $415.00 mmoles $255.00 mmole $52.50 Labeled NucleosideMonophosphates

NH NH NH NH O S O O S S O S NH NH NH NH O O O 2 O NH Labeled Nucleoside Monophosphates Labeled H N H N H N 2 H N NH O (800) 863-6801 (800) O O O Na O O Li O O O O Li O O Li Na P Li P + P + + P + P P + + O O O O O O O O O O O O OH OH OH OH OH OH O O O O O O N N N N N N N N N OH N N OH OH OH N N O N N O NH N NH N N O NH NH 2 NH N 2 N 2 N NH NH NH NH 2 2 2 MW= 672.70 (freeacid) MW= 656.75 (freeacid) MW= 688.70 (freeacid) MW= 672.69 (freeacid) MW= 462.40 (freeacid) MW= 446.40 (freeacid) ε ε ε ε ε ε = 15300@258nm = 15400@259nm = 13700@253nm = 13700@252nm = 13700@253nm = 13700@253nm C C C C 26 C 26 26 26 C >90% purity >90% purity >90% purity >90% purity >90% purity >90% purity H Sodium salt Lithium salt Lithium salt Lithium salt Lithium salt Lithium salt H H H 16 16 41 H 41 34 39 H N N N N 27 27 8 N 8 8 O 8 N O O O 6 10 6 O 9 8 9 O

PS PS PS PS

8 7 P P 77 Nucleotides 78 Nucleotides K-1007 K-1006 K-1005 K-1004 terminators. DNA as well as RNA Weoffer protocols. termination chain for useful nucleotides contain kits These Chain TerminationKits K-1003 K-1002 K-1001 applications. other and translation nick for useful nucleotides, alpha-thiol combine kits These Alpha-ThiolKits Nucleotide triphosphates. nucleoside on section preceding the in found be can products individual the describing data physical and Structures TriLink'sguarantee. quality high with come and requirements development assay your all for perfect are kits These triphosphates. nucleoside modified popular our of combinations various contain that kits nucleotide offer we convenience, Foryour Nucleotide Kits Contains 1.0 µmole each of 5 nucleotides: 3'-Deoxynucleotide Kit (RNA chain terminators) Contains 1.0 µmole each of 4 nucleotides: 3'-Amino-2',3'-Dideoxynucleotide Kit (DNA chainterminators) $622.50 Contains 1.0 µmole each of 5 nucleotides: 3'-Azido-2',3'-Dideoxynucleotide Kit (DNA chainterminators) $487.50 Contains 1.0 µmole each of 4 nucleotides: 2',3'-Dideoxynucleotide Kit (DNA chainterminators) 2',3'-Dideoxynucleoside Alpha-Thiol NucleotideKit Contains 1.0 µmole each of 4 nucleotides: Ribonucleoside Alpha-Thiol NucleotideKit Contains 1.0 µmole each of 4 nucleotides: 2'-Deoxynucleoside Alpha-Thiol NucleotideKit Contains 1.0 µmole each of 4 nucleotides: N-4005 2',3'-Dideoxycytidine-5'-Triphosphate N-4001 2',3'-Dideoxyadenosine-5'-Triphosphate N-8005 N-8001 -Dideoxyadenosine-5'-O-(1-Thiotriphosphate) N-8009 2',3'-Dideoxyadenosine-5'-O-(1-Thiotriphosphate) N-8006 Cytidine-5'-O-(1-Thiotriphosphate) N-8002 2'-Deoxycytidine-5'-O-(1-Thiotriphosphate) N-3001 N-4010 3'-Amino-2',3'-dideoxyadenosine-5'-Triphosphate N-4007 3'-Azido-2',3'-dideoxyadenosine-5'-Triphosphate N-4003 2'-3'-Dideoxyuridine-5'-Triphosphate N-4002 2',3'-Dideoxyguanosine-5'-Triphosphate N-8010 2',3'-Dideoxycytidine-5'-O-(1-Thiotriphosphate) N-3002 3'-Deoxyguanosine-5'-Triphosphate N-8008 Uridine-5'-O-(1-Thiotriphosphate) N-8007 Guanosine-5'-O-(1-Thiotriphosphate) N-8003 2'-Deoxyguanosine-5'-O-(1-Thiotriphosphate) N-4011 3'-Amino-2',3'-dideoxycytidine-5'-Triphosphate N-4008 3'-Azido-2',3'-dideoxyguanosine-5'-Triphosphate N-8012 3'-Deoxythymidine-5'-O-(1-Thiotriphosphate) N-8011 2',3'-Dideoxyguanosine-5'-O-(1-Thiotriphosphate) N-3003 3'-Deoxycytidine-5'-Triphosphate N-8004 2'-Deoxythymidine-5'-O-(1-Thiotriphosphate) N-4012 3'-Amino-2',3'-dideoxyguanosine-5'-Triphosphate N-4014 3'-Azido-2',3'-dideoxycytidine-5'-Triphosphate N-4009 3'-Azido-3'-deoxythymidine-5'-Triphosphate N-3004 3'-Deoxy-5-methyluridine-5'-Triphosphate N-4013 3'-Amino-3'-deoxythymidine-5'-Triphosphate N-4015 3'-Azido-2'-3'-dideoxyuridine-5'-Triphosphate N-3005 3'-Deoxyuridine-5'-Triphosphate Adenosine-5'-O-(1-Thiotriphosphate) Adenosine-5'-O-(1-Thiotriphosphate) 2'-Deoxyadenosine-5'-O-(1-Thiotriphosphate) 3'-Deoxyadenosine-5'-Triphosphate Nucleotide Kits Nucleotide www.trilinkbiotech.com

$387.50 $160.00 $260.00 $135.00 $135.00 K-1008 K-1006 molecules. RNA and DNA of labeling post-synthetic for useful nucleotides contain kits These Post-SyntheticModification Reagents K-1013 K-1012 K-1011 K-1010 K-1009 K-1008 ribonucleotides. modified 2' contain kits These 2' SugarModifiedRNAKits [email protected] Contains 1.0 µmole each of 3 nucleotides: 2'-Amino-2'-Deoxynucleotide Kit (For RNA) Contains 1.0 µmole each of 4 nucleotides: 3'-Amino-2',3'-Dideoxynucleotide Kit Contains 1 kit of each of the following: 2' Sugar Super Modifier Kit Contains 1.0 µmole each of 4 nucleotides: 2'-O-Methyl-Nucleotide Kit Contains 1.0 µmole each of 5 nucleotides: 2'-Fluoro-2'-Deoxynucleotide Kit Contains 1.0 µmole each of 3 nucleotides: 2'-Azido-2'-Deoxynucleotide Kit Contains 1.0 µmole each of 3 nucleotides: Ara-Nucleotide Kit Contains 1.0 µmole each of 3 nucleotides: 2'-Amino-2'-Deoxynucleotide Kit N-1046 N-4010 3'-Amino-2',3'-dideoxyadenosine-5'-Triphosphate K-1008 N-1015 2'-O-Methyladenosine-5'-Triphosphate N-1007 2'-Azido-2'-deoxyadenosine-5'-Triphosphate N-1028 2'-Azido-2'-deoxycytidine-5'-Triphosphate N-1045 N-1048 N-1046 N-1026 2'-Amino-2'-deoxycytidine-5'-Triphosphate N-4011 3'-Amino-2',3'-dideoxycytidine-5'-Triphosphate K-1009 N-1016 2'-O-Methylcytidine-5'-Triphosphate N-1008 2'-Fluoro-2'-deoxycytidine-5'-Triphosphate N-1029 2'-Azido-2'-deoxyuridine-5'-Triphosphate N-1033 Aracytidine-5'-Triphosphate N-1026 2'-Amino-2'-deoxycytidine-5'-Triphosphate N-1027 2'-Amino-2'-deoxyuridine-5'-Triphosphate N-4012 3'-Amino-2',3'-dideoxyguanosine-5'-Triphosphate K-1010 N-1017 2'-O-Methylguanosine-5'-Triphosphate N-1009 2'-Fluoro-2'-deoxyguanosine-5'-Triphosphate N-1034 Arauridine-5'-Triphosphate N-1027 2'-Amino-2'-deoxyuridine-5'-Triphosphate N-4013 3'-Amino-3'-deoxythymidine-5'-Triphosphate K-1011 N-1018 2'-O-Methyluridine-5'-Triphosphate N-1010 2'-Fluoro-2'-deoxyuridine-5'-Triphosphate K-1012 N-1055 2'-Fluorothymidine-5'-Triphosphate 2'-Amino-2'-deoxyadenosine-5'-Triphosphate Kit 2'-Amino-2'-Deoxynucleotide 2'-Fluoro-2'-deoxyadenosine-5'-Triphosphate Araadenosine-5'-Triphosphate 2'-Amino-2'-deoxyadenosine-5'-Triphosphate Ara-nucleotide Kit Ara-nucleotide 2'-Azido-2'-Deoxynucleotide Kit 2'-Azido-2'-Deoxynucleotide 2'-Fluoro-2'-Deoxynucleotide Kit 2'-O-Methyl-Nucleotide Kit

(800) 863-6801 (800)

Nucleotide Kits $207.50 $622.50 $1135.00 $110.00 $357.50 $232.50 $227.50 $207.50 79 Nucleotides 80 Nucleotides Hybridization Modulation Kits Hybridization K-1016 K-1015 development. assay for thermostability increasing or RNA and DNA modified of properties hybridization studying for useful duplexes, of thermostability the increase to properties have that nucleotides contain kits These Nucleotide Kits K-1017 Determining Relative Thermostability byMeltingTemperature conduct the Dissolve enougho buffe of0.2ODunitsperm Repeat w oligo in1m To determi Tm i on thecu obtain acur Plot the the slopeisatits oligonucleotide r ataconcentration s atthe a targetsequence,non-modif at 260nm gr eatest rv ith modified re fo L of e, or sults to mi ve ne how llowing sim . d- pH7.0 . The . when poin f each t aproposedmodification L

Contains 1.0 µmole each of 9 nucleotides: Contains 1.0 µmole each of 5 nucleotides: DNA Thermostability Enhancer Kit Contains 1.0 µmole each of 4 nucleotides: RNA Thermostability Enhancer Kit 2'-Modified Thermostability Enhancer Kit N-1018 2'-O-Methyluridine-5'-Triphosphate N-1017 2'-O-Methylguanosine-5'-Triphosphate N-1016 2'-O-Methylcytidine-5'-Triphosphate N-1015 2'-O-Methyladenosine-5'-Triphosphate N-1055 2'-Fluorothymidine-5'-Triphosphate N-1010 2'-Fluoro-2'-deoxyuridine-5'-Triphosphate N-1009 2'-Fluoro-2'-deoxyguanosine-5'-Triphosphate N-1008 2'-Fluoro-2'-deoxycytidine-5'-Triphosphate N-1007 2'-Fluoro-2'-deoxyadenosine-5'-Triphosphate N-2003 N-1001 N-2004 2-Aminopurine-2'-deoxyriboside-5'-Triphosphate N-1002 2-Amino-6-chloropurineriboside-5'-Triphosphate N-2016 5-Propynyl-2'-deoxycytidine-5'-Triphosphate N-1014 5-Methylcytidine-5'-Triphosphate N-2017 5-Propynyl-2'-deoxyuridine-5'-Triphosphate N-1024 5-Methyluridine-5'-Triphosphate N-2026 5-Methyl-2'-deoxycytidine-5'-Triphosphate ple meltingtemperatureexperimentusingthreeoligonucleotides Repeat untila 1 degr and r Raise thete time tof to m the TargetandControl absor Mix ~0.5 following atransition. (F mi inal absor ead theabso ee C,allowsufficient x ak ied controlprobe,andthemodi 2-Amino-2'-deoxyadenosine-5'-Triphosphate 2-Aminoadenosine-5'-Triphosphate 0.2 sh bance isr solution. e anequi ould ully equilibrate at mL 2 mp bance of bance b fa 60 n e will rily constant eachof eratur appro m) eached mo rb effectthethermostability ofaduplex th ance x. la e e r

. www.trilinkbiotech.com spect cap andplaceinaU Read theabsorbanc boiling m then allow Heat the tem mi room Place 1 xture inacuvette fi rophotom peratur ed probe. at 260nm tem slow omentar mi mL tocool peratur ly e cont xture to . ofth eter . ily w ro e e , l. V it e , h , Increasin : g Abs.

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$467.50 $382.50 $330.00 T M ofModified Duplex

81 Overview of CleanAmpTM Products

The Next Generation in Hot Start PCR

Solutions Start Here. TriLink's CleanAmpTM Product Line is an innovative new approach to Hot Start PCR. We have applied our expertise in modified nucleic acid chemistry to resolve common PCR problems. This advancement in Hot Start technology employs two widely ignored components of the PCR reagent mix: the primers and the dNTPs. Two distinct nucleic acid thermolabile protecting groups identified by TriLink's R&D team make the CleanAmpTM Primer and dNTP technology possible.

CleanAmpTM Products are activated during the initial heat cycle of Hot Start PCR. By remaining unreactive at lower temperatures, CleanAmpTM Products significantly reduce background amplification. A reduction or elimination of primer dimer and mis-priming can be achieved by adding CleanAmpTM Products into your PCR reaction.

Reasons to choose CleanAmpTM Products: • Take advantage of a less expensive solution to high stringency PCR • Achieve a significant reduction or elimination of off-target amplification • Increase your amplicon yield and specificity in most cases • Enjoy little change to current protocols when introducing CleanAmpTM Products • Discover very reasonable licensing terms

San Diego, California © istockphoto.com/Jancouver

Learn More About CleanAmpTM Products! CleanAmp TM Overview of CleanAmpTM Products ...... 83 CleanAmpTM dNTPs ...... 84 Visit www.trilinkbiotech.com/cleanamp CleanAmpTM Primers...... 86 CleanAmpTM Amidites ...... 88 Check out all the great information in this catalog, including product details and benefits, application notes and CleanAmpTM dNTP Application Note ...... 90 an oligonucleotide synthesis protocol. CleanAmpTM Primer Application Note...... 93 TM Procedure for Synthesis of CleanAmp Primers...... 99 Read our recent publications: TM

CleanAmp • Lebedev AV, Paul N, Yee J, Kellum J, Timoshchuk VA, Hogrefe RI, Zon G. Chemically modified primers for improved hot start PCR. Collection Symposium Series. 2008;10:398–399. • Lebedev AV, Paul N, Yee J, Timoshchuk VA, Shum J, Miyagi K, Kellum J, Hogrefe RI, Zon G. Hot start PCR with heat-activatable primers: a novel approach for improved PCR performance. Nucleic Acids Research. 2008 Nov; 36:e131. • Shum J, Paul N. Chemically modified primers for improved multiplex polymerase chain reaction. Analytical Biochemistry. In Press. • Koukhareva I, Haoqiang H, Yee J, Paul N, Hogrefe RI, Lebedev AV. A new approach to improved Hot Start PCR: Nucleoside 5'-triphosphates with thermolabile 3'-protecting group. Collection Symposium Series, 10, 259-263. 2008. • Koukhareva I, Haoqiang H, Yee J, Shum J, Paul N, Hogrefe RI, Lebedev AV. Heat activatable 3'-modified dNTPs: Synthesis and application for Hot Start PCR. Nucleic Acids Symposium Series, 52, 259-260. 2008.

Ask our technical support team about your project today.

82 www.trilinkbiotech.com 82 [email protected] (800) 863-6801 83 CleanAmpTM dNTPs

Improve Yield and Specificity

CleanAmpTM dNTPs vastly reduce mis-priming, primer dimer formation and other common deleterious effects (Figure 1). This improvement in the specificity of target amplification allows for enhanced detection of low input template concentrations (Figure 2).

dNTPs dNTPs dNTPs dNTPs TM TM TM TM

UnmodifiedCleanAmp dNTPsUnmodifiedCleanAmp dNTPsUnmodifiedCleanAmp dNTPsUnmodifiedCleanAmp dNTPs 35

30 Cq (dRn) 25 Unmodified dNTPs CleanAmpTM dNTPs 20 1 10 100 1000 10000

533 bp 715 bp 365 bp 653 bp Initial quantity (copies)

TM Figure 1: CleanAmpTM dNTPs improve amplification Figure 2: CleanAmp dNTPs improve the yield and specificity for a number of targets in PCR. limit of detection in real-time PCR by two orders of magnitude.

TM TM TM TM R TM R TM

San Diego, California © istockphoto.com/Jancouver A Flexible Solution Taq Pfu Pfu (exo-)DynazymeDeep VentTth Tfi EconoTaq Taq Pfu Pfu (exo-)DynazymeDeep VentTth Tfi EconoTaq

CleanAmp

TM CleanAmpTM dNTPs are compatible with a number of thermostable DNA polymerases, allowing for 365 bp dNTPs greater flexibility in PCR design (Figure 3). The use of CleanAmpTM dNTPs in the experiment shown in Figure 3 eliminated or reduced primer dimer TM TM CleanAmp dNTPs are the most versatile solution in TriLink's line of PCR enhancing products. CleanAmp dNTPs formations. Moreover, all of the DNA polymerases Unmodified dNTPs CleanAmpTM dNTPs offer a universal approach to improved Hot Start PCR. Replacement of the standard dNTPs, the essential DNA

were able to produce the desired amplicon TM TM TM Figure 3: CleanAmp dNTPs are compatible with a number of polymerase substrate, with CleanAmp dNTPs, offers the same advantages as more costly Hot Start enzymes. TM CleanAmp demonstrating the versatility of CleanAmp dNTPs. commercially available DNA polymerases suited for PCR.

CleanAmpTM dNTPs offer precise control at the start of PCR thermal cycling by blocking DNA polymerase nucleotide incorporation until heat activated. The temperature-dependent control of DNA polymerase extension vastly reduces mis-priming, primer dimer formation and other common deleterious effects. To learn more about CleanAmpTM dNTPs see pages 90-92 or go to www.trilinkbiotech.com/cleanamp. CleanAmpTM dNTPs:

• Offer significant cost savings over other Hot Start technologies Ordering: • Allow easy introduction into PCR reactions • Reduce or eliminate primer dimer formation CleanAmpTM dNTPs can be ordered online at www.trilinkbiotech.com/cleanamp. • Reduce or eliminate mis-priming They are sold as a mix of dATP, dCTP, dGTP and dTTP at 10 mM. • License the technology at reasonable rates N-9501-2 2 µmole each (4 x 2) $90 N-9501-10 10 µmole each (4 x 10) $350

All CleanAmpTM dNTPs are analyzed by AX-HPLC, RP-HPLC, 31P NMR, 1H NMR, Mass Spec and are tested in a functional assay.

84 www.trilinkbiotech.com 84 [email protected] (800) 863-6801 85 CleanAmpTM Primers

A Novel Technology

CleanAmpTM Primers specifically amplify your target by virtually eliminating extension off primer dimer (Figure 1A) and mis-priming (Figure 1B) events. Furthermore, CleanAmpTM Primers provide robust amplicon formation and are compatible with a number of standard DNA polymerases, such as Taq. (Figure 2)

TM TM R

M Precision M UnmodifiedTurbo Precision UnmodifiedTurbo (A) (B) Taq Pfu Pfu (exo-) DyNAzyme Deep Vent Tth Tfi

CleanAmpTM Turbo Primers

Figure 1: CleanAmp™ Primers improve PCR Figure 2: CleanAmpTM Primers are performance in systems prone to (A) primer dimer beneficial when used with a number formation and (B) mis-priming. Both CleanAmp™ of thermostable DNA polymerases. Turbo and Precision Primers improve the specificity The use of CleanAmpTM Turbo Primers of amplification, with Turbo Primers providing provides robust amplification of the the greatest amplicon yield and Precision Primers desired amplicon with primer dimer providing the highest level of specificity. formation reduced or eliminated.

To learn more about CleanAmpTM Primers see pages 93-98 or go to www.trilinkbiotech.com/cleanamp. San Diego, California © istockphoto.com/Jancouver

CleanAmp TM Primers Choose the Right Primer for You CleanAmpTM Primers are available in two forms that differ in the rate of thermal activation into the corresponding TM CleanAmp Primers contain thermolabile chemical modifications that allow for primer-based Hot Start activation unmodified primer. CleanAmpTM Turbo Primers activate more quickly than CleanAmpTM Precision Primers. in PCR. These modifications prevent primer extension at the lower temperatures of PCR set-up and manipulation. The differential rate of activation of the two product types is beneficial for different applications and PCR needs: A Hot Start thermal activation step removes the modification and generates the corresponding unmodified primer, which supports amplification of the desired target. TM CleanAmp CleanAmpTM Turbo Primers CleanAmpTM Precision Primers These novel primers allow you to: • Fast cycling • Standard cycling • Save money by using a hot start solution that is only pennies per reaction. • Multiplex PCR • One-step reverse-transcription PCR • Introduce CleanAmp™ Primer modifications into any sequence. • Improves amplicon yield • Improves specificity and limit of detection • Depend on a guaranteed 10-15 OD yield from every synthesis. • Reduces mis-priming/primer dimer formation • Greatest reduction in mis-priming/primer dimer formation • Receive primers in a high concentration stock solution. • Scale-up to commercially viable amounts. • License the technology at reasonable rates.

Ordering:

Order online at www.trilinkbiotech.com/cleanamp. Primers are $250/pair, sold as phosphodiester DNA, 15-40 bases with a guaranteed minimum yield of 10 ODs. Please inquire for special orders.

86 www.trilinkbiotech.com 86 [email protected] (800) 863-6801 87 TM CleanAmp 89 Amidites Primers. TM TM esters are much more TM modification in place. TM CleanAmp Primer from a reverse phase cartridge. Primer TM (800) 863-6801 Primers are discussed below. Understanding these are discussed below. Primers TM Primers in-house using standard solid phase synthesis solid phase synthesis in-house using standard Primers TM Amidites TM Primer synthesis can be found on pages 99-101. synthesis can be found on pages If followed Primer Primers. TM TM Primers can be acheived in aqueous buffers, we were very fortunate Primers TM

Primer synthesis visit: www.trilinkbiotech.com/cleanamp. Primer TM Technology for Less Technology Amidites and CleanAmp TM TM Primers by RP-HPLC to ensure that specifications are met. to ensure that specifications are met. by RP-HPLC Primers TM Amidites or Primer above room temperature at any time during synthesis. above room temperature at any time during synthesis. Amidites or Primer TM Amidites can be used on any DNA synthesizer. Make as much or as little material as you as little material as Make as much or DNA synthesizer. can be used on any Amidites TM Primer is eluted, an important part of the protocol is the HPLC assay of the product. For the is eluted, an important part of the protocol is the HPLC assay of the product. For Primer TM [email protected] Primer protecting group is a phosphotriester, like most other phosphorus protecting groups used like most protecting group is a phosphotriester, Primer TM Amidites allow you to manufacture CleanAmp allow you to manufacture Amidites TM

stable than the ß-cyanoethyl commonly used. Therefore we readily identified conditions that removed the group oligonucleotide, while leaving the CleanAmp protecting groups from the rest of the For full details on successful CleanAmp For use a centrifugal concentrator to dry your sample completely. your sample completely. use a centrifugal concentrator to dry Don’t Group of the Phosphorus Protecting Base Lability The CleanAmp best success in a PCR reaction, the amount of unprotected primer should be less than 1%. best success in a PCR reaction, the amount of unprotected primer should be no Primers, case of Precision allow more than 1% unmodified material in the sample mixture, and in the Don’t more than 20% of the singly modified species. Do assay all of your CleanAmp characteristics and carefully following the protocols provided will result in high quality CleanAmp carefully following the protocols provided characteristics and The Benefits of CleanAmp The Benefits CleanAmp with CleanAmp Successful Syntheses the CleanAmp Several properties of precisely, it is very simple to prepare CleanAmp it is very simple precisely, Assaying the Final Product Assaying the Final Once the CleanAmp Do use fast deprotecting phosphoramidites. at room temperature for deprotection. Do use methanolic potassium carbonate deprotection schemes. use standard phosphoramidites or standard Don’t Use of DMSO as the Storage Solution Although reasonable stability of CleanAmp for oligonucleotide synthesis. Phosphotriesters are labile to base, however the CleanAmp for oligonucleotide synthesis. Phosphotriesters to discover that DMSO is an excellent stabilizing solution, allowing us to store these modified primers at room to discover that DMSO is an excellent stabilizing solution, allowing us to store temperature for extended times. Do use pure DMSO. TEA (1:1) to elute your CleanAmp use the standard mix of ACN/aq. Don’t need, when you need it. Protocols for CleanAmp it. Protocols need, when you need procedures. CleanAmp procedures. Thermolability of the Phosphorus Protecting Group Thermolability of the Phosphorus Protecting Hot Start activation required the development of a phosphorus protecting Devising a primer that would undergo at 95ºC. This had to be carefully balanced with the ability to prepare, group that would have the proper lability and not the oligonucleotide. It is critical that the compound is handled appropriately store and handle deliver, any time. subjected to elevated temperatures at heat your CleanAmp Don’t

TM San Diego, California © istockphoto.com/AVTG Amidites can also be purchased TM (100 μmoles) X-9050-100 $100 (100 μmoles) X-9060-100 $100 (100 μmoles) X-9070-100 $100 (100 μmoles) X-9080-100 $100 www.trilinkbiotech.com Amidites - PAC-dA-CE - PAC-dA-CE - PAC-dG-CE - Ac-dC-CE - dT-CE Amidites can be ordered online, by phone or email. TM TM TM TM TM Amidites are available through our website, as well as through Glen Research. Amidites are available through our website, TM CleanAmp CleanAmp CleanAmp Please inquire for bulk quantities. CleanAmp CleanAmp through Glen Research at www.glenres.com. Ordering: CleanAmp Amidites are offered purely for research and development use. They may not be used to make CleanAmp™ Primers for commercial use, either as a Primers Amidites are offered purely for research and development use. They may not be used to make CleanAmp™ TM

CleanAmp If you have oligonucleotide synthesis capabilities in-house, you may prefer making your own CleanAmp If you have oligonucleotide synthesis capabilities custom synthesized product or as part of another product, without license from TriLink. Licenses are available at reasonable costs if you desire to commercialize custom synthesized product or as part of another product, without license from TriLink. this product. See Licensing Information section for the disclaimer of license statement pertaining to PCR products. Primers. CleanAmp Primers.

CleanAmp

TM 88 90

CleanAmpTM

Target

as theyareable to decreasetheformationofprimer dimer dNTP with awiderangeofcommonly-usedDNA polymerases. Start PCRthatisamendabletouseinstandardprotocols, dNTPs provideanovel,cost-effective andefficientroutetoHot activation hasshowngreatpromisefortheuseofCleanAmp tures (95°C)). This temperature-dependentcontrolofdNTP buffer at~pH8(25°C)tobecome6elevatedtempera- an increaseintemperaturewillcausea Tris-based reaction based PCRbuffers astemperatureisincreased(forexample, of protectinggroupcanbeattributedtotheacidification Tris- which isnowasuitableDNA polymerasesubstrate. The loss group isremovedtoformthecorrespondingstandarddNTP, heated totheelevatedtemperaturesofPCR,protecting PCR reactionpreparation(Figure1).Whentheis primer extensionatthelessstringent,lowertemperaturesof ranyl protectinggroupsontothe3'-hydroxylofadNTP blocks PCR. The introductionoftemperaturesensitivetetrahydrofu- groups thatallowfordNTP-mediatedHotStartactivationin porated inanyreactionbysimplesubstitution.CleanAmp in allPCRexperiments,modifieddNTPscaneasilybeincor Start activationapproachinPCR.SincedNTPsareubiquitous 5´-triphosphates (dNTPs)thataremodifiedtoallowforaHot that hadnotbeenexploredistheconceptofdeoxynucleoside- can alsosignificantlyincreasethecostofreactions.Onearea While thesemodificationstoPCRsetupcanbeeffective, they use ofchemicallymodifiedprimersthatareunextendable(2,3). DNA polymerase,theadditionofaccessoryproteinsand the physicalseparationofreactioncomponents,inhibition been madeinthistechnology, includingstrategiesthatrequire cycling temperaturesarereached.Manydevelopmentshave from formingprimerextensionproductsuntilhigherthermo- process, thecomponentsofaPCRreactionareprevented off-target amplificationinPCRaretermedHotStart(3).Inthis intended target(2). nents withinthereactionandinhibitingamplificationofthe the overalleffectiveness ofPCRbyconsumingkeycompo- inevitable (1).Competingoff-target amplificationsdecrease experiment, whereprimerinteractionandextensionarealmost plifications aregenerallyduetotheexcessofprimersineach dimer formationdominatesthereaction. These off-target am- PCR, especiallywhennonspecificamplificationsuchasprimer monly usedtechniques.However, inherentflawscanplague Chain Reaction(PCR)isoneofthemostpowerfulandcom- Figure 1:HotStart Activation ofCleanAmp fers, pH8to9at25°C. dNTPs withDNA polymerasesthatutilize Tris-based PCRbuf-

By Tony Le,JonathanShumandNatashaPaul,Ph.D.; TriLink BioTechnologies CleanAmp

3’ 5’

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POPOPO H POPOPO H dN*TP CleanAmp uct. The followingexperimentsexploretheeffectiveness of products whileincreasingthespecificityofdesiredprod tions primer/template systempronetoprimerdimer. PCRCondi- 10-100 foldincrease insensitivityrelativetostandard dNTPs, Eliminate PrimerDimerFormation,Increase Yield Start DNA polymerases. used incombinationwithavarietyofnon-HotStartandHot plotted inastandard curve,CleanAmp at alltemplateconcentrations. Whentheresulting datawas CleanAmp of standarddNTPsfailedtoamplify thetargetband,while 3). When5to50copiesoftemplate wereemployed,theuse ies using a varietyoftemplateconcentrations rangingfrom5-5,000cop- The detectionoftheLambdagDNA amplicon wastestedover nant primerdimerformationatlower templateconcentrations. plays reducedsensitivityinreal-timedetectionduetopredomi- Real-time detectionoftheabove-mentioned533bptargetdis- a greaterrangeoftemplateconcentrationsinreal-timePCR. seen withadNTP concentrationof0.4mMandaMgCl using 0.2mMconcentration,themostconsistentresultsare cleaner PCRreaction. Although theseresultswereobtained off-target ampliconsaresignificantlyreduced,resultingina nate. WhenCleanAmp amplicon iscompletelyabsentandprimerdimerspredomi- most significantatlowertemplateconcentrationsasthetarget tions, theeffect onspecificityandsensitivityofdetectionis non-target sequences(Figure2).UnderstandardPCRcondi- natural dNTPsarecompromisedbycompetingamplificationof trations (4). After 40thermalcycles,reactionsthatemployed dimer formationinPCR,especiallyatlowtemplateconcen- template systemfromLambdagenomicDNA ispronetoprimer amplicon yieldinendpointPCR. A problematic533bpprimer/ or eliminateprimerdimerformationwhileincreasingtarget demonstrate thecompatibilityofCleanAmp dNTPs andHotStartDNA polymerases.Studieswillalso dimer artifactsbycomparingtheirperformancetounmodified CleanAmp desired amplicon'syieldisincreasedaswell.Byintroducing dNTPs, notonlyareprimerdimerssignificantlyreduced,the Figure 2:EndpointPCRevaluationofCleanAmp centration of2.5mM. Template Concentrations Improve SpecificityOveraGreaterRangeof 1 CleanAmp CleanAmp onpage92. Taq TM TM TM dNTPssuccessfullyyieldedthedesired product dNTPsintoaPCRsetup,primerdimersandother to primer systemsprone primer/template in dNTPs Man® probedetectioninreal-time PCR(Figure TM TM dNTPsprovideamplificationspecificityover dNTP modificationssignificantlylower TM dNTPsaresubstitutedforstandard TM dNTPsafforded a TM dNTPswhen 533 bp TM dNTPsina 2 con- - in two separate primer/template systems. in twoseparateprimer/templatesystems. against unmodifieddNTPsatdifferent templateconcentrations experimental designforreal-timetargetdetection. dNTPs givetheusermoreoptionsandfewerrestrictionsin at lowertemplate concentrations. The standarddeviationat dNTPs providegreater experimentalprecision,especially For boththeHIV-1 andLambdaDNA targets,CleanAmp™ consistent whenusingCleanAmp 1 copyofHIV-1 gDNA and50copiesofLambdagDNA were Figure 4:SYBRGreenreal-timePCRassay whereresultsof Lambda gDNA usingCleanAmp Figure 3:Real-timePCRdetectionof5-5,000copies centrations. Byincreasingthelimitofdetection,CleanAmp thus allowingfordetectionoverawiderrangeoftemplatecon- template concentrationsarelimiting(3,5).WithCleanAmp iterations toachievereproducibledata,especiallywhen Increase PCRReproducibility page 92. chart form.PCRConditions A. Amplification plotsofthereal-timedata.B.Real-timedatain ment showninFigure4,CleanAmp run, allowinganalysiswithconfidence.Inthereal-timeexperi dNTPs, PCRdatafluctuatesverylittlewithinanexperimental

FluorescenceFluorescence (dRn) (dRn) -0.4 0.4 0.8 1.2 0 0 Real-time PCRresultsoftenvaryandrequiremultiple 50 copies Lambda50 copies genomic DNA Lambda GenomicLambda Target DNA (533 bp) 02 040 30 20 10 Amplification Plot Amplification

-0.5 Fluorescence (dRn) Cq (dRn) 0.5 1.5 2.5 3.5 4.5 20 25 30 35 40 0 1 2 3 4 Cycles template genomic template genomic Lambda Cycles HIV DNA DNA DNA DNA 0 1 -1 Fluorescence (dRn) -0.2 -0.1 0.1 0.2 0.3 0.4 0.5 0.6 0 Reproducibility of Real- of Reproducibility 1 [email protected] Copies 5000 02 040 30 20 10 500 CleanAmp Reproducibility dNTP: (HIV-1) 50 25 (Standard Values) of deviation Ct 5 5 1 1 5 Unmodified dNTP

9 01010 10000 1000 100 10 13 CleanAmp Unmodified dNTPs Initial quantity(copies) 17 Amplification Plot Cycles (std.(std. dev.) dev.) 1,2 21 Standard Curve CleanAmp Unmodified dNTPs dNTP dNTP dNTP 0.22 0.32 1.57 0.05 0.240.24 3.623.62

onpage92. 25

Fluorescence (dRn) Cycles TM Fluorescence (dRn) -0.4 TM dNTPs 33 0.4 0.8 0 CleanAmp dNTPs Time Data TM 37 dNTPs.PCRConditions 0 TM dNTPs. TM dNTPs 1 copyHIV CleanAmpCleanAmp dNTPsweretested HIV-1 Genomic Target DNA (365 bp) (std.(std. dev.) dev.) 0.07 0.09 0.11 0.04 0.080.08 0.110.11 02 040 30 20 10 -   1 genomic1 DNA Amplification Plot Amplification dNTP dNTP dNTP Cycles Cycles 5 50 500 5000 TM 1 on TM - CleanAmp merases wereabletoproducethedesired365bpamplicon. reduced primerdimerformations,andalloftheDNA poly- using standarddNTPs,DeepVentR ases thatmaybeamenableforusewithCleanAmp merases inPCR,thereareotherthermostableDNA polymer- demonstrating theirversatility. evaluated alongsideotherHotStarttechnologies,CleanAmp by providingagreaterrelativeampliconyield.Overall,when CleanAmp plate system,reactionscontaining suppressed off-target ampliconformationinthis primer/tem- dimer formation. Although allHotStartapproachessuccessfully DNA polymerases,inadditionto Versatility ofCleanAmp of second-guessingPCRresults. CleanAmp merase withCleanAmp Polymerases Achieve BetterResultsComparedtoHotStartDNA decreases considerablywhenCleanAmp these lowconcentrationsishighunderstandardconditions,yet with eitherstandardorCleanAmp whether theycouldsuccessfullyamplifya365bptargetband were testedinPCRwithendpointagarosegelanalysistolearn To investigatefurther, sevennon-HotStartDNA polymerases

Figure 5:EvaluationoftheperformanceCleanAmp 365 bp CleanAmp standard deviationofreactionswithdNTPsislow, the reaction.Evenathightemplateconcentrations,where of CleanAmp the remainingDNA polymerases.Incontrast,theintroduction Furthermore, off-target amplificationisevidentformostof dimer while polymerases. PCRConditions in amplificationreactionswithavarietyofthermostableDNA for specificdetails. CleanAmp (3). WhentestedagainstothercommonHotStarttechnologies, for theirabilitytoamplifya365bptargetfromHIV-1 (Figure6) with variousHotStartDNA polymerasesandnaturaldNTPs further optimization. further optimization. SeetheCleanAmp target amplification.Otherprimer/templatesystemsmayrequire dNTPs displaycomparableoff-target resultswhile enhancing CleanAmp Though In theseexperiments,reactionscontaining TM TM TM TM TM Tth andTfiDNA showedonlyslightamplification. dNTPsoutperformedtheHotStarttechnologies dNTPsprovidedacomparablereductioninprimer dNTPsworkwelltogetherwithabroadselectionof dNTPs,moretimeisspentanalyzingdatainstead dNTPsstillproduceamoreconsistentresult.With TM Taq (800) 863-6801 (800) dNTPsintotheseexperimentseliminatedor isoneofthemostcommonlyusedDNA poly- TM dNTP Application Note dNTPApplication TM dNTPswerecomparedtoreactions TM dNTPs 2 onpage92. Taq TM Taq dNTPs(Figure5).When TM DNA polymerase,further amplifiedonlyprimer DNA polymeraseand TM TM dNTPManual Product dNTPsareusedin Taq TM DNA poly- dNTPs. TM dNTPs

91 CleanAmp TM 92 CleanAmpTM conjunction withCleanAmp yield, yethadsomeprimerdimerbyproducts.Whenusedin and PrecisionPrimersbothincreasedrelativeamplicon reduced oreliminated.Similarly, TriLink's CleanAmp used in combination with CleanAmp™ dNTPs. used incombinationwithCleanAmp™dNTPs. ment isfurtherenhancedwhentheHotStarttechnologiesare specificity andefficiencyofPCRreactions,butthisimprove When usedindividually, eachHotStart approachimprovesthe Primers, whereoff-target amplificationwasentirelyeliminated. Conditions performance wasseen,especiallywithCleanAmp of CleanAmp but primerdimerformationwasstillevident.Withtheaddition amplicon yieldimprovedcomparedto and HotStart-IT®Taq susceptibility toprimerdimerformation.WhenPlatinum® conducted witha Lambda gDNA template system, known for its ment inPCRspecificity. InFigure7,experimentswere amplicon yield(normalizedto HIV-1 genomicDNA.B.Graphicalrepresentationoftherelative PCR analysisofamplificationreactionscontaining5copies mercially availableHotStartDNA polymerases. A. Endpoint Figure 6:ComparisonofCleanAmp™dNTPstoothercom- gies, CleanAmp Improve PerformancewithOtherHotStartTechnologies CleanAmp When usedincombinationwithotherHotStarttechnolo-

2 Relative Amplicon Yield onpage92. 0.2 0.4 0.6 0.8 1.2 1 0 TM dNTPs,off-target amplificationissignificantly

TM dNTP

dNTPsallowforanevengreaterenhance-

TM Taq + HS + Taq

dNTP Application Note dNTPApplication

wereusedwithstandarddNTPs,relative

Taq* Platinum TM Hot Start Technologies Start Hot dNTPs,animprovementinPCR

Taq Gold*

+CleanAmp AmpliTaq®

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365 bp dimer primer

dNTPs).PCR HS* TM DyNAzyme Precision TM www.trilinkbiotech.com Turbo Taq - PCR Conditions at afractionoftheprice. performance thanHotStartDNA polymerasescanbeachieved plification whileenhancingtargetyield.Comparableorbetter real-time experiments. NOTE: TaqMan® probe(0.1µM)andROXwereusedfor 2. PCR:1XPCRbuffer (20mM Tris (pH8.4),50mMKCl,2.5 . Koukhareva,I.,Haoqiang,H., Yee, J.,Shum,Paul,N., 7. Koukhareva,I.,Haoqiang,H., Yee, J.,Paul,N.,Hogrefe, 6. Svenstrup,H.F., Jensen,J.S.,Bjornelius,E.,Lidbrink,P., 5. Lebedev, A.V., Paul,N., Yee, J., Timoshchuk, V.A., Shum, 4. Chou,Q.,Russell,M.,Birch,D.E.,Raymond,J.andBloch, 3. Dang,C.andJayasena,S.D.(1996)Oligonucleotideinhibi- 2. Brownie,J.,Shawcross,S., Theaker, J.,Whitcombe,D., 1. References PCR: 1XPCRbuffer (20mM Tris (pH8.4),50mMKCl,2.5 1. the relativeoff-target amplification.PCRConditions 5 copiesofHIV-1 genomicDNA.B.Graphicalrepresentationof A. EndpointPCRanalysisofamplificationreactionscontaining polymerases usedwithandwithoutCleanAmp™dNTPs. Figure 7:ComparisonofcommerciallyavailableHotStartDNA Summary [95°C (40sec),56°C(3072°C(2min)]35X.(7min). DNA polymerase(var. U),50 µL. Thermal cycling:95°C(10min); MgCl2), Primers(0.4µM)0.2mMdNTPs,5copiesHIV-1 gDNA, (10 min);[95°C(40sec),57°C(3072°C(60sec)]40X. gDNA, 1.25UTaq MgCl2), Primers(0.2µM),0.2mMdNTPs,5,000copiesLambda PCR. Nucleic Acids SymposiumSeries, 52,259-260. 3'-modified dNTPs: Synthesisand ApplicationforHotStart Hogrefe, R.I.andLebedev, A.V. (2008) Heat Activatable 259-260. bile 3'-ProtectingGroup.Collection SymposiumSeries,10, Hot StartPCR:Nucleoside5'-Triphosphates with Thermola- R.I. andLebedev, A.V. (2008) A New Approach to Improved plasma genitalium.JClinMicrobiol,43,3121-3128. quantitative real-timePCRassayfordetectionofMyco- Birkelund, S.andChristiansen,G.(2005)Developmentofa 36, e131. proach forimprovedPCRperformance.Nucleic Acids Res, Hot startPCRwithheat-activatableprimers:anovelap- J., Miyagi,K.,Kellum,Hogrefe,R.I.andZon,G.(2008) Nucleic Acids Res,20,1717-1723. dimerization improveslow-copy-numberamplifications. W. (1992)Preventionofpre-PCRmis-primingandprimer number targetsbyPCR.JMolBiol,264,268-278. tors of Taq DNA polymerasefacilitatedetection oflowcopy 25, 3235-3241. of primer-dimeraccumulationinPCR.Nucleic Acids Res, Ferrie, R.,Newton,C.andLittle,S.(1997) The elimination CleanAmp TM dNTPsreduceoreliminateoff-target am- DNA polymerase,50µL. Thermal cycling:95°C 1 onpage92. needs. Herein,weinvestigatetheutilityofCleanAmp tion ofunmodifiedprimercanbecontrolledtosuityourreaction primers orthefaster-releasing Turbo Primers,therateofforma- after activationathighertemperatures(Figure1). tion, butalsodisplaytheflexibilitytoallowintendedextension discriminating temperaturesofreactionset-upandmanipula- fications preventDNA polymeraseextensionatthelower, less tion andextensionduringPCR. The thermolabileprimermodi- into PCRprimersallowsforgreatercontrolofprimerhybridiza- Figure 1:HotStart Activation ofCleanAmp DNA polymerases. modifications arecompatiblewithmanycommonlyused oligonucleotide synthesisprotocols.Inaddition,CleanAmp™ introduced toanyprimersequenceusingstandardsolidphase Hot StartactivationinPCR. These modificationscanbeeasily thermolabile protectinggroups,representasimpleapproachto DNA polymerase.Incontrast,primers,whichhaveCleanAmp extensive manipulations,suchasinthepreparationof tions canaddsignificantcosttothereactionbyneedfor proteins. ManyofthespecializedDNA polymerasecomposi- inhibition oftheDNA polymerase,andtheuseofaccessory which includephysicalseparationofreactioncomponents, primer dimerformationbytheuseofHotStarttechnologies, template islimited. eliminate off-target amplification,especiallywhenavailable (1) Therefore, itiscriticaltosubstantiallydecreaseoreven an increaseinundesiredprimerhybridizationandextension. priming isexacerbatedaslesstemplateavailablecausing concentrations, theproblemofprimerdimerformationandmis- dNTPs, fromamplifyingthedesiredtemplate. At lowertemplate substrates, suchastheprimers,DNA polymerase,andthe lower theefficiencyofPCRbyeffectively sequesteringPCR specific regionsonthetemplate.Suchoff-target amplifications of primerstooneanotherornon- caused bythehybridization tions. These includeprimerdimerandmis-primingproducts and reproducibilityareoftenplaguedbyoff-target amplifica interest. Although PCRisapowerfultechnique,itssensitivity place molecularbiologicalmethodtoamplifyaDNA targetof PCR and fast cycling PCR. PCR andfastcyclingPCR. advanced techniquessuchasmultiplexPCR,one-stepRT- Primer modificationsinstandardPCRprotocolsandmore By JonathanShum,Joyclyn Yee, ElenaHidalgo Ashrafi, andNatashaPaul,Ph.D.; TriLink BioTechnologies CleanAmp

Furthermore, byusingeithertheslow-releasingPrecision The introductionofthermolabileCleanAmp Attempts havebeenmadetoalleviatetheproblemof The PolymeraseChainReaction(PCR)isacommon- [email protected] TM

PrimerNote Application TM Primers TM modifications TM - TM CleanAmp improved ampliconyieldrelativetounmodifiedprimers. evident overawiderangeofinputtemplateconcentrationswith the greatestbenefit. Thisreductioninoff-target amplificationis of themis-primingproducts,withPrecisionPrimersproviding comparison tounmodifiedprimers, Turbo Primersreducemuch fidelity andefficiencyofamplificationthedesiredtarget.In tion wereremovedat30,35and40cycles. primer/template systempronetoprimerdimer. Aliquots ofreac- CleanAmp General Applications template concentrations usingSYBRGreen from Lambdagenomic DNA wasexploredoverarangeofinput In Figure3,thelowerlimitofdetection ofa533bpamplicon template concentrationascompared tounmodifiedprimers. successfully amplifythecorrectamplicon at10-100foldlower Figure 2:EndpointPCRevaluationofCleanAmp PCR conditions:1Xbuffer (20mM Tris (pH8.4),50mMKCl,2.5MgCl amplicon formation.CleanAmp plification, complicatingreal-time PCRdetectionofthedesired trations, off-target amplificationscompetewiththedesired am difficulty encounteredinPCR.Often,atlowtemplateconcen a largerangeoftemplateconcentrations. is required. found tohavethegreatestutilitywhenpureampliconformation cantly reducingprimerdimerformation.PrecisionPrimerswere were abletoefficientlyformthedesiredamplicon,whilesignifi (Figure 2C). These studiesdemonstratedthat Turbo Primers at 30thermalcyclesbutfullyrecoveredafter40 dimer formation,robusttargetamplificationwasslightlydelayed the Precisionprotectinggroupssignificantlyreducedprimer primer dimerformation.However, whiletheslowerreleaseof Primers yieldedonlythedesiredamplicon,withnodetectable seen after40cycles.Inthesamesystem,useofPrecision 2B). For Turbo Primers,onlyaslightamountofprimerdimeris target yieldascomparedtotheunmodifiedprimers(Figure reduced primerdimerformationandpromotedanevengreater By contrast,theintroductionof Turbo Primerssignificantly with theformationofdesired365bpamplicon(Figure2A). to bepronerobustprimerdimerformation,whichcompetes 2). Amplifications usingunmodifiedPCRprimerswerefound removing aliquotsafter30,35and40thermalcycles(Figure (1) Inthesestudies,thereactionprogressionwasmonitoredby in theamplificationofaregionHIV-1 tatgenomicDNA. lower, ifnoteliminateofftargetamplification time PCR.Forthe unmodifiedprimers,therange ofdetection sec), 72°C(1min)]40X,(7min). (1.25U), 50µL. Thermal cyclingconditions:95°C(10min);[95°C(40sec),56°C(30 Primers (0.5µM),dNTPs(0.2mM),5copiesHIV-1 gDNA,Taq CleanAmp Primer dimerformationhasbeenfoundtobeproblematic Mis-priming canalsobeasignificanthindrancetothe Detection ofatargetatlowconcentrationsisanother TM TM Primersprovideamplificationspecificityover Turbo andPrecisionPrimerssignificantly 1X PCR buffer (20 PCR conditions: 1X PCR buffer (20 PCR conditions: 95 Thermal cycling conditions: dNTPs PCR cycle # PCR cycle # (800) 863-6801 (800) o o C (10 min); [95 (0.2mM), 5 copies HIV TM Primer Application Note PrimerApplication o o Unmodified Unmodified C (40 sec), 56 mM mM Primers Tris Tris A BC A BC (pH8.4), 50 (pH8.4), 50 -1 gDNA o o TM CleanAmp™ C (30 sec), 72 sec), (30 C Primershavebeenfoundto Primers Turbo , Taq mM mM DNA polymerase (1.25U), 50 KCl KCl o o , 2.5 , 2.5 C (1 min)]40X, 72 CleanAmp™ Precision mM mM Primers ® MgCl MgCl detectioninreal- 403530403530403530 403530403530403530 DNA polymerase 2 ), Primers (0.5 o o C (7 min) (7 C TM 365bpamplicon primer dimer primer 385 Primersina µ bp L. amplicon µ M), M),

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93 CleanAmp TM 94 CleanAmpTM efficiently reducing primerdimerformationandmis-priming. can beemployed withoutcompromisingamplicon yield,while to theperformanceofCleanAmp as achemicallymodifiedversionof with oneofaseriesHotStartDNA polymerases,such In thesestudies,theperformanceofunmodifiedprimers Figure 3:SYBRGreen PCR conditions:1Xbuffer (20mM Tris (pH8.4),50mMKCl,2.5MgCl amplicon formationandotheroff-target formation. or less,itwouldbeimpossibletodifferentiate betweendesired incided withthenotemplatecontrol(NTC)curveat500copies started above500copies.Becausetheamplificationcurveco CleanAmp examined. These findingsaresignificant,astheyindicate that was muchhigherthaneachofthe HotStartpolymerases polymerase gavethegreatestbenefit, astheampliconyield 4A, B).Moreover, Turbo Primersandunmodified fied Taq or muchloweryieldthanreactions thatemployedunmodi- fied Taq CleanAmp stream applications,suchassinglemoleculedetection. indicative oftheirutilityinanumberhigh-sensitivitydown- This increasedlimitofdetectionusingPrecisionPrimersis the greatestlevelofdetection,detectingaslowfivecopies. able fromtheNTCcurve.PrecisionPrimersultimatelyprovide crease indetection,asthe50copyconcentrationisdistinguish- Turbo andPrecisionPrimers. copies ofLambdagDNA wereassayedusingunmodified, CleanAmp ments werethencarriedouttocomparetheperformanceof efit relativetoreactionswithunmodifiedprimers,experi Technologies Precision CleanAmp (40 sec),57°C(3072°C(1min)]40X. reactions performedinduplicate. Thermal cyclingconditions:95°C(10min);[95°C (0.2 mM),0-50,000copiesLambdagDNA,1.25U CleanAmp The useof Turbo Primersprovidesatleastaten-foldin- Since CleanAmp Fluorescence Fluorescence Fluorescence -1 -1 -1 -1 -1 -1 DNA polymerasewith Precision Primers(Figure DNA polymerase. Amplicon wasformedwith equal 1 3 5 1 3 5 1 1 3 3 5 5 1 1 3 3 5 5 0 0 0 0 0 Fluorescence TM TM -1 -1 1 1 3 3 5 5 TM Primersandunmodified PrimerstothatofotherHotStarttechnologies. PrecisionPrecision Turbo Turbo Unmodified Unmodified 0 PrimersoutperformotherHotStart TM Primers(0.5µM),SYBRGreen(.15X),ROX(30nM),dNTPs 02 040 40 30 30 20 20 10 10 02 040 30 20 10 02 040 40 30 30 20 20 10 10 TM 02 040 30 20 10 Primer Application Note PrimerApplication TM ® Primersprovidesignificantben Cycles Cycles Cycles Cycles Cycles Cycles real-timePCRassaywhere0-50,000 Cycles Cycles TM Primerswithunmodi- Taq Taq Taq (2),wascompared DNA polymerase,25µL; DNA polymerase NTC NTC copies copies 50,000 50,000 copies copies 5,000 5,000 500 copies 50 copies 50 copies 5 copies NTC NTC copies copies 50,000 50,000 copies 5,000 500 copies 500 copies 50 copies 50 copies 5 copies NTC copies 50,000 copies copies 5,000 5,000 500 copies 50 copies 5 copies 5 copies 500 copies 50 copies Taq NTC NTC copies 50,000 copies copies 5,000 5,000 500 copies 500 copies 50 copies 50 copies 5 copies 5 copies www.trilinkbiotech.com DNA - - 2 ), - polymerase werekeptconstant.Overall,CleanAmp exception ofDeepVent efficient amplificationoftheDNA target.Inallcases,withthe Each oftheDNA polymerasesexaminedwasabletosupport ability torobustlyformthedesired365bpamplicon(Figure5). merases devoidofHotStartactivationwereevaluatedfortheir PCR experiments.Intheendpointreactions,sevenDNA poly- could beemployedwithotherDNA polymerasesinendpoint 56°C (30 sec), 72°C (1 min)] 35X, 72°C (7 min). 56°C (30sec),72°C(1min)]35X,(7min). (2.5U), to reactionscontaining B. Graphicalrepresentationoftherelativeampliconyield,normalized amplification reactionscontainingfivecopiesofHIV-1 genomicDNA. available HotStartDNA polymerases. A. EndpointPCRanalysisof CleanAmp Figure 4:ComparisonofCleanAmp factor istheincreased propensityforprimerdimer formation(5), inherent problems thatinhibitrobustamplification. Onemajor culture tests(4). be moresensitiveandlesstimeconsuming thantraditionalcell as viralscreens(3),wherePCRbased assayshaveprovento different medicaldiagnosticandscientificapplications,such of interest. This applicationhasbeenanessentialtoolformany multiplex PCR,employsadistinct primerpairforeachamplicon multiple targetsinasinglereaction. This approach,knownas PCR Applications Multiplex Figure 5:EvaluationoftheperformanceCleanAmp PCR conditions:1Xbuffer (20mM Tris (pH8.4),50mMKCl,2.5MgCl potential fortheimplementationofCleanAmp polymerases inendpointPCR. This compatibilitygivesgreat are versatileastheycanbeusedwithotherthermostableDNA polymerases. in amplificationreactionswithavarietyofthermostableDNA number of applications. number ofapplications. determine whetherPrecisionand Turbo CleanAmp DNA polymerases Turbo Primers. Primers (0.4µM),dNTPs(0.2mM),5copiesHIV-1 gDNA,Taq (1.25U), * A denot spo es Although multiplex PCRhasmanyadvantages,there are One promisingapplicationofPCRistheabilitytoamplify Taq Tfi(20U), 50µL. Thermal cyclingconditions:95°C(10min);[95°C(40sec), 1X PCR buffer (20 PCR conditions: 1X PCR buffer (20 PCR conditions: mM 95 Thermal cycling conditions: Dynazyme mM 95 Thermal cycling conditions: Dynazyme

Pfu (2.5U),(exo-)Dynazyme Taq + Turbo Primers ly o o C (10 min), [95 C (10 min), [95 me dNTPs dNTPs DNA polymerasewasusedasapointofreferenceto Taq + Precision Primers ra

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er AmpliTaq Gold * o o C (40 sec), 56 C (40 sec), 56 mM mM , Deep Vent , Deep Vent t fi s St Dyna zy me™ II ed HotStart-IT* ar Tris Tris t Taq TM TM -1 -1 Deep Vent ™ ac DyNAzyme HS * (pH8.4), 50 (pH8.4), 50 ti gDNA gDNA va TM am Tth DNA polymeraseplusCleanAmp 385 ti ™(2.5U) ™(2.5U) on andTfipolymerase,theunitsofDNA o o plicon C (30 sec), 72 sec), (30 C C (30 sec), 72 sec), (30 C , , Tfi Taq Taq bp , , (1.25U), (1.25U), mM mM Taq Tth Tth B TM KCl KCl (2.5U), (2.5U), (2.5U), (2.5U), CleanAmp CleanAmp Pfu Primerstoothercommercially Precision Precision Precision Precision Primers Primers o o Pfu Pfu , 2.5 , 2.5 C (1 min)] 35X, 72 35X, min)] (1 C C (1 min)] 35X, 72 35X, min)] (1 C Pfu (exo-) Fraction of amplicon formation 0.2 0.4 0.6 0.8 1.2 TM Tfi Tfi (2.5U), (2.5U), (2.5U), (2.5U), 0 1 mM mM

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™ DyNAzyme template concentration is not an option. template concentrationisnotanoption. in clinicalsettings,wheretemplatesampleislimited,increasing increased tocompensateforinefficientamplification.However, multiplexed PCRassay, oftentemplateconcentrationmustbe template concentrations Multiplexed targetamplificationatlow dimer formation. the CleanAmp™ Turbo Primers,improvingwithreducedprimer vealed minimaloptimizationofthedesignandconcentration sensitive toprimerdimerformationwasused.Findingsre- system whereampliconyieldandPCRefficiencyareextremely single targetreactionswasappliedtoamultiplexPCRassay. A Primers toreduceothercompetingoff-target amplification,in than theshorter targets whenusingunmodifiedprimers, with more, thelonger targetsappearedtoamplifyless efficiently unmodified primers couldonlydetect5,000copies. Further efficiently detecting50copiesof Lambda genomicDNA,where when using Turbo Primers(Figure6A),with Turbo Primers reaction wasdetectedata100-fold lowerinputoftemplate compared tounmodifiedprimers, ampliconformationinatriplet Turbo Primershavedemonstratedmuchpromise.When was normalizedto Turbo. in Figure6A.Foreachtemplateconcentration,ampliconyield con yieldandprimerdimerformationforexperimentperformed a varying amountof template. B. Quantitative analysisof ampli amounts. A. Performance of Turbo and unmodified primers with Figure 6:Multiplexreactionsoverawiderangeoftemplate PCR conditions:1Xbuffer (20mM Tris (pH8.4),50mMKCl,2.5MgCl dominate thereaction.Below, theabilityofCleanAmp probability ofsuccess,shouldoff-target ampliconformation similar(7). This isatimeconsumingprocess,whichhaslow optimized, suchthatamplificationefficienciesofalltargetsare in primerdesign,individualpairconcentrationsmustbe tion canbeachallenge.Coupledwiththisdecreasedflexibility of interestandexhibitalowleveloff-target ampliconforma- design ofmultipleprimerpairsthatarebothspecificforatarget the preferentialamplificationofcertaintargets(6). Thereforethe quences inthereaction. Another challengeofmultiplexPCRis which canresultfromthelargernumberofuniqueprimerse- gets inmultiplexPCRisevaluated. The abilityofCleanAmp ers toimprovethespecificityofampliconformationforalltar (30 sec),72°C(2min)]35X,(7min). polymerase, 50µL.Thermalcyclingconditions:95°C(10min);[95°C(40sec),56°C Primers (0.5µM),0.2mMdNTPs,var. copiesLambdagDNA,1.25U Percent Primer Relative Amplicon

Dimer 100 120 Relative Amplicon Yield B. A. 0.5 1.5 20 40 60 80 When individualassaysarecombinedintoasingle, In thesecaseswheregreatersensitivity isnecessary, Relative0 AmpliconYield Yield 0 1 0.5 1.5 0 1 # copies of template # copies of 05050 50000 5000 500 50 05050 50000 5000 500 50 Unmodified Unmodified template 533bp 600bp 05050 50000 5000 500 50 Unmodified Unmodified 50 500500050000 533bp Template (copies) 600bp 962bp Turbo Turbo Template (copies) Template 962bp Turbo Turbo Template (copies) Template [email protected] 05050050,000 5,000 500 50 05050050,000 5,000 500 50 x- x- -x -x x- x- -x -x x x - - Turbo Unmodified x x - - Turbo Unmodified x x - - PercentTurbo PrimerUnmodified x x - -

Dimer 100 120 x x - - 20 40 60 80 0 x x - - x x - - x x - - 05050 50000 5000 500 50 x x - - Template (copies) x x - - x x - - x x - - x x - - NTC x x - - NTC x x - - x x - - Taq TM DNA Prim- - TM 2 - ), Turbo Unmodified -

Cycles 15 20 25 30 35 40 step RT-PCR isnotwithoutitsown inherentproblems. and the PCRstep.However,both thereverse transcription one- advantage foraone-step protocolisthatreplicates willrepeat nique thatreducesthechancesof contamination(11). Another RT-PCR protocolprovidesastreamlined,high-throughput tech- col canintroduceopportunitiesfor contamination. A one-step extra manipulationproceduresinherent toatwo-stepproto- step followedbyanelevatedtemperature PCRstep(10). The protocol thatinvolvesalowertemperature reversetranscription files(8,9). Thetypical RT-PCR reactionconsistsofatwo-step gold standardforvalidationofmicroarraygeneexpressionpro- cantly. ReversetranscriptionPCR(RT-PCR) hasbecomethe massive amountofgeneexpressionresultshasgrownsignifi RT-PCROne-step Applications limit ofdetection. endpoint andreal-timeassays,witha100-foldincreaseinthe fication. Furthermoregreatersensitivityisachievedforboth primer concentrationsproducerobust,non-preferentialampli- provide greatflexibilityinassaydesign,asawiderangeof amplicon yieldandlowerprimerdimerformation. Turbo Primers PCR providesseveraladvantages,whichincludegreater ers varyingconcentrationoftemplate. template amounts.Comparisonof Turbo andunmodifiedprim Figure 7:Real-timeMultiplexPCRdetectionofawiderange gets withsimilarefficiency. AdditionallytheuseofCleanAmp concentrations examined, Turbo Primersamplifiedallthreetar of templatewereemployed.Ontheotherhand,atall the longest962bpampliconnotforminguntil50,000copies primers is delayed or not present. primers isdelayedornotpresent. ful amplification,whereasamplicondetectionwithunmodified primers. Turbo Primersprovideearlierdetectionofsuccess- L600 target,noCqisobservedat50copiesfortheunmodified tration decreased(Figure7).Insomecases,suchasinthe unmodified and Turbo Primersincreasedastemplateconcen Primers. Inthisduplexreaction,thedifference inCqbetween PCR conditions:1Xbuffer (20mM Tris (pH8.4),50mMKCl,2.5MgCl amplification ismuchmoresensitiveusingCleanAmp experiments, atlowtemplateconcentrations,thedetectionof man® probedetectionwasperformed.Muchliketheendpoint ible inquantitativereal-timePCR,aduplexreactionusing Taq- multiplex PCR CleanAmp™ Turbo Primersoutperforminreal-time template concentrationsatincreasedsensitivity. three ampliconsbeingformedoverabroadrangeofinput Turbo Primersislessrestrictedbytargetsizelimitations,all formation (Figure6B).Insummary, efficientamplificationby Primers improvedPCRperformancebyreducingprimerdimer 95°C (10 min); [95°C (40 sec), 56°C (30 sec), 72°C (2 min)] 35X, 72°C (7 min). 95°C (10min);[95°C(40sec),56°C(3072°C(2min)]35X,(7min). µM), ROX(30nM),1.25UTaq Primers (0.5µM),0.2mMdNTPs,var. copiesLambdagDNA, TaqMan .+11E0 1.E+05 1.E+03 1.E+01 CleanAmp

Amount of template (copies) CyclesCq 15 20 25 30 35 40 .+11E0 1.E+05 1.E+03 1.E+01 With theadventofmicroarrays,needtovalidate Overall, theuseofCleanAmp To confirmthattherangeofdetectionwasalsoreproduc Amount of template (800) 863-6801 (800) (copies) TM Primer Application Note PrimerApplication DNA polymerase,50µL.Thermalcyclingconditions: L533 L533 Unmodified Turbo II

TM CyclesCq Unmodified Turbo II 15 20 25 30 35 40 Turbo Primersinmultiplex .+11E0 1.E+05 1.E+03 1.E+01 Amount of template (copies) ® probe (0.1 TM L600 L600 Turbo 2 TM ), - - - - -

Unmodified Turbo II

95 CleanAmp TM 96 CleanAmpTM RT-PCR istoemployCleanAmp Primers andslower-releasingCleanAmp CleanAmp gene ofinterest,thePCRprimerswereunmodified,contained Virus Reverse Transcriptase (M-MLV RT) (Invitrogen).Forthe Transcriptase (Invitrogen)andMoloneyMurineLeukemia reverse-transcription PCRusingSuperScript Script primer. Reactionscontained U 0.16 mMdNTPs,0-0.25µgHumanbraintotalRNA,25Ureversetranscriptase,0.3 CleanAmp methods forapplicationssuchasmultiplexone-stepRT-PCR. amplifications andalsoenableothermoreuniversal RT priming shown thatwithvarious RNA tissuesources,one-stepRT-PCR employing either totalRNA orpoly(A)+RNA.Experimentshave two typesofCleanAmp prepared aseithera)standard,unmodifiedprimersorb)oneof RT enzymes. To illustratethis,PCR primersequenceswere extension duringtheRT stepwithanumberofcommonlyused At highertemperature,theCleanAmp specific ampliconformationfromextensionofPCRprimers. ing reversetranscription. This reduceslower-temperature,non- dance. Amplicon formation wasenrichedwhenCleanAmp desired amplicon(264bp)being formed atlowrelativeabun- sulted informationofseveralnon-specific amplicons,withthe for bothRT enzymes. The useofunmodifiedPCRprimersre MLV RT) (Invitrogen) (Figure8),withsimilarresultswerefound Moloney MurineLeukemiaVirus Reverse Transcriptase (M- tested: SuperScript®IIReverse Transcriptase (Invitrogen) and responding unmodifiedprimer. Thefollowing RT enzymeswere differ intherateoftemperature-inducedformationcor- Figure 8:EvaluationofCleanAmp Tris (pH8.4),50mMKCl,1.5MgCl Reactionconditions:1XPCR buffer (20mM sec), 72°C(30sec)]30X,(5min). Thermal cyclingconditions:42°C(30min),95°C(10[95°Csec),60°C is required. inhibition ofsuchprimerextensionatlowertemperatures scription. To improvethesensitivityandspecificityof RT-PCR, products atthelessstringenttemperaturesofreversetran- extension ofprimerstoformprimer-dimerand/ornon-specific reverse transcriptase(14)orDNA polymerase(15)mediated as two-step(12,13). The lackofsensitivitycanbetheresult In manycasesone-stepRT-PCR reactionsarenotassensitive CleanAmp Precision modification.ReversetranscriptionutilizedapolydT CleanAmp ers displayedimprovedspecificity. lesser degree.However, reactionscontaining PrecisionPrim- Primers, ampliconformationwas also enrichedtoaslightly regardless ofreversetranscriptase(RT)enzyme Turbo Primerswereused.InthecaseofCleanAmp PCR. CleanAmp allowing forgreaterspecificityofprimerextensionduring Taq DNA polymerase,25µL. One approachtoimprovingthespecificityofone-step CleanAmp CleanAmp ® IIorM-MLV Reverse Transcriptase. TM TM TM Primerpair, onlytheRT primercanelongatedur- Turbo modificationorcontainedCleanAmp Primersimprovesensitivityandspecificity TM TM TM Primerscanalso beutilizedwithreactions PrimersdecreasecompetingPCRprimer TM Primersprovideasolutiontonon-specific Primer Application Note PrimerApplication TM Primers(16,17).CleanAmp Taq 2 ), Primers(0.5µM),polydT primer(1µM), TM DNA polymeraseandSuper- Primers.Byintroducinga TM Primersinone-step TM Primersareactivated, TM ++-+-+-+- -+-+ + ++-+-+-+- -+-+ + + -++- - - ++- --- + -++- - - ++- --- ++++++------++++++------++-+-+-+-+-+-+ ++-+-+-+-+-+-+ ----++----++ ----++----++ + ---++- - -++ - - + ---++- - -++ - - PrecisionPrimers M- SuperScript M- SuperScript Unmodified primers Unmodified primers CleanAmp CleanAmp 0.25 CleanAmp 0.25 CleanAmp ® MLV RT (25 U) MLV RT (25 U) IIReverse amplicon m m 264 bp g Human liver total RNA total liver Human g g Human liver total RNA total liver Human g TM TM TM TM ® ® Precision Primers Precision Precision Primers Precision TM Turbo Primers Turbo Primers II RT (25 U) II RT (25 U) www.trilinkbiotech.com TM Precision Turbo TM TM - 18 Turbo andPrecisionPrimers. The useofCleanAmp unmodified primers.CleanAmp ers enhancedthespecificityofreactioncomparedto not recommendtheuseofpolydT Turbo andCleanAmp utilized apolydT CleanAmp™ Precisionmodifications.Reversetranscription and GeneB),thePCRprimerswereunmodifiedorcontained in singleplexandduplex.Forthegenesofinterest(Gene A reverse-transcription PCRinHumanLiver Total RNA (Clontech) primers producedoff-target amplificationproducts.CleanAmp (Figure 9).Inbothsingleplexandduplexreactions,unmodified B (205bp)wereemployedinsingleplexandduplexreactions port multiplexedone-stepRT-PCR. Gene A (264bp)andGene CleanAmp RT-PCR protocols. sensitivity andspecificityinone-stependpointreal-time displayed improvedspecificitywhenCleanAmp providing thehighestenrichmentinmultiplexamplification. CleanAmp the mostsignificantincreaseinampliconformation.Overall, Figure 9:EvaluationofCleanAmp MgCl Reaction conditions:1XPCRbuffer (20mM Tris (pH8.4),50mMKCl,1.5 brain totalRNA,25Ureversetranscriptase,0.3 mary, CleanAmp ficity ofsingleplexreactions,CleanAmp have beenevaluatedinreal-timeqRT-PCR usingCleanAmp were employed.Furthermore,one-stepRT-PCR experiments RT-PCR of CleanAmp tiplex one-stepRT-PCR reactions. This isalsoakeyadvantage priming methodallowsforamorestreamlinedapproachtomul- synthesis stepsinsteadofgene-specificprimer. Thisuniversal dT modified DNA polymerasesor RT enzymes.Furthermore,poly are recommended. There isnoneedtousemore expensive reverse transcriptases,suchasM-MLV reversetranscriptase, polymerases, suchas formation inone-stepRT-PCR protocols. Non-HotStartDNA vated inthePCRstep,therebyreducingnon-specificproduct ers fromextensionduringtheRT stepuntilthermallyacti- merase andM-MLV Reverse Transcriptase. have somedrawbacks includingadecreaseinsensitivity anda throughput. However, these fastercyclingPCRprotocolsalso PCR amplification step,allowingforfasterresults andgreater ing, efforts havebeendirectedatreducingthedurationof Fast PCRThermalCycling Applications ment overunmodifiedprimerswhen usedinone-step RT-PCR. 18 2 orrandomprimerscanbeemployedduringthecDNA ), Primers(0.5µM),polydT primer(1µM),0.16mMdNTPs,0-0.25µgHuman CleanAmp This uniquethermolabilemodificationprotectsPCRprim In PCRapplicationssuchasdiagnostics ormedicaltest- Ge TM A TM ne Primersdemonstratedmarkedimprovementof Primersimprovespecificityofduplexone-step TM Primersbecauseotherone-stepRT-PCR kitsdo TM 18 TM Primers were examined for theirabilitytosup- Primerswereexamined primer. Reactionscontained Ge PrecisionPrimersareasignificantimprove B ne TM Taq PrecisionPrimersimprovedthespeci- A Ge DNA polymerase,andstandard an ne B d ++- -+-+ + ++-+ -+-+ s TM Un Cl Turbo Primersdisplayed e TM 18 mo anA orrandomprimers.Insum- Primersinone-step 0 pampl 205 bp Ge ampl 264 bp Ge di Taq mp fi ne ne TM ed DNA polymerase,25µL. TM B A PrecisionPrimers Pr Pr im ec ic ic er is on on s TM io Taq Primers n Pr DNA poly- TM im Prim- er s TM - TM - In comparison,theuseofCleanAmp before eachofthesubsequentlower templateconcentrations. plate DNA wasaccurately detected,theNTCsampleamplified amplification oftheNTCsample. Although 125 copiesoftem pretation oftheamplificationplots wascomplicatedbystrong (Figure 10).Whenunmodifiedprimers wereemployed,inter 66 Figure 10: A. Amplification plotsofCleanAmp PCR conditions:1Xbuffer (20mM Tris (pH8.4),50mMKCl,2.5MgCl B. A. CleanAmp demonstrated. primer concentrationusingfastcyclingPCRprotocolswillbe Turbo Primersinpreventingoff-target amplificationathigh time experiments,especiallywithSYBRGreen amplification, whichcanincreasethefalsepositiverateinreal- increase inprimerconcentrationoftenleadstohigheroff-target The maincomplicationencounteredwiththissolutionisthatan creased sensitivityistoincreaseprimerconcentration. loss inreproducibility(18).Onesuggestedsolutiontode- were plottedinstandard curves(Figure10B),CleanAmp mal cyclesofthe experiment.Inaddition,whenthe Cqvalues curve didnotcross thethresholdline(Cq)during the 40ther- for detectiondowntoasinglecopy. The NTCamplification tion infastcyclingPCR time SYBRGreen off-target amplificationandtoimprovethesensitivityofreal- Turbo Primersandunmodified primers. merase, 25µL 1:2000SYBRGreen™I. Thermal cyclingconditions:[95 Primers (1.5µM),0.8mMdNTPs,0-125copiesHIVgDNA,3.75U when usingfastcyclingPCR.Herein,theutilityofCleanAmp concentration couldleadultimatelytoanincreaseinsensitivity Therefore, preventionofoff-target amplificationathighprimer tions. B.CorrespondingstandardcurvecomparingCleanAmp primer dimerformationoverarangeoftemplateconcentra- and unmodifiedprimersinaprimer/templatesystemproneto

o C (30sec)]40X. Turbo Primerswereevaluated fortheirabilitytoreduce TM

Fluorescence Fluorescence Turbo Primersdecreaseoff-targetamplifica -1 -1 1 3 1 3 Fluorescence -1 0 0 1 3 Unmodified ® Turbo 0 assaysusingatypicalfastcyclingprotocol CyclesCq [email protected] 20 25 30 35 0.1 Amount of template (copies) 02 040 30 20 10 02 040 30 20 10 02 040 30 20 10 Unmodified 1 Cycles Cycles yl s Cycle 0101000 100 10 TM Turbo II Turbo Turbo Primersallowed TM ® TurboPrimers detection(18). Taq DNA poly- o C (10sec), NTC 1 copy 5 copies 25 copies 125 copies TM 2 - - TM ), - TM B. Corresponding standard curve comparing CleanAmp standard curvecomparing B. Corresponding dimer formationoverarangeoftemplate concentrations. Velocity the 5copysamplewithFullVelocity Master Mix. CleanAmp specificity overalargerangeoftemplateconcentrations. Primers areusedanddemonstrates Turbo Primer'sincreased Figure 11: A. Amplification plotsofCleanAmp B. A. µM). Thermal cyclingconditions:95 modified Primers):1XFull Velocity™ MasterMix(Stratagene/Agilent),Primers(0.15 3.75 UTaq 2.5 mMMgCl PCR conditions(Turbo Primers):1XPCRbuffer (20mM Tris (pH8.4), 50mMKCl, detection in a fast cycling protocol. detection inafastcyclingprotocol. lower Cq's. This allowedforevengreaterspeedofamplicon each technology, reactionsthatemployed Turbo Primershad trations evaluated. Although theefficienciesweresimilarfor approaches displayedgoodlinearityovertherangeofconcen- the experimentalresultswereplottedinastandardcurve,both lower levelofoff-target amplificationby Turbo Primers.When plified beforetheNTCcurve. Thesefindingsarelikelyduetoa when Turbo Primerswereemployed,5copiesoftemplateam- Primers andFull Velocity and FullVelocity technologies denaturation timenecessaryfortheFullVelocity pare thetwoproductsinsameexperiment,a5minuteinitial 245 bptargetfromLambdagenomicDNA. To accuratelycom- fast cyclingPCR. The experimentinvolvedamplificationofa a commerciallyavailablekitwhichisformulatedspecificallyfor suppression ofprimerdimerformationwhenCleanAmp fied primers. Thischaracteristicismostlikelyrelatedtothe Turbo Primersexhibitedgreaterlinearitycomparedtounmodi- Primers hadslightlygreatersensitivitythantheFullVelocity was usedwiththesecyclingconditions,wefoundthat Turbo CleanAmp As depictedinFigure11A, theNTCcurveoverlappedwith Next, CleanAmp TM DNA polymerase,25µL 1:2000SYBR Green™ I.PCRconditions(Un- SYBRGreen 2 ), Primers(0.3µM),0.8mMdNTPs,0-10,000copiesLambdagDNA, TM Turbo Primersoutperformotherfastcycling (800) 863-6801 (800) Fluoresence (dRn) Fluorescence (dRn) CyclesCq -1 -1 TM 20 25 30 35 40 1 3 1 3 Amount(copies)oftemplate inaprimer/templatesystemprone toprimer 0 TM 0 1 Turbo Full Velocity Primer Application Note PrimerApplication TM ® Turbo PrimerswerecomparedwithFull Full Velocity 0100 10 QPCR Mastermix (Stratagene/Agilent), QPCRMastermix(Stratagene/Agilent), 02 040 30 20 10 02 040 30 20 10 TM -1 1 3 o SYBR Green C (5min)[95 0 ™ Ct Turbo II Turbo Ct 001000 1000 20 TM o C (10sec),66 MasterMix.However, 0 ® QPCRMasterMix. 40 TM TurboPrimers o C (30sec)]40X. TM Mastermix NTC copies 5,000 500 copies 50 copies 5 copies TM TM Turbo Turbo

97 CleanAmp TM 98 CleanAmpTM www.trilinkbiotech.com/cleanamp. has beendemonstratedthatCleanAmp achieved, duetoimprovementinspecificityandsensitivity. It eliminate off-target amplification.Greaterampliconyieldisalso Which CleanAmp other technologiesinmultipleapplications. a broadrangeofapplicationsCleanAmp control ofprimerhybridizationandextensionduringPCR.Over Summary For themostcurrentCleanAmp CleanAmp dimer formation Reduced mis-priming/primer Improved ampliconyield Multiplex PCR Fast cycling Turbo Primers In summation,CleanAmp TM TM Primerisbestformyapplication? Primer Application Note PrimerApplication TM TM Primersallowforgreater formation mis-priming/primer dimer Greatest reductionin of detection Improved specificity and limit transcription PCR One-step reverse- Standard cycling Precision Primers Primer Application Notevisit: TM TM Primersoutperform Primersreduceor www.trilinkbiotech.com 8 Hilscher, C.,Vahrson, W. andDittmer, D.P.18. (2005)Faster Louwrier, A. and A. vanderValk. 2005. Thermally revers- 17. Kielar, D.,D.W., T. Langmann,C. Aslanidis, M.Probst, 16. Chou,Q.,M.Russell,D.E.Birch,J.RaymondandW. 15. Peters,I.R.,C.R.Helps,E.J.HallandM.J.Day. 2004. 14. Suslov, O.andD.A.Steindler. 2005.PCRinhibitionby 13. LekanneDeprez,R.H., A.C. Fijnvandraat, J.M.Ruijter 12. Wacker, M.J.andM.P. Godard.2005. Analysis ofOne- 11. Bustin,S.A.and T. Nolan.2004.PitfallsofQuantitative 10. 9. Canales,R.D., Y. Luo,J.C.Willey, B. Austermiller, C.C. 8. Cinque,P., Bossolasco,S.andLundkvist, A. (2003) 7. Henegariu,O.,Heerema,N.A.,Dlouhy, S.R.,Vance, G.H. 6. Brownie,J.,Shawcross,S., Theaker, J.,Whitcombe,D., 5. Gilbride,K.A.,Lee,D.Y. andBeaudette,L.A.(2006) 4. DeBiasi,R.L.andKennethL. Tyler, K.L.(2004)Molecular 3. Moretti, T., B.KoonsandBudowle.1998.Enhance- 2. Chou,Q.,M.Russell,D.E.Birch,J.RaymondandW. 1. References e182. and showincreasedvariability. Nucleic Acids Res,33, quantitative real-timePCRprotocols maylosesensitivity Technology 36:947-952. for applicationinhotstartPCR.Enzyme andMicrobial ible inactivationof Taq polymerasein an organicsolvent Chemistry 47:2089-2097. tissues byreal-timereversetranscription-PCR.Clinical tion ofhuman ABCA1 mRNA invariouscelltypes and M. NaruszewiczandG.Schmitz.2001.Rapidquantifica tions. Nucleic Acids Research20:1717-1723. primer dimerizationimproveslow-copy-numberamplifica Bloch. 1992.Preventionofpre-PCRmis-primingand 286:203-217. tive assaydesign.JournalofImmunologicalMethods Real-time RT-PCR: considerations forefficientandsensi fication efficiency. Nucleic AcidsResearch33:e181. reverse transcriptaseleadstoanoverestimationofampli- Analytical Biochemistry307:63-69. SYBR greenIdependsoncDNA synthesisconditions. quantitative real-timepolymerasechainreactionusing and A.F.M. Moorman. 2002.Sensitivityandaccuracyof Script III.Journalofbiomoleculartechniques16:266-271. Step and Two-Step Real-Time RT-PCR UsingSuper- action. Journalofbiomoleculartechniques15:155-166. Real-Time Reverse-Transcription PolymeraseChainRe- Bioinformatics 7:23. cleotide arraydataviaquantitativereal-timePCR.BMC and K.F. Kerr. 2006.Evaluationofmethodsforoligonu- Qin, L., R.P. Beyer, F.N. Hudson,N.J. Linford, D.E. Morris 24:1115-1122. tive geneexpressionplatforms.NatureBiotechnology 2006. EvaluationofDNA microarrayresultswithquantita- Barbacioru, C.Boysen,K.Hunkapiller, R.V. Jensen,etal. of thecentralnervoussystem.JClinMicrobiol,26,1-28. Molecular analysisofcerebrospinalfluidinviraldiseases and Step-by-StepProtocol.BioTechniques, 23,504-511. and Vogt, P.H. (1997)MultiplexPCR:CriticalParameters Res., 25,3235-3241. tion ofprimer-dimeraccumulationinPCR.Nucl. Acids Ferrie, R.,Newton,C.andLittle,S.(1997) The elimina- process control.JMicrobiolMethods,66,1-20. crobial communities,detectingpathogens,andreal-time Molecular techniquesinwastewater:Understandingmi- biol Rev, 17,903–925. Methods forDiagnosisofViral Encephalitis.ClinMicro- pliTaq ment ofPCRamplificationyieldandspecificityusing Am- tions. Nucleic Acids Res20:1717-1723. primer dimerizationimproveslow-copy-numberamplifica Bloch. 1992.Preventionofpre-PCRmis-primingand GoldDNA polymerase.Biotechniques25:716-722. - - - - . SynthesisofCleanAmp A. Procedure: . Cleavage/Deprotectionof CleanAmp B. C. SepPakisolationandpurification procedure: Procedurefor SynthesisofCleanAmp

. Purchase,ormanuallyfill,a1µmolesynthesiscolumnwithUnyLinker 4. Addactivatedmolecularsieves(20beads/mL)toeachamiditebottle,flushwithdryargongas,recapthesealusing 3. . Follow anautomatedsynthesisprotocolforfastdeprotectingphosphoramiditemonomersasrecommendedbytheinstru- 6. . Prepare0.067MsolutionsofeachCleanAmp 2. Preparesolutionsofeachfastdeprotectingphosphoramiditemonomerinanhydrousacetonitrile(ACN)using 1. . Itisrecommendedtoproceedwiththecleavage/deprotectionofCleanAmp 9. Whensynthesisiscompleted,drythecolumnusingargonflow. 8. Atthelastcouplingcycle,leaveDMT group“On”. 7. . LoadtheDNA synthesizerwiththereagentslistedinReagentstableappropriatewellaccordingtomanufac- 5. . Equilibratecartridge with10mL ofacetonitrile,followed by10mL of50%acetonitrile in100mM TEAA andfinally by15 2. Fita60mL syringetoSepPakcartridge;part#20515. 1. Processthesolutionimmediately usingSepPakisolation/purification,otherwisekeepthesolutionat –80°C to–20°C. 7. Measurethetotalcrudeyield usingUVspectrophotometer. 6. Wash supportwith2mL of1M Triethyl Ammonium AAcetatel (TEAA)andaddtothedeprotectionsolution. 5. Letsupportsettleandtransfer supernatantsolutionintoa50mL conicaltube. 4. Placethevialonarotarymixer for20hrsatroomtemperature. 3. Add6mL offreshlyprepared50mMpotassiumcarbonatesolutioninMeOH. 2. Transfer support(witholigonucleotide)fromthecolumntoascrewcap8mL glassvial. 1. dry dichloromethane(DCM):ACN(1:1)inastandardDNA synthesizerbottle. parafilm andkeepitovernightatroomtemperaturebeforeuse. the appropriatesolvent. ment manufacturerwiththefollowingexception:increasecoupling timeforCleanAmp Loadtheancillaryreagents.Itiscriticalthatappropriatecappingreagentsareusedforfastdeprotectingmono- b. precipitation oftheamidite. instructions. We recommend usingaconcentrationof0.067M. manufacturer’s minutes. of thesynthesis,otherwisekeepdrycolumnat–20°C. standard DNA synthesizerbottle.Prepare0.067MsolutionofCleanAmp . Loadtheamidites.UseaspareportforCleanAmp a. instructions. turer’s mL of25mM TEAA. mers. “Cap A” mustbethephenoxyacetylanhydrideversionofreagent.DONOT USE ACETIC ANHYDRIDE. CleanAmp Reagents DMSO anhydrous GlenResearch TriLink BioTechnologies,2% TFA Inc. inwater(Trifluoroacetic Acid) 1M TEAA, pH7.2(Triethylammonium Acetate) 50 mMPotassiumcarbonateinMethanol HPLC GradeWater GlenResearch Cap BreagentwithN-Methyl-Imidazole Cap A reagentwithPhenoxyaceticanhydride Activated MolecularSieves FisherScientific Dichloromethane Acetonitrile, driedto≤20ppmwater(ACN) atdpoetn N hshrmdtsGlenResearch Synthesis ColumncontainingUnyLinker Fast deprotectingDNA phosphoramidites To properlydissolvea100 umolebottleofaCleanAmp [email protected] TM TM DNA phosphoramidites oligonucleotidesonsolidsupportat1µmolescale: TM oligonucleotides: Procedure for SynthesisofCleanAmp TM TM phosphoramidite(exceptforCleanAmp Vendor Fisher Scientific TriLink BioTechnologies, Inc. Fisher Scientific Glen Research TriLink BioTechnologies, Inc. Fisher Scientific Glen Research TriLink BioTechnologies, Inc. TM monomer(s). TM monomertoobtaina0.067Msolution,use1.50mL of TM TM support. Primers (800) 863-6801 (800) TM -dG phosphoramiditemonomerinmixtureof TM oligonucleotideimmediatelyaftercompletion Higher concentrationsmayleadto TM -dG)indryacetonitrilea TM phosphoramiditestoten TM

Primers

99 CleanAmp TM 100 CleanAmpTM *See nextpageforrepresentative RP-HPLCanalysisofCleanAmp www.trilinkbiotech.com/cleanamp. For themostcurrentCleanAmp HPLC Analysis ofCleanAmp Procedure for SynthesisofCleanAmp 2 Cap,vortexandspindownfractions.Readfraction2only. Ifyieldislessthan10OD 12. . Determinethemobilityofunprotectedoligonucle- 3. 2. Method: . If specifications arenotmet,thebenefitsof 5. Turbo shouldhavelessthan1%ofthefullydeprot- 4. 5 Storetheoligonucleotideat4°Corless. Although itisverystableatroomtemperatureintheDMSOsolution,longterm 15. . Preparesampleforloading: 3. . Analyzea0.2OD 1. 4 AnalyzepurifiedCleanAmp 14. DeterminemolarconcentrationofpurifiedoligonucleotideinDMSOsolutionusingtheabsorbance readingandthecalcu 13. . Wash SepPakwithapurificationbuffer: 9. Pass10mL of1M TEAA throughthecartridgeover5minutestoneutralizeacid. 8. ImmediatelyrinseSepPakwith20mL HPLCwater. 7. Pass10mL of2% TFA throughthecartridgeover3minutes.Observeappearanceoforangecoloredband. 6. Rinsethecartridgewith20mL ofwater. 5. 11. Rinsecartridgewith20mL ofwater. Collectrinse. 10. . Loadthesolutiontocartridgewithaflowrateof1-2mL/min andpourintothe60mL loadingsyringeoncartridge.Col 4. ElutesamplesusingDMSO. . ForCleanAmp a. . ForCleanAmp b. . Add200µL DMSOtosyringeandeluteintoafreshmicrotube(fraction3). e. Add200µL DMSOtosyringeandeluteintoafreshmicrotube(fraction2)usingplunger. Add another200µL to d. Removesyringefromcartridge,pull outplunger, reattachthe syringetothecartridge,andpushairintosamegradu- c. Add200µL DMSOtosyringeandeluteintoamicrotube,labeledfraction1. b. Attacha3mL sliptipsyringetothecartridge. a. fraction 3. Add justenoughofthematerialinfraction3to2achieve10OD otide byheatinganother0.2OD Observeat260nm. e. FlowRate:1mL/minute d. Gradient:0-100%Bover40 minutes c. Buffer A: 100mM TEAA; Buffer B: ACN b. Column:Waters µBondapak8mmRP cartridge a. less demandingthanothers. system isveryrobustandsomePCRapplicationsare CleanAmp doubly modifiedPrecisionoligonucleotide. which elutesbetweentheunmodifiedproductand have lessthan20%ofthesinglymodifiedmaterial, less than2%ofthatcontaminant.Precisionshould ected oligonucleotidewhilePrecisionshouldcontain storage isimprovedatlowertemperatures. the purifiedsample,andnomorethan20%ofsinglymodifiedspeciesifaPrecisionPrimerwasprepared. . Dilutethesolutionto50mL with1M TEAA. c. Rinsethedeprotectionvesselwithanother2mL ofwater, passingthatthroughthefilterandintotubewith b. Add2mL of1M TEAA tothesampleandpipettesolutioninto5mL syringefittedwithaluerlockand0.44 a. lated extinctioncoefficient.Ifnecessary, dilutewithDMSOtoobtaina0.200mMsolution. CleanAmp minutes at95°C,whichwillcompletelyremovethe the CleanAmp contamination byoligonucleotidesnotprotected rial byreversephaseHPLCtodeterminethelevelof lect andreadtheabsorbanceofflow-throughtoensureoligoisboundcartridge. DMSO tosyringeandeluteintothesamegraduatedtubeforatotalof400µL DMSOinfraction2. ated tubetocompletelyremovealltheDMSO. (WAT 027324)orcomparable. sample. micron filterdisc.Filterthesolutionintoa50mL conicaltube. TM TM technology may still be experienced. may still technology The modificationfromtheoligonucleotide. TM moiety. 260 TM TM TM unitsampleoftheisolatedmate- PrecisionOligos,apply15mL of25% ACN, 100mM TEAA tocartridge.Collectrinse. Turbo Oligos,apply15mL of15% ACN, 100mM TEAA tocartridge.Collectrinse. TM Primerprotocolvisit: TM oligonucleotidesbyRP-HPLCtoensurethatnomorethan1-2%ofunmodifiedprimerisin 260 unitsamplefor40 www.trilinkbiotech.com TM Primers TM Turbo andPrecisionPrimers. Single 4-oxo-tetradecylmodification CleanAmp TM Turbo Primers 260 units,readtheabsorbanceof Double 4-oxo-tetradecylmodification CleanAmp 260 units. TM Precision Primers - - Injection :30µL :0-100%Bover40min :100mMTEAApH=7-7.5 Gradient A Buffer :Waters uBondapakC18,10µm,125A,300x3.9 mm RP3-HPLC CleanAmp Reverse-phase HPLCanalysis:

TM Primers [email protected] CleanAmp CleanAmp Procedure for SynthesisofCleanAmp TM Flow Rate : 1mL/min Flow Rate :Acetonitrile B Buffer TM Precision Primers Turbo Primers (800) 863-6801 (800) Temperature :RT

Primers

101 CleanAmp TM 102 Technical Information

Enzymatic Activity of Selected Nucleoside 5'-TriphosphatesNucleoside Selected of Analogs Activity Their Enzymatic and SELEX Processthe of TriphosphateOverview Nucleoside An Modified Applications: WeightCalculations Molecular TriphosphatesNucleoside FormulaCoefficient ...... Extinction Oligonucleotide An Introduction to Extinction Coefficients and Molecular Weights of Oligonucleotides Weightsof Molecular and Coefficients Extinction to Introduction An Tablesand Data Oligo When is my Oligonucleotide PureEnough? Oligonucleotide my is When Duplexes ...... Oligonucleotide Modified of Thermostability Yields Synthetic Oligonucleotide Understanding Acids ...... Nucleic Pseudo-complementary Quenchers...... Technical Information Randomer Oligonucleotides Randomer Licensing Your Licensing Compound ...... TriLink'sPublications,PresentationsCollaborations Patents Research and ...... ResearchRewards Research Programs Triphosphate Nucleoside Bibliography Trademark, Patent andLicensingInformation ...... An Antisense Oligonucleotide PrimerOligonucleotide Antisense An Reagents ...... Phosphoramidite Synthesized Custom of Use Applications Oligonucleotide Linker,Methods ...... Pre-ActivatedCarboxyl and A Applications DADE: CPGs ...... Amino Phthalimidyl the for Methods Deprotection of Comparison ProductConjugated of Yield Enhances Reagent Amine Protected3' Phthalimidyl Utility ...... Their and Reagents Spacer and Linker DNA Oligonucleotides ...... Modified Troubleshootingof Synthesis the Synthesis ...... Oligonucleotide of History Short A Reagents and Synthesis DNA 31 P Chemical Shifts in NMR Spectra of Nucleotide Derivatives Nucleotide of Spectra NMR in Shifts Chemical P ...... www.trilinkbiotech.com ...... La Jolla,California©istockphoto.com/aimintang ...... 141 140 139 139 138 135 132 126 134 130 128 120 153 154 153 153 152 150 147 119 117 116 114 112 109 103 [email protected] [email protected] systems. ofbiological overall understanding the which wascriticalinadvancing access tooligonucleotides, synthesizer intheearly1980's.Many labsnowhadroutine DNAfirst phosphoramidite the (ABI), commercialized which formed collaboration, Applied Biosystems, Incorporated phosphoramidite oligonucleotide synthesis chemistry. This of CalTech, whentheysetouttoautomateCaruthers'new of theUniversityandProfessorLeroyHood Colorado, between ProfessorMarvinCaruthers to thecollaboration the eraofhumangenomecanbedated investigating synthesis wasareality.oligonucleotide of The beginning automated before reliable, require manyadvancements to research laboratories. easily available it markedthefirstchemistry attempt tomakeoligonucleotide Smithsonian. Although itusedchemistrythatisnowoutdated, the first DNA synthesizer, which canbeviewedinthe Vegathis fieldandalsointroduced pioneered Biotechnologies synthesizers werethefirst automated systemsavailable. Consequently,faster thanthatofoligonucleotides. peptide the synthesisofpeptides(orsmallproteins)developed Becausethechemistrywassomewhatsimpler,biomolecules. that sciencetocreatenew and cloningtechniquesallowed systems, through toolssuchasrecombinantprotein synthesis tools. It was theabilitytosimulateandmodify biological of precise level, itwasultimatelyfueledbythedevelopment systems atthemolecular aboutbiological knowledge growing Although thebiotechindustrycameintoexistencethrough proteins andnucleicacids. bio-macromolecules: elucidated an interestintryingtosyntheticallypreparesomeofthenewly chemistry. It was onlynaturalthatchemistswouldsoonhave and the interfacebetweenbiology science ofinvestigating result. the biological biology,Thus wasbornmolecular the the linkbetweenchemistry revealed ofgeneticsand DNA's doublehelixstructure(Watson andCrick,1953)that by Watsonpaper published andCrickin1953describing code wasknownbythemid-1940's,itlandmark acids andtheirbiologicalfunctionascarriersofthegenetic these molecules. make-upofnucleic Although thegeneral became interestedinsynthesizing why researchersinitially of thelength. regardless synthesized nucleicacids, to includeallchemically broadened in length.Inthepast20years,meaninghas nucleotides strictest sense, isashortpieceofnucleicacid lessthan50 inthe1950's. biotech milestones, beginning synthesis, thisarticlewilldiscusscertain oligonucleotide to thelaunchof thatledspecifically scientific contributions of recent chronicle In anattempttopresentacondensed development of groundbreaking diagnosticsand therapeutics. huge levelsofcapitalinvestmentnecessarytofinancethe to securethe funded research,whichinturnmadeitpossible academic institutionstoownandpatentfederally allowed was thepassageofact. the Bayh-Dole This legislation this novelchemistry Another eventcrucialtoestablishing synthesis. key steppingstonestothebirthofoligonucleotide fermentation, pasteurization,andvaccinedevelopment,were such as interwoven. Forexample,earlybiotechinnovations the historyofmodernbiotechnology, for thetwoaretightly synthesis, itwouldbenecessarytoexamine oligonucleotide By RichardHogrefe, Ph.D.; TriLink BioTechnologies Synthesis A ShortHistoryofOligonucleotide However, to thechemistrywould general accessibility of tohavesomeunderstanding Next, itisnecessary inthe First, anoligonucleotide, for theuninitiated, In ordertooffer anexhaustivereport on thehistoryof (DCC), tocouplethe5'hydroxyl ofanother3'-protected carbodiimide reagent, suchasdicyclohexyl condensation activated usinga 3' phosphatesweresubsequently to thephosphomonoester. These 5'protectednucleoside thatthenhydrolyzed using phosphorochloridates nucleoside 3' phosphatesofthe5'protected chloride, heprepared phosphoryl one step,oxidation.Inplaceofthe hydrolysable oftheaddition scheme usedtodaywiththeexception synthesis (Figure2),isthesamecyclic oligonucleotide (Khorana, et. al., 1956). whenactivated that coupledtothedesirednucleoside other wasthefirstnucleoside use ofastablephosphorylated (Schaller,publications e.t al., 1963;Smith,et. al., 1961). The from Khorana'sinitial chemists, virtuallyunmodified synthesis, isstillwidelyusedtodaybyoligonucleotide oligonucleotide protection schemenecessaryforsequential more thanjustafewbaseslong.Oneconcept,theon-off the convenient synthesisofoligonucleotides made possible He introducedtwoconceptsto oligonucleotides. thefieldthat H. GobindKhoranabecameinterestedinthesynthesisof bythenameof researcher attheUniversityofChicago Contribution The Khorana hydrolysis. Figure1showsthebasicscheme. wasnotstable,being susceptibleto intermediate chloride well, albeitslowly.reasonably Additionally, the phosphoryl 5' hydroxylofa3'protectedthymidine. The chemistryworked dichloride. phenylphosphoryl This wasthenreactedwiththe ofa5'benzoylprotected thymidine,using chloride phosphoryl wasmadebyfirstthe3' thymidine nucleosides preparing and (Michelson nucleotide Todd, 1955). and Michelson Todd reportedthepreparationofadithymidinyl occurred in1955when synthesis ofanoligonucleotide First Dinucleotide oligonucleotide synthesis. of theinfluentialresearcherswhoweredirectparents article willexamineingreaterdetailthecontributionsofsome sequence thehumangenome. sections of The followingthis to sophisticated sequencingmethods,whichmadeitpossible the more including of amyriadapplications, development Kary Mullisinthe1980sprovedtobecatalystforrapid Figure 1: Phosphoryl chloridate method described by Michelson and Todd. This protocol, called the phosphodiester method of This protocol,calledthephosphodiester In thelate1950'sacreativeandforward-thinking link betweentwo In theirreport,thephosphate The firstaccountof published thedirectedchemical The inventionofPolymeraseChainReaction(PCR)by 800-863-6801 O O O O P R O Cl DNA Synthesis andReagentsDNA O O N N H O + O H O O O O N N H O O O O O R O O O P O N O O N H O O O N

H O 103 Information Technical 104 Technical Information periods), sostrongacidsshouldbeavoided. (pH 4-5forextendedperiods,pH1-3fairlyshort conditions to undermildlymoderatelyacidic susceptible depurination contaminants. To raisethebarevenfurther, purines are from will belowandtheproductitselfmayirresolvable almost quantitatively. Otherwise,theyieldofdesiredproduct the protectinggroupsmustberemovable more challenging, protecting groupat the desiredtime. To makematters group schemethatallowstheselectiveremovalofaspecific an efficient, cyclic,step-wisesynthesisisagoodprotecting protecting groups. area ofnucleoside The keytodeveloping becomes evenmoreremarkable. an active tRNA ofpreparing the accomplishment molecule of thetask, reagents. Whenoneconsidersthemagnitude be purifiedorprecipitatedbetweenstepstoremoveexcess the processveryslow, hadto because the oligonucleotide even morechallenging. phasechemistrymade The solution also increased,makingpurification percentage ofbranching However, lengthincreased,the as theoligonucleotide contaminants wereremoved. process inwhichthebranched was necessarytofollowaveryarduousmulti-steppurification of thepreviouscouplingswasamajorproblem. As aresult,it linkages phosphate attheinternucleotide protected, branching shortcomings. Becausethephosphateitselfwasnot (Khorana, 1970). 72-mer tRNAin Nature molecule,whichwaspublished produced atrulyremarkablefeat: the synthesisofanactive nucleoside. at This methodwasrevolutionary thetimeand Synthesis andReagentsDNA Different Trityls have a Range of Absorbances Figure 3: Khorana’s Phosphodiester Coupling Method Figure 2: O Khorana's mostlastingcontribution,however, was inthe Like mostarchetypes,themethoddidhave O O O P O O O O O N O O N H O + (+) (+) (+) O H O O O O O N N H O Trityl Cation Orange Mono Red Di Yellow me N DCC thox me thoxytri N ytri tyl Cation O tyl Cation O O O O O O O P www.trilinkbiotech.com O N O O N H O O O N N H O his sightsontoa newlyemergingfield,biomacromolecule significant playerinthatfield,but intheearly1960'sheturned University inthelate1940'sasa boronchemist.Hewasa Epoch The Letsinger chemistry. in thefield:solidphasesynthesis andphosphite-triester two importantsteps Northwestern Universityanddeveloped in thisarticle,andRobertLetsinger, who workednearby at names includeMarvinCaruthers,whowillbediscussedlater graduate students, post-docs, orvisitingscholars. Those chemistrypassedas of thegreatnamesinoligonucleotide and throughhislabs,wheremany through hispublications to benzoyl. removed compared exception ofacetyl-protectedcytidine,whichismorereadily today,are themostcommonlyusedgroups with thepossible Brown, et. al., 1979)(Figure4). Although othersexist,these and cytidine(Schaller,benzoyl foradenosine et. al., 1963; and standard protectinggroups;isobutyrylforguanosine exocyclic aminesthataretodayknownasthe nucleosidyl DMT step. it totheprevious andcomparing by measuringtheabsorbanceofreleased can befollowed (Figure 3). The efficiencyaddition ofeachcyclenucleoside a yellowcolor, while theparenttrityl(Tr) itselfisdeepred optically. The monomethoxytrityl(MMT) carbocation has very lowconcentrationsitcanstillbeaccuratelymeasured has ahighextinctioncoefficient, whichmeansthatevenat colorinmildacidthat carbocation hasaverystrongorange tool. an extremelyusefuldiagnostic The dimethoxytrityl(DMT) have adistinctivecolorwhenionized, whichhasturnedinto mildly acidicconditions.Likemanycarbocations,thetrityls to remainstableasapositivelychargedspeciesundervery three phenylgroupsissufficient toallowthemethylcarbon systemformedbythe of thepielectroncloud back bonding that actuallylikestoformacarbocation. molecules The under acidicconditionsliesinthefact it isoneofthefew trityl family.unique such asleuvenylandFMOC,the but noneareaspopular Severaloptionsareavailable, unbeatable. mild acidhasbeen stability andeasyremovalwith of goodgeneral combination chemistry today.in oligonucleotide 1961), isubiquitous The the dimethoxytrityl(DMT)protectinggroup(Smith,et. al., Exocyclic Amine Protecting Groups Figure 4: Professor Letsinger beganhiscareeratNorthwestern Professor Letsinger many withhiswork,both influenced Professor Khorana Khorana alsointroducedtheprotectinggroupsfor The reasonthistriphenylmethylethercleavessoreadily The solutionKhoranaoffered for5'hydroxylprotection, N R N R N N H H N R N R N N H O N O N O N N N O O N O N H H N O N- N-Is N-B N- Benz Ac obutyryl enzoy etyl oy Cyt l l Adenos Cyti G idin uanos dine e in e in e [email protected] [email protected] usingthesamesupport(Letsinger trimer oligonucleotides synthesis ofdimerand the solid-phase paper describing the first in somesolvents.In1965hepublished of swelling polymer thathadtheunfortunateproperty divinylbenzene consisted ofwhatwascalleda"popcorn"polymer, a styrene- and Kornet,1963;Letsinger, et. al., 1964). The support in 1963and 1964(Letsinger in paperspublished described Synthesis Solid Phase the rootofmethod. Marvin Caruthers'phosphoramidite departure, theP(III)method,whichis based phosphite-triester method. Finally,phosphodiester he introducedaradical of synthesis,animportantimprovementonKhorana's above. Secondly, method he introducedthephosphotriester field. First, he introducedsolidphasechemistry asstated advancements that benefited anentireindustry. Khorana, thusconvertingastrokeofhardluckintoscientific taught by synthesis procedures of theoligonucleotide using solidphasesynthesisforpeptidestotheimprovement synthesis. Herapidlyconvertedhismethod oligonucleotide and focushisattentiononanothernascentchemistry: work. This unexpectedscooppromptedLetsingertoregroup peptides first and eventuallywontheNobelPrizeforhis the solidphasesynthesisof his seminalpaperdescribing their findingsassoonpossible.BobMerrifieldsubmitted andstrugglingtopublish and neckintheprocessofdiscovery using solidphasetechnology. At the timetheywereneck the synthesisofpeptides Merrifield, wasalsoinvestigating this lead. only researcherfollowing Another scientist,Bob important step forward.However,incredibly he wasn'tthe filteringsystemthatprovedtobean it addedaninternal units sequentially. topeptidesynthesis, Whenapplied withacyclicchemistryschemeofadding through technology mainly forthesupportofcatalysts.Letsingerutilizedflow- scheme usingsolidphasechemistrythathadoriginated chemistry inthemid-1960's. oligonucleotide However, a twistof fate moved Letsingerfrompeptideto synthesis. At that time,thetargetwaspeptidesynthesis. Coupling of dC to Polymer Support Figure 5: Letsinger's first support forpeptidesynthesis was Letsinger madethreemajorcontributionstothe Dr. apeptidesynthesis Letsinger wasdeveloping O O O O O H O O O O O O O NH N O H 2 N N N H O O O O N O H H N N O O M M N Cl ild ild H A B O O cid as e S Poly uppor me t r Poly S uppor me Poly S uppor t r me t r critical drawbacks. critical drawbacks. Among themwasthe fact that despite the mapping, PCR,andtargetvalidation. of gene the industrywasprimedforemerging techniques with theexactsequencedesired. prepared Thus equipped, oligonucleotides geneswithradio-labeled the abilitytoprobe and created non-chemists topreparesimpleoligonucleotides, instrument of itsera. popular These instrumentsallowed was basedonphosphotriesterchemistryandthemost Biosearch. HeintroducedtheSAMI in thelate1970's,which Another earlyentrantwasRonCookandhiscompany by Vegathe earlyinstrumentsdeveloped Biotechnologies. DNAand semi-automated by synthesizers,exemplified led tothe creationofthefirst methodology viableautomated easilywithsolidphase of achemistrythatworkedrelatively to reproducesuccessfullyinmanylabs. The combination and Ogilvie,1969). by farthemostpopular(DevineandReese,1986;Letsinger (MSNT) were (MSCl) and mesityl sulfonylnitrotriazole chloride step, wastheselectionofaproperactivator. Mesitylsulfonyl inexcessof95%per of thereaction,whichreachedlevels It turned out,however, that thekey topushingtheefficiency deprotection mixture. amorecomplicated used butitrequired (Letsinger andOgilvie,1969). was also An o-chlorophenyl removedwithammoniumhydroxide group thatiseasily protecting groupmostcommonlyusedwastheß-cyanoethyl approach. the phosphodiester that plagued the branching The method wastheprotectionofphosphategrouptoprevent (Letsinger, et. al., 1969)(Figure6). The keyadvanceofthis synthesis on thephosphotriestermethodofoligonucleotide Phosphotriester Chemistry own sectioninthishistory. fell tohisformerstudent,Caruthers,whowillshortlyhave that swelling wassodetrimentaltothechemistry. That role formulations, butneverfoundasolutiontotheproblemof Caruthers. Letsingerexploredanumberof polymer the graduatestudentinstrumentalinthatworkwasMarvin the 3' hydroxyl tothesupport,asisdonetoday. Infact, the bestapproachto solid phasesynthesiswastoattach phase synthesistechnique.Hequicklydeterminedthat (Figure 5). with base thus forminganamidebondthatwascleavable made tothesupport, whichwasactivatedanacidchloride, synthesis. for oligonucleotide nucleoside The attachmentwas thesupportbound then removedwithmildacidtoprepare and the5'withaDMTposition group. The DMT groupwas 3' hydroxylofthedCwasprotectedwithabenzoylgroup amide bondthatwascleavedwithammoniumhydroxide. The base itselftomodifiedsupportandformingan acid chloride (dC) wasattachedthroughtheamineat the 4positionof report,2'deoxycytidine and Mahadevan,1965).Intheinitial Phosphotriester ApproachFigure 6: However, chemistrystill sufferedthe phosphotriester from This wasthefirst chemistry that was simpleenough thefirst In thelate1960's,Letsingerpublished paper Through the1960'shecontinuedtoexploresolid O 800-863-6801 T EA+ O O O P O O O O DNA Synthesis andReagentsDNA O N N N H O + Solid S O H upport O O O O N N H O O S MSNT, Bas N O N N e NO 2 - O N O Solid Suppor O O O O O O P O N O t O N H O O O N

H O 105 Information Technical 106 Technical Information standard DNA bepreparedfaster, but the doorwasopened chemistry asasignificant stepforward.Notonly could within seconds. base veryefficientlyoxidizes phosphorus andquantitatively Fortunately, a verysimplemixtureofiodine,waterandsome to intermediate theacidrequired to removetheDMT group. cycle becauseoftheinstability thephosphite-triester backbone. ateachstepofthe This oxidationwasrequired into aphosphotriesterwasneededinordertostabilizethe lone pairofelectrons. tetrahedron configurationintotheplanarmuchmorethan The doublybondedoxygenhindersthetransitionfrom intermediate isformed. in Figure8,atrigonalbipyrimidal of intermediates theP(III) species versusP(V). As shown difference intheenergyofformationsfortransitional factor ofthereactionrateturnsouttobe determining electronegativity.based onit'senhanced However, the the P(V)wouldbemorereactivetoattackbyanucleophile oxygen, absence ofdata,thatbecausethedoublybonded the P(V)speciesisnotintuitive.Onewouldsuspect,in phosphomonochloridite intermediate. for couplingduetothehighlyreactivenatureofnucleoside this chemistrywasthesignificantreductionintimerequired prepare thenaturalP(V)backbone. The majoradvantageof step inthesynthesiscycle, additional oxidation,inorderto chemistry.P(V) phosphoryl The schemerequiredan intheP(III) reactive phosphorus state,insteadoftheclassic 1976) (Figure7). This chemistryisbasedontheuseof chemistry (Letsinger, et. al., 1975;LetsingerandLunsford, method ofoligonucleotide the phosphite-triester describing Phosphite-Triester Chemistry an hourandahalf. which resultedincycletimesthatcommonlyranlongerthan in length. time, Another problemwastheextensivecoupling lessthan20bases to theroutinesynthesisofoligonucleotides 97%, andoftenfailedtoreach95%. This limitedthemethod step-wise efficiencybe raisedabove couldneverreproducibly years ofworkbyanumberresearchgroups,theaverage Synthesis andReagentsDNA Figure 8: Phosphite-Triester Method Figure 7: The researchcommunitywasquick toacceptthisnew The fact that theP(III) intermediate ismorereactivethan the first In themid-1970's,Letsingerpublishedpapers O O O Oxidation of the phosphite-triester intermediate Oxidation ofthephosphite-triester Nu O Trigonal Bipyrimidal Intermediate O O P O N Cl N H O + Solid R S O H upport O O O R P O N N " H 2. Iodi 1. Bas O R ne/ e wa ' te r O R" O Solid O O O S ' O O uppor O P www.trilinkbiotech.com O N O t O N H O O O N N H O solubilized 5' protected nucleoside to asolutionofRO-PCl 5' protected nucleoside solubilized called fortheslowadditionofaveryslightexcess controllable reaction. combat thatonlyledtotheoppositeeffect and anevenless threshold. Increasingtheconcentrationofreagentsto reagent belowacritical the concentrationofactivenucleoside effect in thattoomuch3'-3'adductwouldbeformed,reducing harmful had alike-wise of thephosphodichloridite equivalents used toreduceformationofthe3'-3'adduct.Usingtoofew was thereasonthat an excessofthereagent couldnotbe had timetocouple. before thedesiredintermediate That reagent wouldveryefficiently capoff the growingchain in solution. that remained phosphodichloridite This unused the amountofunused of desiredmaterialandincreased product diddoubledamageinthatitreducedtheamount formation of3'-3'dimer(Figure9). The formationofthisside- the while reducing the formationofdesiredintermediate the 5'insuchamannerastomaximize protected nucleoside hadtobeadded activating reagent phosphodichloridite was verytricky.the formationofactiveintermediate The made justpriortoeachcoupling. Another issuewasthat was noteasytostoreandthereforebest intermediate intermediate. Ittohydrolysis. was verysusceptible The reactive natureofphosphomonochloridite the nucleoside drawbacks. chemistrydidindeedhavemajor form ofthephosphite-triester by Vegadevelopment However, Biotechnologies. the early onthischemistryandanotherwasin synthesizer based of formerLetsingerstudents,marketedanautomated Biologics,acompanypartiallycomprised oligonucleotides. for theinvestigationintoavarietyofbackbonemodified trityl alcohol that selectivelyremovedanyexcessRO-PCl trityl alcohol with ascavengersysteminvolving method waslatercoupled in themix(Hogrefe,1987). phosphodichloridite This improved making toomuchof the 3'-3'adductorleavingtoomuch temperature, itformedausefulactivereagentwithout at room reagenttothenucleoside the phosphodichloridite student thatshowediftherewasarapidintroductionof Adiscovery wasmadebyagraduate serendipitous time. about bythefastercoupling advantages brought during this activation,removednearlyallofthe conditions reagent justpriortoeachcoupling,andtheneedforarduous the active for preparing of the requirement the combination at extremelycoldtemperatures(-78ºC). As itturnedout, instrument was also developed by Vegainstrument wasalso developed in Biotechnologies times of15minutesorless. synthesizer withcoupling This of apracticalautomatedDNAthe development finally allowed from thereactionmixture.It was thisnewprotocol,which 3’-3’ AdductFigure 9: The mostsignificantproblemwasthehighly This problemwasnotsolveduntiltheearly1980's. The protocols designed to optimizethisreaction The protocolsdesigned O O O O O O O O O P O O N O O N N H N O H O 2 2 Figure 10: Synthesis Cycle [email protected] [email protected] efficiency,time andimprovedcoupling reduced washing as ofthesupport. step andeasiertoaccessallportions This easier to rinse thesupportofoldreagentsprior tothenext freer flowofreagentsoverthesupport.It by allowingwas efficiencysupports beingusedatthetime,thusincreasing with thepolymer problem experienced solved theswelling pore glass(CPG). by controlled much latersupplanted This Originally, chromatography gradesilicawasused.It was synthesis (MatteucciandCaruthers,1981). oligonucleotide matrices assupportsfor the useofinorganic he published intermediate. In1981, activenucleoside the phosphitylated of swelling theorganicpolymersupportsandinstabilityof Caruthers workedoutthesolutiontotwomajorproblems, Overthenextdecade in thesynthesisofoligonucleotides. at the Universityof Colorado, wherehecontinuedhisresearch Khorana, whowasnowatMIT. thefaculty In1973,hejoined Right Chemistry, Caruthers: RightTime market. methodintothe the entryofphosphodichloridite clouded method. It phosphoramidite solvedmanyoftheproblemsthat by anewchemistrydiscoveredMarvinCaruthers,the sold. method wassooneclipsed The phosphodichloridite instruments describedearlierinthissection,itwasnever was asignificantimprovementoverthephosphotriester with Letsinger.collaboration Although thisparticularinstrument Capping Step After Caruthers leftLetsinger'slabhedidapost-docwith (C CP Base aps unr CP Base G O G CP Base O O eacted 5'te O G O O O O R O Base O H Py O O P ri O O rmin dine O O O i. )

O Detritylation Step Pyridine DM I 2 /H T 2 O CP Base G O Cycle Synthesis O R Base O O P O O CP Base G O all traces of acid and reduce adventitious water.all tracesofacidandreduceadventitious Nowcome to remove Next, thesupportiswashedwellwithacetonitrile acid in dichloromethane. acid ortrichloroacetic dicholoracetic group isnormallydonewithamildsolution(2-3%)ofeither bound tothesupport. nucleoside The removaloftheDMT cycle starts with thedeprotectionof5'hydroxyl method offered byLetsingerwastheactiviationstep. The synthesiscycleandtheexisting the phosphoramidite manufacture anddistributionofDNA synthesisreagents. a weakacid,tetrazole. thecommercialscale This allowed was thenactivatedjustpriortocouplingbysimplyadding stable solidandstoreduntilneeded. The phosphoramidite couldbemadeinadvance,isolatedasa or phosphoramidite intermediate nucleoside molecule. Nowthephosphitylated tothepropertiesof it resultedinverysignificantchanges synthesis because of routineoligonucleotide the development Caruthers, 1983).However, this subtlechangewaspivotalto an amine(BeaucageandCaruthers,1981;McBride the exchangeofoneleavinggroup,achloride,foranother, contributionappearsalmosttrivial:simply,method, Caruthers' Phosphoramidite Approach The or undesiredcapping. the desiredreactionthroughhydrolysis,prematureoxidation, interferedwith leftonthesupport invariably any contamination O DM O Oxidation Step T As showninFigure10,theonlydifference between Letsinger'soverallphosphite-triester As farasmodifying 800-863-6801 O + Cl DM DNA Synthesis andReagentsDNA 2 C T O O H CP Base CP G Base CP R G O Base Base O O G R O Base NR P O O Coupling Step O O O ' O P O 2 O O O + H O O Te H O DM traz DM T ol

e T 107 Information Technical 108 Technical Information Caruthers. As the fieldadvances, TriLink followsthetradition by progressivescientists suchasKhorana,Letsinger, and methods usedtodayarethefruits ofdecadesresearch Conclusion make useofit, the biologists. automated synthesizerbecameuseful tothosewhocouldbest successfully.method, the with the phosphoramidite Coupled and requiredahighdegreeofexperiencetoreproduce method wastricky in themid-1980's,phosphodichloridite to runsuccessfully.impossible At state its mostadvanced with thechemistriesoftime,nearly and whencombined chemists. experienced The instrumentsweretemperamental was largelyinthehandsof the synthesisofoligonucleotides method, of thephosphoramidite born. Priortotheintroduction and introduced toHood:aninstrumentwasdesigned ABI was for almost twodecades. unchanged and N-methylimidazole. anhydride amixtureofacetic with freehydroxylsarecappedusing Letsinger. unreacted strandsending Then thefewremaining described by reagent originally the sameiodine/water/base nucleoside phosphochloridite. what wouldbeobtainedifexcesstetrazolewasaddedtoa suggests thatanaromaticamineisboundtoit, similar to 1989). after activation The chemicalshiftofthephosphorus for thiswasfoundthrough that intermediate reactswiththe5'hydroxyl. The evidence the nearbyrecentlygeneratedtetrazolide.It is actuallythis by rapidreplacementoftheprotonatedaminewith followed mechanism isthattheaminebecomesprotonated(slowstep), interesting, andcontroversial. The currentlyaccepted protecting group(Figure11), theactualmechanismis to achieveefficient activationwithoutlossofthe5'hydroxyl acidic enoughtoactivatethephosphoramidite. does notremovetheDMT groupfromthereagent,yetitisstill aromatic ring. that The pKaofthisacidishighenoughit allows theformationoffavoredanionic a thermodynamically that actuallyactsasamildacid. of The donationtheproton activation isdonewithtetrazole,anunusualsecondaryamine to the5'hydroxylofboundnucleoside. and coupling The the mostimportantsteps,activationofphosphoramidite Synthesis andReagentsDNA Figure Tetrazole Without a doubt, the oligonucleotide synthesis Without adoubt,theoligonucleotide With aviablechemistryinhand,Carutherswas This cycle,elegantinitssimplicity, has remained virtually usingessentially After thesupportisoxidized coupling, Besides thedelicatebalanceinactivatorpKaneeded 11: Mechanism of Activation of Phosphoramidite by N N N N N u N N 31 u u O P NMRexperiments(Berneretal, N O O u O P O P N O O P N N N O N P N + N RO N H O N R H N N N H N N www.trilinkbiotech.com 21. 20. 19. 18. 17. 16. 15. 14. 13. 12. 11. 10. 9. 8. 7. 6. 5. 4. 3. 2. 1. References way.we've comealong still neededforthiswork,buttouseanoldcliché, indeed industry.for thediagnostic oligonucleotides Chemistsare markets; andimprovedsynthesisqualityofdyemodified methodsforthescreeningandPCR better high-throughput for therapeuticapplications; of modifiedoligonucleotides largerquantities lie intheneedforincreasingly challenges of seekingbetterwaysmakingoligonucleotides. Today the Watson JD,CrickFH. (1953)Nature171,73. (1961) J. Am. Chem. Soc. 84,430-440. Smith M, Rammler DH,GoldbergIH, Khorana HG. J. Am. Chem. Soc. 85,3821. Schaller H,Weimann G, Lerch B, Khorana HG. (1963) Michelson AM, Todd AR. (1955)J. Chem. Soc., 2632. 24, 245-8. McBride LJ,CaruthersMH.(1983) Tetrahedron Lett. 103, 3185-91. J. Matteucci MD,CaruthersMH.(1981) Am. Chem. Soc. 98, 3655-61. Letsinger RL,LunsfordWB. (1976)J. Am. Chem. Soc. (1975) J. Am. Chem. Soc. 97,3278-9. Letsinger RL,FinnanJL,HeavnerGA, Lunsfold WB. Chem. Soc.91(12), 3360-5. Letsinger RL,OgilivieKK, Miller PS.(1969) J. Am. 91(12), 3350-5. Letsinger RL,OgilivieKK. (1969)J. Am. Chem. Soc. 87(15), 3526-7. V.Letsinger RL,Mahadevan J. (1965) Am. Chem. Soc. (1964) J. Am. Chem. Soc. 86(23),5163-5. Letsinger RL,KornetMJ,V,Mahadevan Jerina DM. 85(19), 3045-6. Letsinger RL,KornetMJ. (1963)J. Am. Chem. Soc. Köster H,SinhaND.(1984)USPatentNo.4725677. Khorana HG. (1970)Nature227,27-34. 1523. Chem. &Ind.London, Khorana HG, Tener GM, Moffatt JG, PolEH.(1956) University,IL. Evanston, Hogrefe RI.Ph.D. (1987) Dissertation.Northwestern 5529-32. CB.(1986) KG, Reese Devine TetrahedronLett. 27(45), Methods Enzymol.68,109. R, RyanMJ, Brown EL,BelagajeKhoranaHG. (1979) Acids Research17(3),853-64. Berner S,L,SeligerH.(1989)Nucleic Muehlegger 22, 1859-62. Beaucage SL,CaruthersMH.(1981) Tetrahedron Lett. [email protected] [email protected] 1. Observations ple, evenwhilestored dryunderanhydrousconditions. synthonsrapidlylosttheirability tocou- the phosphoramidite it wasfound that dimer synthonstoprepareoligonucleotides, Problem Case History1:Synthesis arepresented. how tosolvesomesynthesisproblems synthesis schemehadtoberedeveloped. nucleicacids,theentire In somecases,suchaswithpeptide and theclassicmethodsofsynthesisabound. oligonucleotides of issuesbetweenthenewgenerations pounds. Compatibility for thepreparationofthesecom- thetic chemistsresponsible methylphosphonates. chiral Genta's MOEs, and ISIS's MMIsand phosphoramidates, chemistries: Gilead'spropynyls,Lynx'smore sophisticated with oligonucleotides have introduced of themajorplayers of drugs.Most them. Nowweareenteringthethirdgeneration duce moreradicaldeparturesfromnatureinordertocontrol more sophisticated, thedemandsonchemistsincreasetopro- such astheaptamersofGileadandISIS. led to modifications thediscoveryofnewmodesaction, being themostdrugs. popular ofthesecondgeneration These overcome theseobstacleswasintroduced;phosphorothioates to the abilityofoligonucleotide of modificationsenhancing drugcandidate. cleotides wouldnotbecomeaviable A series 5. 4. 3. 2. 1. agree thatthefollowingareprimaryconsiderations: severity ofthevariousissues,andsolutions,mostwould problems tosolve. about the Although thereisdisagreement nescent reagentsandmicrochipscanenhancesensitivity. reporter groups,suchasfluorophores,chemilumi Conjugating properties. to controlhybridization and backbonemodifications base, sugar modifications, including number ofoligonucleotide order tosolvetheseproblems,researchershaveturneda to falsepositives.In to non-targets,leading binding increased towards duplexformation,thereisan driving theequilibrium targetswithhighaffinity.that willbindavailable However, in high sensitivity, one musthavearobustsystemwithprobes are almostmutuallyexclusive.Inordertohave requirements background noise).Unfortunately,consequently these two stringency andsensitivity(and with twoprimarychallenges: tries beingdeveloped. industry isconfronted The diagnostic differentoverlap inthechemis- needs,thereisconsiderable applications. to getandtherapeutic the desiredpropertiesfordiagnostic "tweeking" isnecessary that somedegreeofoligonucleotide Most researchersfind oligonucleotides. modified developing fairly limited,agreatdealoftimeandmoneyisspenton is oligonucleotides of unmodified Because theapplicability synthesishasgrowntremendously.ing oligonucleotide By RichardHogrefe, Ph.D.; TriLink BioTechnologies Troubleshootingof Modified the Synthesis Amidite waspure by the useofchirallypuremethylphosphonate In developing A briefreviewofseveralcasehistoriesdemonstrating With eachnewtwist, the problemsmultiplyforsyn- processes becomes of biological As ourunderstanding It oligonu- was obviousfromtheoutsetthatunmodified (toxicity) Metabolic properties Tissuetargeting andcellular Circulatory lifetime high stringency-invivo propertiesto Strong hybridization targetwhilemaintaining In vivostability The therapeuticindustryhasaslightlybroaderrangeof and therapeuticindustrieshave Although thediagnostic regard Over thepastdecade,levelofsophistication 1 H and 31 P NMR,HPLC, MS. - - Conclusion: Definitely adifferenceDefinitely Conclusion: between CU oligos. AG and AG oligo wasacleansingle band of CUoligowithnomajorband, banding Result: Significant issue. vs. pyrimidine purine Purpose: as usual,analyzeongel. 1. Synthesizealternating AG and CURNA oligos,deprotect Experiments 4. 3. 2. 1. Observations the silylgroup. nium fluoride(TBAF) was thereagent ofchoiceforremoval with thesesynthons. At the timeofthiswork,tetrabutylammo inactive productsbeingproduced complaints aboutbiologically and cost in" principlesavings.However, there areanumber of reagents forthesynthesisofRNA,mainlybecause"first Problem Case History2:Deprotection pare ourownmolecularsieves. lose theresultsofmanyhardweeksinlab.We nowpre- reagent.It any newormodified isbettertobesafe,than lar sievesfortwodayspriortouse.Infact, this isdonewith Follow Up Molecular sievesareaviablesolution. Conclusion: efficiencyactive reagent.Coupling >95%. Result: After 2 daysoversieves,NMR showedover50% efficiency. Purpose: Try toremovewater, and therebyincreasecoupling Test byNMRandcoupling. 3. Treatsieves. dimersynthonwithhighquality molecular It's water.Conclusion: product. (>10%). Mostoftheamiditeconvertedtoside- hydrolyzed Result: Very littleactivematerialobservedwithdimersynthon intermediate. ifsynthonconvertscleanlytotetrazolide Purpose: Determine DMT-T amiditestandard. 2. Activate synthonwithtetrazoleinNMRtube.Compareto from beginning. synthesis ofsynthon,orwouldhavebeenpresent original withproduct.Notfrom Contaminant co-migrates Conclusion: Result: Noimprovement. Purpose: Tryimpurity (salts,etc.) toremoveundetectable (95% originally). 1. Repurifyonasilicadimerthatcoupledat25%efficiency Experiments 4. 3. 2. Hopkins University, madethisobservation.) originally when runon 40cmgels.(Prof. banding PaulMiller, Johns pure on20cmgelsshowedextra Oligos thatappeared quality oflonger strands(40mer+)oftenpoor.Biological purine rich. rich sequenceswerenotashigh Quality ofpyrimidine Variable qualityofRNA. 2'-O-silyl RNA monomershavebecomethemostpopular All thedimersynthonsarenowtreatedwith3Åmolecu- ciencies. effiin synthonwasobserved,despitelossofcoupling - described above,nochange Using analyticaltechniques efficiency.successful inrecovering anddrydowntimeswerenot Extensive co-evaporations couple ofdays,insomecasesto20%orless. (>95%), onlytorapidlyloseefficiency overthenext after Synthons coupledwellimmediatelypurification 800-863-6801 Repeat Miller's observation, Repeat Miller'sobservation, and to beginexploring DNA Synthesis andReagentsDNA Oligonucleotides

- 109 Information Technical 110 Technical Information are notinusefor alongperiodoftime. of The combination We usethe small TBAF (5 mL)bottlesfrom Aldrich sothatthey which willnotdeprotect undernormal conditions.) pyrimidines contain morethan5%water, some containasmuch8%, Karl Fisherpriortouse.(We foundthat most brandnewbottles proper dryness,andremovethe need totest each bottleby Follow Up Molecular sievescometotherescueagain. Conclusion: about ½ofthedimerbythattime. water deprotected dimers within6hours. untreated The original TBAF with 20% the CT deprotected 2% waterafter3days.Itcompletely UT and Result: The TBAF treated with molecularsievesdriedtoonly reagent itself,orthereaction? water, but actuallyrecoverwetreagent. Is water affecting the Purpose: ability todeprotect. 20% waterwithsieves,compare aliquot ofreagentcontaining 4. Treat TBAF sievestoreducewater.with molecular Treat again. It's water Conclusion: of desilylation. reduction inrate up to20%water withnoobservable handling 5% waterinthe TBAF. completely impervious, Purines appear with morethan with therateofdesilylationrapidlydeclining verysensitivetowatercontentof Result: Pyrimidines TBAF, phosphate. ment withaneighboring tion ofbasesinamodelsystemrealisticenviron individual Purpose: water. Follow removalofsilylgroup byHPLC. groups intact. Treat with TBAF amountsof containing variable 3. PreparedimersCT, UT, AT andGT andisolatewithsilyl first contained over10%water, the freshbottlecontained~6%. TBAF reagents weredeterminedbyKarlFisher titration. The Incomplete removalofsilyl.WaterConclusion: contentofboth into singleband. Result: Multiplebandscollapsed was problem. ifincompletedeprotection Purpose: Determine 2. Retreatwithfresh CU oligo TBAF. Water in Amidite Water Testto for Experiment NMR 31P Synthesis andReagentsDNA A Amid A mi Act. R Amid Activa Ox mi di Activa idized Species di ite +Tetr Ph te We treatthe TBAF uponarrivalwithsievestoensure te it eagen os e ted reagent +T phit ted reagen et Determine ifwecannotonlyprotect TBAF against Quantify affect of waterin TBAF on rateofdeprotec- e- t +N azol razo tr iester 200 200 200 200 200 200 e le ucleos t id e Good Synthon Poor Synthon 100 100 100 100 1 100 00 www.trilinkbiotech.com 0 ppm 0 ppm 0 ppm 0 ppm 0 ppm 0 ppm - 4. 3. 2. 1. Observations limited ouroptions. then fixit. To topitoff, problemthat we alsohadasolubility was tofirst determine whatcausedtheunknown modification, problem, EDA adductformationwithbenzoyl-C,thesecond as well. showedothermodifications gos tosinglenucleosides oligo tothesupport. Notonlythat,ofMPbut degradation oli- N by PaulMiller. The problemwasthatEDA transaminated (EDA)asanalternativewasestablished of ethylenediamine usingammoniumhydroxide. deprotection conditions The use degradeundernormal sensitive tobaseandwillcompletely is notrivialmatter. Oneproblemisthatthebackbonevery Problem Case History3:Deprotection Tetrabutylammonium Fluoride. The Effect of ExcessWaterof ontheRateofDesilylation etal.inthepaper: This workwaspublishedbyHogrefe sievespriortouse. tive reagentwithhighqualitymolecular of nofaileddeprotectionsoverseveralhundredsyntheses. fresh reagentwelldriedwithsieveshasledtoatrackrecord tried O also modified,somethingnotreportedintheliterature.We change, ibuinsteadofbzprotectionondC.However, dG is Conclusion: The first problem wasneatlysolvedwithasimple Unfortunately, ½theproduct othermethodsdid. b yielded modification inmethodsaandc, unidentified butnotwithb. EDAyielded systems. dC-Bz adductineverycase.dGyielded Result: dA,dC-ibuand Tdeprotect cleanlyinall oligomers Purpose: a.first hyrdrazine, thenEDA/ethanol(Miller) 1. Experiments nium hydroxide.SomehowtheNH of theoligoinaq.ammo- to lossofproductdueinsolubility ammonium hydroxidewasnotveryeffective atscaleandled to scaleup.Useofabreiftreatmentwith was notamenable the benzoylgrouppriortotreatmentwithEDA. This method up. Millerusedaprimarytreatmentwithhydrazinetoremove not onRP-HPLC. This problemwasexacerbatedduringscale could seethesecompoundaslaterelutingspeciesongel,but replaced with acetonitrile (ACN) oneatatimeandthepro - with acetonitrile replaced acid, oxidizer,dichloroacetic etc., were amidites, caps, monomer isnot elongated intoanoligo.Eachofthe reagents, cycles, the the supportpriortorunningcoupling capping then (the otherbasesweredoneaswell). By deblocking, over supportbounddG-ibu 3. Cycle20artificialcouplings (NMI). to becleavedbyN-methylimidazole to usebecauseMPrequired enough arebaselabile oligomers (DMAP), whichwe were suspected dimethylaminopyridine during deprotection. Weing itsusceptibletotransamination dG-ibu must bemodifiedduringsynthesis,mak- Conclusion: Result: Nomodification. of dGinisolatedenvironment. Purpose: Lookatmodification 2. Treat dG-ibuanddG-ibu-DPCwithmethodsa,bc. exacerbated theproblem. 4 -benzoyl cytidine, leading to undesired EDA toundesired -benzoyl cytidine, leading adducts.We Other pre-treatments, Other pre-treatments, suchasNH EDA isthebestsolventforMP oligos. were> Other modifications 10%,but still significant. was foundtooccurwitharateofup15%perC. of benzoyl-CwithEDADuring scaleuptransamination We here.Onewastofixaknown hadtwoproblems The synthesis of methylphosphonate oligonucleotides The synthesisofmethylphosphonate to say,Needless we nowtreateverywatersensi- Deprotect using: rounded by T's using C-bz,C-ibu,G-ibu,and A-bz. Synthesize 9mersconsistingofdA,dC,dGor T sur- nol, etc., for theremovalofbz-CpriortoEDA failed. 6 protected dG-ibu (diphenylcarbamoyl, DPC), butthat protecteddG-ibu(diphenylcarbamoyl, Determine extentof problem, comparemethods. alone c. EDA/ethanol b. first NH b. 4 OH, then EDA/ethanol(Sarin) 4 OH permanently bound the OH permanently 3 saturatedisopropa - NH the DMAP-dGadduct.Furthermore,itwasfoundthat2% [email protected] [email protected] Result: Sameresults aswithRP. Purpose: Try otherHPLCapproaches. umns. 2. Attempt sameexperimentwithnormalphaseand AX col - Conclusion: The peptidestickstotheoligo. toeveryoligo. of peptidebound Result: 3molecules what ratio. Purpose: Try todetermine ifpeptidesticksto oligo, andin tide andoligo. Analyze byReversePhase-HPLC andquantitateareaofpep- 1. Mixpeptideandoligotogether(10/1peptide/oligo). Experiments 2. 1. Observations active. It had thesameactivityprofileasfree peptide. acid (TFA)/ACN astheeluent. The oligowasfoundtobetoo sized andpurifiedusingRP-HPLCwith0.1% Trifluoro acetic to theterminusofanoligo.It date forconjugationwassynthe- activity ofitsownwasdeemedaviablecandi- with biological Problem Purification 4: History Case Procedure. UsingaNovelOne-Pot Oligonucleotides Methylphosphonate by Hogrefeetal.inthepaper: published side product. nated thetransamination This workwasalso was thattheputativesolution,O adducts. This wasreportedbyEadie. Whatsurprising It Conclusion: isnotthatibu-dGformedEDAsurprising methods aandc, but notinb. Result: DMAP formedadductswithibu-dGandDPC-ibu-dGin in conjunctionwithotherreagents. and simulated oligosynthesisconditions,bothindependently Purpose: supports weredeprotectedbymethodsa,b,andcabove. tion of replacing allbutoneofthereagentswith ACN. The cess repeated. The processwasthenrepeatedwiththevaria- ment with2%NH oligo withthemildNH able lowrateof1%/hour. nificantly moremodified. The NH b, whichcalledforapre-treatmentof NH Wemerely hadtoapplyourknowledge. couldn'tusemethod Follow Up product. back todesired revert themodification of dC faster, cleaner andmoresimplethan before. Also, theuse were acceptable. And wehadaonepotmethodthat was deprotection. Ourdeprotectionswentcleanlyandouryields lowed withonevolumeofEDA after30mintocompletethe short treatmentof10minwith30%NH experiment gaveustheneededclue. It was foundthatavery be discoveredtoavoidNMIorDMAP. Fortunately, one last routine wouldhaveto anewcapping deprotecting reagent the bestsolventforthatstep,buttouseitasprinciple very lowyieldsonscale-up.EDAbecause ityielded is 4 OH solution degraded the MPOH solutiondegraded backboneataveryaccept- gramicidin. This tookseveralmonthstocomplete. the peptidepeakwascomparedtoaninternalstandard, oligo, theothertodeterminepeptide. The intensityof different solventshadtobeused,onedeterminethe there wasthepeptide. To quantitatethepeptide,two of lowermassregion.Sureenough, closer examination oligo clean onmassspec, Conjugated untilwerequested separated verywellindependently.oligonucleotide oligo was cleanonHPLC. Conjugated Freepeptideand In thesearchforanuptakeenhancer, a certainpeptide We solvedtheproblembyfirst treating thesupportbound Now thatweknewtheculprit,andhowtofixit, we ibu insteadof dC To studytheeffect of eachreagentonibu-dGunder 4 OH in ACN/EtOH wassufficient torevert bz 4 andthisdeprotectionmethodelimi OH solutiontorevertdGadducts,fol- 6 4 DPCprotecteddG, was sig- OH pre-treatmentservedto 4 OH, or a30mintreat- 4 Deprotection of OH beforeEDA, - with a T.with suchthattheydon'tend problem wastodesign ouroligomers that didnotsolve ourproblem. The simple solutiontothis the retentiontimesofvariousoligomers.However,predicting a formula our consultant,LCResources,was abletodevelop that phase HPLCwassowelldefined bybasecomposition Follow Up if then-1terminatesin T, etc. resolve fromann-1product,ifitterminatesin A, C, orG; n-2 Conclusion: AMPa 5'terminal oligocontaining T willnot phase HPLC. Result: T's do noteffect retention timeofoligosusingnormal on sequence. properties of chromatographic Purpose: Exploredependence tions. different containing 2. Prepareanumberofcompounds dele Conclusion: of nandn-1. There wasnoseparation Result: n-1didnotresolve.n-2,etc. did resolve. if failureproductsresolvefromproduct. Purpose: Determine and subjectedto analysis. and n-1,n-2,n-3oligoswereprepared 1. Fulllengtholigo Experiments 2. 1. Observations fore subjectedtoRP-HPLCanalysis. and there- wasgoingtobeconjugated particular compound and wasusedexclusiveofothertechniques.However, one worked very, earlyon very wellforanumberofcompounds scheme usingnormalphasechromatography.purification It you see. for somethingnew,looking and secondly, what you believe gel orCEwell).Whatmorecanyoudo?First off, you keep mined byHPLC(MP oligos,havingnocharge,donotrunon on theintegratedareaofputativeproductpeakasdeter- youbasedpurity (MP) oligonucleotides, Methylphosphonate opment ofmassspectroscopy(MS)fortheanalysis lesson hereistobelievewhatyousee.Priorthedevel- methods areneededalongwithpurificationmethods. The Problem Case History 5: Purification 5: History Case Not athoughtforthetimid. gels? you evercontemplatedpilotplantscaleelectrophoresis Have happy abouttheresult,wewerenotentirelydispleased. pound wasfoundtobecompletelyinactive. Although not Follow Up Electrophoresis istherouteofchoice. Conclusion: Result: Removedpeptide. positively charged. Purpose: Try toripthecomplexapart using fact that peptideis 4. Try electrophoresis. Conclusion: This complexisreally, really tight! Result: Nothingworked. Purpose: Try toripthecomplexapart. blender. tool knowntoman,shortofaWaringtion, andseparation 3. Try everymolecularweightcutoff filter, solid phaseextrac- Conclusion: This complexisreallytight! The chromatographic propertiesofMPThe chromatographic oligosonnormal product. 30% n-1 thecompoundcontained us. Lowandbehold, At this time,ESI-MSofMPto oligosbecameavailable tem, even thoughitrepeated. was passedon,but discounted asanartifact of thesys- seen infrontofthemainproductpeak. This information Ato 30%of newpeakcorrespondingthetotalareawas After anew much trialandtribulation,Gentadeveloped The lessonfromthelastcasewasthatanalytical After successfully removingthefreepeptide,com- 800-863-6801 DNA Synthesis andReagentsDNA

- 111 Information Technical 112 Technical Information readily cleaved from the solid support readily cleavedfrom thesolidsupport usingstandard depro solid phasesynthesis.It is alsomandatorythatthemoietybe group thatisstablethroughoutthe multi-stepoligonucleotide ing themcandidatesforantisense research(8). have beenfoundtobemoreresistant mak- to endonucleases In additiontothis,some3'-amino modifiedoligonucleotides fluorescent chromophores,biotin, enzymesandpolypeptides. used topost-syntheticallyintroduceavarietyofproductslike the 3'-endisintroductionofa3'-aminogroupwhichcanbe at a verypowerfultool.Oneofthemoreusefulmodifications has become in concert withvarious5'-modifications nucleotide 3'-Amino-CPG interactions (7). undesired must bespacedfarenoughfromthecorresponding ers (C6andC12)aretypicallyusedwhentheoligonucleotide posesnoproblems. the oligonucleotide The longerchainlink- (C3: (CH otide havebeenprepared. The shortercarbonchainlinkers between thereactiveordiagnosticmoietyandoligonucle C3, C6vsC12 Amino Linkers (3,4) biotin(5)andEDTA (6). fluorescent dyes be attachedtothe5'-primaryamineincluding (2). removed usingacidicconditions A varietyofmoietiescan otide fromfailuresequences. The MMT groupissubsequently to chromatography readilyisolatetheoligonucle mance liquid and canbeusedasa'handle'inRP-highperfor- conditions and deprotection ing groupisstabletothebasiccleavage and deprotectedoligonucleotide. MMTThe acidlabile protect- oligoisdirectlyisolatedfromthecleaved the amino-modified 1). The baselabile TFAin caseswhere moietyisemployed (Figure coupled used inanautomatedsynthesizerormanually TFA protectedandMMTcan be protected5'-amino-modifiers monomethoxytrityl (2).Boththe fluoroacetate (1)oracidlabile tri- protectedwithbaselabile using amino-phosphoramidites which canbeintroduced the 5'-terminusofoligonucleotide, is aprimaryamineat Currently themostcommonmodification also grows. oligonucleotides to readilypreparefunctionalized systems increaseintheirimportance,theneedformethods (TFA/MMT)5'-Amino Linkers By DavidCombs,Ph.D.; TriLink BioTechnologies DNA LinkerandSpacerReagentsand TheirUtility Synthesis andReagentsDNA and sold through Glen Research Figure 1: TFAC6-Amino Linker Catalog #10-1916 Glen Research Linker MMT C12-Amino Catalog #10-1912 Glen Research Linker MMT C6-Amino Catalog #10-1906 Glen Research Incorporation ofa3'-aminofunction requiresaprotecting at the3'-endofanoligo Introduction ofmodifications A wideselectionof linker lengthsfor incorporation diagnostic As DNA-basedmicroarraysandmulti-label 2 ) 3 5' Amino Linker Structures manufactured by TriLink ) canbeusedininstanceswheretheproximityof N N N O O P P O P O O O N N N H N H N OM O e C F 3 H N www.trilinkbiotech.com OM e - - - - efficiency. reagent givingoligoproductwithahigherconjugation reliable found that was amore the phthalimidyl-3'-C6-Amino-CPG was done.It with thephthalimidyl-3'-C6-Amino-CPG was modifier C7(purchasedfromGlen Research,Sterling,VA.) CPG support. unit betweenthelinkerand as the bridging tional advantage asanaminoprotectinggroupwiththeaddi- group phthalimidyl These newsupportstakeadvantageofthestability years ago(Figure4)(11,12) alongwithanovelC3analog. through GlenResearchwhichwasfirstseveral published purification made considerably easier. otide synthesistheimpuritieswillbewashedawayandfinal is that if theprotectinggroupisremovedduringoligonucle functionality (10). The advantageofsuchaprotectinggroup solid supportswhileactingasaprotectinggroupfortheamino oped usingcarbamatemoietiestoattachthelinker amine andreducedyields(9). of whichleadstocappingthe tions oftheoligonucleotide, the FMOCprotectinggroupislabiletosynthesiscondi- group uponremoval. A drawbacktousingthisreagentisthat at theDMTis elaborated oligonucleotide protectedhydroxy cinate bondthroughthesecondaryhydroxylgroupwhile C7 CPG (Figure 2). The linkerisboundtotheCPGviaasuc- is thebranchedFMOC-protectedaminoC3and modification which introducethe3'-aminomodification. tection conditions. A havebeendeveloped numberoflinkers step synthesis. (8) (Figure3).Howeverthisreagent requiresacostlymultiple derivative givingsuperiorresults oped, withthephthalimidyl tion anumberofotherprotectinggroupshavebeendevel Figure 3: Figure 2: FMOC NPEOC ing group with phthalimidylprotect- CPG Modifier C3 Amino 3' CPG Modifier C7 Amino FMOC CPG Modifier C3 Amino FMOC A comparisonofthecommonly used3'-amino We currentlysellaC6variantof3'-aminoCPGsupport A numberofotherinterestingreagents havebeendevel One ofthesupports usedto3'-amino introduce To overcomethisinherent problemof the FMOC protec- Other 3' Amino Linkers 3' Amino Linker Structures, FMOC protected FM FM CP CP CP OC OC G G G H N H N O H N O H N DMTO O O succ NO O ODM O in 2 O yl O -lca T H N N ODM a-CP ON O O T G O H succ n ODM in yl -lca T

n a-CP ODM G T - - - [email protected] [email protected] single to lead and reactions β-elimination undergo to known and is used tomimicabasicsiteswithinanoligonucleotide sequence whenthebaseisunknown. The dSpacercanbe yls of(16)orreplaceabasewithin the oligonucleotide mimic thethreecarbonspacingbetween3'-and5'-hydrox- support andboundoligo(15). The C3spacercanbeusedto steric interaction between byreducing bound oligonucleotide dye (14). to asupport They canalsoincreasehybridization and thefluorescent theoligonucleotide interaction between and reduces tags atgreaterdistancesfromtheoligonucleotide spacers toplace and/or additional 3' and5'-amino-modifiers with spacers canbeusedinconjunction otides. Inaddition sectionsofoligonucle- atoms andaretypicallyusedtobridge able (Figure5). These spaceramiditesdiffer inthenumberof 5'-Spacers fication. 80% aceticacidor2%aqueoustrifluoroaceticafterpuri- with must befreedusingdetritylationconditions the aldehyde gies. However, modifierC2isprotectedand the 5'-aldehyde standardsolidphase synthesis methodolo using incorporated modifiers are (C6vsC2).Bothaldehyde in thelinkerlength functional groupbutvary via abenzaldehyde the aldehyde ety onthe5'–terminusareavailable. The amiditesintroduce reduced toformastablelinkage. amines to form Schiff's base,buttheSchiff's basemustbe (13). zides, respectively The 5'-Aldehydescanalsoreactwith toformstablehyrozonesandsemicarba- and semicarbazides moiety canbereactedwithavarietyofsubstitutedhydrozinos substitution. hyde modifierisanelectrophilic The aldehyde substitutions, thealde modifications, whicharenucleophilic Incontrasttothe5'-aminoand'-thiol is the5'-aldehylde. at the5'-endthatcanbefurthermodifiedpost synthetically Modification 5'-Aldehyde Figure 5: and sold through Glen Research Figure 4: Pth C3-Amino Linker CPG Pth C3-AminoLinker Catalog #20-2954 Glen Research CPG Pth C6-AminoLinker Catalog #20-2956 Glen Research dSpacer Catalog #10-1914 Glen Research Linker Amidite C3/Propyl Catalog #10-1913 Glen Research Spacer 9 Catalog #10-1909 Glen Research A numberofdifferent areavail- spacerphosphoramidites Twomoi- thatincorporateanaldehyde phosphoramidites reactive functionality Another approachforintroducing Spacers 3' Amino Linker Structures manufactured by TriLink CP CP G DM N DM G T T O O O H N O H N DM O T O O N P O O N P O O O O O N N O O O N N P ODM O T ODM N T - - 20. 19. 18. 17. 16. 15. 14. 13. 12. 11. 10. 9. 8. 7. 6. 5. 4. 3. 2. 1. References (DTT). then freedbytreatmentwithdithiothreitol kept toaidinpurificationoftheoligonucleotide. The thiolis S-S. As withotherreagentsthetritylprotectinggroupcanbe tylation canbeaproblem. with silvernitrate. Although successfullyused,oxidativedetri- labile, unlikeMMT orDMT, it mustberemovedbyoxidation purification. However, because thetritylgroupisnotasacid to aidin usually keptonaftercleavageoftheoligonucleotide C6 is linker thetritylprotectinggroupon5'-thiol-modifier modifier C6S-S(Figure6).LiketheMMT protectedamino C6orthe5'-thiol is typicallydoneusingthe5'-thiol-modifier tometalsurfaces(22). (21),andbound via adisulfidelinkage peroxidase, andpeptides ages, enzymessuchashorseradish derivatives ofthedyetoformthioetherlink- and maleimide rescent probes(20)viareactionsofthethiolwithiodoacetate withavarietyoffluo- powerful tool,thiolscanbeconjugated to this tion canbeusedtointroducebiotin(19).Inaddition otides. Likethe3'-and5'-aminomodifiersthiolmodifica- so doestheinterest in 3'-and5'-thiolmodifiedoligonucle strand scissionofDNA (17,18). 5'-Thiol-Modifiers Figure 6: S-S C6 Modifier Thiol 5' Catalog #10-1936 Glen Research C6 Modifier Thiol 5' Catalog #10-1926 Glen Research Nucleotides & Nucleic Nucleotides Acids 20,1007. S. O.;Guzaev,Doronina, A. P.;Nucleosides, M. (2001) Monoharan, Nucleic Acids 20,539. & Nucleotides Nucleosides, Antopolsky,M.; Azhayev,(2001) A. Diego. Hermanson, G. T. Bioconjugate Techniques, Acedemic Press,San Research, 15,4837. B. S.;Beijer,Sproat, B.S.;Rider, P.; Neuner, P.Nucleic (1987) Acids Biochemistry 27,924. M.W.;Kalnik, C.N.; Grollman, Chang, A. P.;D.J. (1988), Patel, (1987) J. Biol. Chem. 262,10171. Takeshita, M.; Chang, C.N.; Johnson, F.; Will, S.; Grollman, A.P. 12, 900. Trawick,N.; Osiek, B. T. Chem. Bioconjugate J. K.(2001) A.; Bashkin, Nucleic Acids 20,369. Shchepino, M. S.; Korshun, V. A. & (2001) Nucleosides,Nucleotides 5,419,966 (1995). M. W.;Reed, Meyer, R.B.Jr.,R.; C. Petrie, Tobone,U.S. Patent J. C. 3, 85. Chem., Bioconjugate M. W.,R.; Reed, C. Petrie, D. Adam, A.; Meyer,B. Jr. R. (1992) G.; Chemistry4,1649. Eritja, R. & Medicinal (1996) Bioorganic Aviño, A.; Garcia, R.G.; Albericio, F.; Mann, M.; Wilm, M.; Nebauer, Trilink catalog,p.94. Jayaraman, K.; Mulvey, Chem.6,599. D. (1995)Bioconjugate Vu,N.; Rieger,H.; Joyce, M.;Walker,I.; Hill, D.;Goldknopt, T. S.; News Ed.6, 47. Hogrefe, R.I.; Mackie, H.; Powell, M. L. (1988) Amer. Biotechnologies 968. Dreyer, G. B.; Dervan, P. B. (1985) Proc.Natl. Acad. Sci.USA,82, Kempe, T. et Nucleic al. (1985) 13,45. Acids Research. 15, 5305. Zuckerman, R.; Corey, D.; Shultz, P. (1987) Nucleic Acids Research, Connolly, B. A.; Rider, P.Nucleic (1985) 4485. 13, Research, Acids Connolly, B. A. (1987) Nucleic 15,3131. Acids Research, 3rd Ed. John Wiley&Sons,Inc. New York Greene, T. W.: Wuts, G. M. Protective GroupsinOrganicSynthesis An alternative to this reagent is the 5'-thiol-modifier C6 An alternativetothisreagentisthe5'-thiol-modifier Thiol modificationatthe5'-endofoligonucleotide As thenumberofthiolspecificdyescontinuetoincrease 800-863-6801 5' Thiol Linkers DNA Synthesis andReagentsDNA N N O P O P O O N N S S S O

OM OM -

e e 113 Information Technical 114 Technical Information By RichardHogrefe, Ph.D,PaulImperial,Kelly Christianson and Terry Beck; TriLink BioTechnologies Yield Product ofConjugated Phthalimidyl ProtectedReagent 3'AmineModifying Enhances Synthesis andReagentsDNA capped vials. were After deprotectiontheoligonucleotides aqueousammoniumhydroxidein 4 ml fresh concentrated deprotected byovernighttreatment at 55°C with1.5mlof each supportweresynthesized. were The oligonucleotides Expedite 8909andstandardconditions. Three columnsof CAC-GTC-GA-(Linker)-NH phosphodiester oligonucleotide,5'-GTC-ATC-TGA-TAG- each) wereloadedintosynthesis columnsanda20mer available throughGlenResearch,Sterling,VA. are Modifier C-7CPGandthePhthalimidyl-3'-Amino-CPG the reportedprocedurewithslightmodification. The 3' Amino synthesized by TriLink's organicchemistrygroupaccordingto Modifier C-7support. was The Phthalimidyl-3'-Amino-CPG protected aminosupportandcompareditto the 3' Amino wesynthesizedthephthalimide modified oligonucleotides, wasnotseenintheliterature again. reagent innovative (Petrie, several yearsago (Figure 2),waspublished phthalimide within theactuallinktosolidsupportusingabaselabile step oftheDNAcapping synthesiscycle. acetylation duringthe the aminesusceptibletoirreversible group protectingtheprimaryamineisunstablemaking (FMOC) theflourenylmethoxycarbonyl serious drawback; Unfortunately,a nucleosidephosphoramidite. it suffers one with for linkagetoasolidsupportandtheothercoupling on abranchedalkylaminewithtwohydroxylgroups,one through severaldistributors(Figure1).It available isbased is the3' terminus ofanoligonucleotide Amino ModifierC-7 Figure 2: Figure 1: The two types of 3' amino labeling support (1µmole The twotypesof3'aminolabeling Always seekingnewwaystomakehigherquality An interestingalternative,whichplacedtheamine The reagentmostcommonlyusedtomodifythe3' . 1992). However, et al.1992). report, this initial the after O Structure of Phthalimide 3’-Amino Modifier C-6 CPG Structure of 3’-Amino Modifier C-7 CPG O O O O O O 2 -3', was prepared usingan O N H O O O N O O N H O H N CP G CP www.trilinkbiotech.com G 2. 1. References of oligonucleotides. labeling with superiorproperties,itistheobviouschoicefor3'amino efficiencies areverydesirable. As alessexpensivereagent and highconjugation reproducibility where applications reagent foruse inhighthroughputandmicrospotting reliable lot variationsortosynthesisissues.It is thereforeamore efficiencynature, theconjugation isnotsusceptible tolot-to- to the3' Byitsvery Amino ModifierC-7formostapplications. This correspondstothemassofanacetylcappinggroup. major peak(~20%)at+42au(6368). contains anadditional 6). The productisolatedfromthe Amino ModifierC-7support were thenanalyzedbymassspectroscopy(seeFigures5and using thesameRP-HPLCconditions. The oligonucleotides dried, andthenrepurified removed andtheoligonucleotide the entireDMT-on product,thetritylprotectinggroupswere acetonitrile. thefractionscontaining After anddrying pooling acetate (pH7.2)andagradientof triethylammonium C-18 column(8x100mm)using0.05M µBondapak were purifiedbyRP-HPLConaWatersoligonucleotides each support,withthedimethoxytritylslefton. These asabovewassynthesizedwith of thesamesequence 30 OD containing andaliquots precipitated The sampleswereethanol decanted, thebeadsrinsed,andcombinedsolutionsdried. conjugation efficiency,conjugation 1µmolescalecolumn an additional of theaminefunctionalityisrootcause the lower product qualitybetweenthetwosupports. 4 aretypicalofthesetandclearlyillustratedifference in below. The HPLCchromatogramsshowninFigures3and (RP) HPLC. were analyzedbyreverse-phase oligonucleotides dH mixtures werethenrunthrougha10mlG-25columnusing conditions. ester for6hoursunderidentical succinimidyl The conjugation. for separate 1.5mlmicrotubesanddriedinpreparation Phthalimidyl-CPG 2 13 65% 103 C-7 3'-Amino-2a b19 68% 2b109 Modifier Table 1:DABCYL conjugation results 2 O as theeluent toremoveexcessdye. The conjugated Tabone, JohnC.U.S. Patent 5,419,966. 1995. Reed, MichaelW., Meyer, Jr., Rich B., Petrie, Charles R., Chem., Bioconjugate Volume 3,85-87. Synthesis of3'-Amine-Tailed1992, Oligonucleotides, Meyer, Rich B. Jr., An ImprovedCPGSupportforthe Petrie, CharlesR., Reed, MichaelW., Adams, David A., is superior In summation,thePhthalimide-3'Amino-CPG In order to substantiate the hypothesis thatacetylation In ordertosubstantiatethehypothesis are shownin The resultsoftheconjugations Table 1 toDABCYLEach ofthesampleswasconjugated 260 unitsofeachthesixsampleswereplacedinto 1 90 1a Synthesis 1b 91 1c 93 2c (in OD Crude Yield Percent 111 260 units) Conjugated 83% 82% 82% 67% product run at ~16.5 mins. mixture with Phthalimidyl 3'-Amino support. Conjugation [email protected] [email protected] fied oligonucleotide synthesized Figure 5: Figure 3: U m A - Electrospray mass spectroscopy analysis of HPLC puri RP-HPLC chromatogram of crude conjugation Minutes --260nmBand=4 off phthalimide-3 of ' CPG. product run at ~15.4 mins. '-Amino Modifier C7 support. Conjugation mixture with 3 Figure 4: purified oligonucleotide synthesized Figure 6: CPG. U m A 800-863-6801 RP-HPLC chromatogram of crude conjugation Electrospray mass spectroscopy analysis of HPLC DNA Synthesis andReagentsDNA Minutes --260nmBand=4 off

'-Amino Modifier C-7 of 3 115 Information Technical 116 Technical Information Figure 1: spectral analyses. spectral analyses. species asshownintheFigure1wasobservedbymass of of thesyntheses.Noevidenceanypartiallydeprotected with littlen-1evidentinany products alllookedexceptional inmethanol. potassium carbonate offdeprotection yielding the thymidinesupport, whichwas efficiencies. The resultsaregiven asratiostothehighest actual amountdeprotectedandfordifferences incoupling to accountfordifferencesaveraged andnormalized inthe 260 nm. Table 2showstheresultsof these deprotections, at carefully isolatedandtheyielddeterminedbyabsorbance shownin deprotection conditions Table 2. The productwas vials. Aliquots werethensubjected, induplicates,tothe out in1 weighed as wellthecouplingefficiencies. collected sothat the exactstartingscalecould bedetermined, amino-CPG, and T-CPG. The initialdimethoxytritylswere amino-CPG, phthalimidyl-C-6 supports; phthalimidyl-C-3 thymidyl 20merona15mmolescaleeachof the following Results thymidine CPG. of athymidyl20merpreparedfromstandard,esterlinked, rate. 10-minute deprotection for manyhighthroughputsynthesislaboratorieswithitsfast deprotection method.Itthereagentofchoice is becoming system, hydroxide/methylamine) whichisanewerfast modifications. that contain to deprotectoligonucleotides been developed have Manyconditions deprotection conditions. oligonucleotide amino supportsunderavarietyof other, commonly used to comparethecleavageefficiencies ofthephthalimidyl efficiencyquestion regarding ofcleavage. Also, wewanted the hours, wedonotfeelthataddressed we adequately of at55°Cfor15 conditions conc.ammoniumhydroxide article onthesemoleculesoffer datausingdeprotection support byammoniumhydroxide. Although theaccompanying 3'-amino CPGistheefficiencyreaction from ofthecleavage By RichardHogrefe, Ph.D.,PaulImperial, Alexandre Lebedev, Ph.D.andKristinRossum; TriLink BioTechnologies Amino CPGs Methods forthePhthalimidyl Comparison ofDeprotection Synthesis andReagentsDNA Table 1: Coupling Efficiencies Thymidine (1) Support Thymidine (2) Phthalimidyl-C-3-amino (1) Phthalimidyl-C-3-amino (2) Phthalimidyl-C-6-amino (1) Phthalimidyl-C-6-amino (2) The products were analyzed by PAGEThe productswereanalyzed forpurity. The After synthesis, thesupportsweredriedand carefully The experimentwascarriedoutbysynthesizinga As acontrolwecomparedourresultstothedeprotection We alsowantedtoexplorethe AMA (ammonium the useofphthalimidyl One oftheconcernsregarding 5' 5' Partially protected species Oligonucleotid Oligonucleotid mole scale aliquots mmole scalealiquots into4mL screwtop e e 3' 3' O O 98.8% Efficiency Avg.Coupling 98.7% 97.4% 97.8% 96.9% 97.8% O O N H N O H O O O O O O H H www.trilinkbiotech.com found that with oneexception,K Cy5 dye.Inconclusion,we useful forphosphoramidite protected aminosupports. This deprotectionconditionis for usewiththephthalimidyl and shouldnotbeconsidered less product doyieldconsiderably deprotection conditions this reagent morefully.are exploring and wellsuitedfortoday'shighthroughputrequirements.We forthissupport.Ittoprepare choice is convenient an excellent slightly betterresults,the10-minutedeprotectionwith AMA is side product. Although thelongerdeprotectionwith AMA gave that theextraabsorbanceisduetoaromaticlinker released system resulted in the highestyieldwithourlinker. We believe the crudeproductmix.We tofindthatthe werepleased AMA conditions, orwewouldseesomeof those side productsin does notcleaveunderthese aromatic amidelinkapparently tothesupport. bonds cleavewellbeforethelinkage The amide Figure 1,wecanonlyassumethatthetwophthalimidyl properly. derivatives donotextendawayfromthesupport nucleosidyl although onecouldsuggestthatthenon- phenomenon, modified supports.Wefor the havenoprovenexplanation efficiencieslowered withalmostallof the non-nucleosidyl 1% percycleasshownin Table 1.We alsoseesimilar efficiencieswas lostduetothelowercoupling ofabout from thethymidinesupport. 10%ormore An additional efficiencies.after takingintoaccounttheslightlylowercoupling which was80-90%oftheyieldfromthymidinesupport, product, all yieldedequalamountsofaminolabeled conditions the last article. Ingeneral,thevariousammonium hydroxide given thebenefitsshownin seem hardlyworthconsideration ammonium hydroxide,althoughthedifferences of10%to20% amino supportsdonotfullycleaveusing the phthalimidyl Discussion deprotection conditions withgoodresults. deprotection conditions amino supportscanbedeprotectedwithanyofthecommon 3. 2. 1. References Table 2: Deprotection Conditions and Yield Determination 0.05 MK Method 1/1 MeNH 0.4 MNaOHin4/1 NH 3/1 NH NH NH NH (norm.) 1/1 MeNH MeOH/ H 4 4 4 4 OH OH OH OH One disappointment was thatthepotassiumcarbonate One disappointment Since weseenopartiallyprotectedspeciesasshownin The actualyieldswereabout70%of the yield recovered that The resultsconfirmtheanecdotalknowledge J. Researchin (2000) Chemical Toxicology 13(2):90-95. Schnetz-Boutaud, N.C.; Mao, H.; Stone, M. P.; Marnett, L. Toxicology10: 1133-1143. Kim, M.;F.Guengerich, P. Researchin (1997) Chemical Tetrahedron Letters36(49):8929-32. Reddy, M.P.;F.; Farooqui, N. B.(1995) Hanna, 4 OH/EtOH 2 2 NH CO O 2 2 /NH /NH ref 2 3 4 inMeOH OH- 30% aq. sol.; MeNH 4 4 OH 55° OH ref 1 55° ref 3 ep ie - - T-20 C-6 C-3 Time Temp 65° 55° RT RT RT RT RT °C 10 11 0.90 1.11 10 min.1.04 1.13 1.18 0.98 1.18 15 hrs1.13 0.72 0.79 0.96 0.72 4 hrs 0.97 0.82 15 hrs0.83 1.00 0.87 48 hrs0.80 0.99 0.92 24 hrs0.92 0.15 0.13 1.00 0.13 15 hrs0.15 0.87 0.91 0.95 0.91 48 hrs0.87 0.97 1.00 0.90 1.00 15 hrs0.97 2 CO 3 2 , the phthalimidyl-3'- , the - 40% aq. soln Pth Pth Std Pth . Figure 2: [email protected] [email protected] of success. fashion thatenhancestheprobability to synthesizeanddoitinacontrolled,singlestep impossible difficult thatwerechemically interesting conjugates ifnot linker, canquicklyscreenthroughanumberof researchers difficult toreproduce.Now, for thesamepriceasa5'amino which addedcostlystepstotheprocessandwasoften linker,or homobifunctional with eitheraheterobifunctional thiol. The onlyalternativewastoreacttheoligonucleotide with aprimaryamineor a conjugatetoanoligonucleotide prepared inordertoprepare derivative hadtobelaboriously Previously, either anactivatedcarboxylicacidormaleimidyl the numberofconjugatesthatcanbereadilyprepared. be coupledtoanyprimaryamine. This greatlyincreases itisnowreadyto to the5'terminusofanoligonucleotide, Figure 1: as well. conjugates it apowerfultoolforthescaleupofoligonucleotide However, advantagesthatmake it hasseveralotherinherent numberofconjugatesfortherapeuticapplication. of alarge It was originallydesignedtoallowhighthroughputscreening and withmuchmoreflexibility.conjugates moreeconomically is uniqueonthemarket.It offers anovelwayofpreparing By RichardHogrefe, Ph.D.; TriLink BioTechnologies CarboxylLinker, APre-Activated DADE: The linkerisusedasshowninFigure2. After coupling TriLink's DADE(decanoicaciddiester)linker(Figure1) O O H N O Structure of DADE linker A typical application of the DADE linker O Ba O O O O O s e O O O O Ba Ba P O O Ba O O O s s s n e e e O O O O Ba P P O O O O O P O O s e n n n Puri Couple DADEto5' Deprotect Oligonucleotid Collect ExcessConjugarf React withLg.Excessof O O O O fy Ba Ba P O ifNeeded O Ba O O O O s s s e e e O O O H 9 P P O O O O O O O Term O N H Conjug e 1'AmineBearin inus ofO P or NextReaction 9 9 O O O N O N H a O r ligonucleotid N Conjug O g a Conjugar N r e 25% acetonitrileinwaterandtheyielddetermined(965OD vacuo. The resultingresiduewasreconstitutedin1mlof in water.acetonitrile The combinedsolutionsweredried was decantedandthebeadsrinsedwith4mL of25% for 5hours at65°C,afterwhichtimethereagent hydroxide and cleavedfromsupportusing2mL ofconc.ammonium c. conjugatewasthendeprotected The oligonucleotide and dried. dichloromethane continuous mixing. The beadswerethenwashedwellwith 4 hoursatroomtemperaturewith The reactionwasallowed in 2mLdissolved with10%triethylamine. ofdichloromethane containing 10 equivalents of 1-octadecylamine (stearylamine) was washedanddried,thebeadswereaddedtoavial b. After oligonucleotide the linkermodifiedsupport-bound ofBeaucagereagent. was carriedoutusing10equivalents with thelinker.synthesis methodsupplied The sulfurization oligonucleotide conjugate wasanalyzed byelectrospraymass oligonucleotide oligonucleotide (5 a. The DADElinkerwascoupledtothesupportbound oligonucleotide. to aphosphorothioate Octadecylamine wasconjugated Phosphorothioate a to Octadecylamine Lipid the of Conjugation 1. Sample Applications of DADE of Sample Applications operation. up conjugation be worthyof consideration foryourhighthroughput orscale- purifications, arereducedoreveneliminated. operations,suchas to completion,theneedfordownstream Bydrivingthereactionfurther in large-scaleconjugations. efficiency inthat much largerexcessesarenowfeasibleeven the obviouscostsavings,thiswillalsoimproveoverall At Besides that timethereactionmixcanbereplenished. kineticallyforat will notbenoticeable leastmanyreactions. the rangeof100foldormore. The useof1%theconjugar if theexcessisin exactly asisforthenextreaction,especially Infact, is thereforerecoverable.thereagentcanbeused used inexcess,theamine,isnotaffected bythereactionand of theconjugate. RP-purification These fattyhandlesfor conjugars canthenactaspurification compounds tooligonucleotides. of verylipophilic conjugation systems, even dichloromethane. This allowstheready allowtheuseofcompletelyorganic phase conjugations mixture. Solid in theconjugation dissolve theoligonucleotide by therequirementthatsomewater(20-50%)isneededto advantages ofitsown. is oftenlimited The choiceofconjugar is stillsupportboundaddsseveral while theoligonucleotide The factchemistry that theconjugationisaccomplished of 200OD product wasisolatedusingthesame methodwithinjection 1 ml/min. with80%efficiency.The conjugationoccurred The in 100mMtriethylammoniumacetate, pH7,ataflowrateof using agradientof(5% to70%over70minutes) acetonitrile (Milford, MA)mBondapakC-18RP-column(4.6 x 305mm) d. The crudeproduct wasanalyzedandpurifiedonaWaters units). All inall,DADEoffers thatitshould enoughadvantages ofthissystemisthatthereagent Another advantage 800-863-6801 260 unitsofthecrudesampleperrun. The isolated DNA Synthesis andReagentsDNA Applications and Methods Applications mmole) using50molesasperthe

260 117 Information Technical 118 Technical Information scale. above. application the highthroughput The onlydifference is This processisessentiallythesame asthatfor described Conjugates Oligonucleotide of up Scale 3. the controls. products areallanalyzedbyPAGE or HPLCandcomparedto by HPLCorPAGE atthistime. We recommendthat the crude samplesaresufficient. Of course, theycanbepurified i. conjugatesarenowready foruseif The oligonucleotide hydroxide asusual. beads todry, then deprotectwithconc. aqueous ammonium by arinsewithacetone. followed the conjugation, Allow the use. Rinsethebeadswithfreshsolvent,sameasusedfor h. Decant the reagentsfrombeadsandstoreforlater yields. reactions toensurehighcoupling the reactionsatleast 4 hours.We recommendovernight reactionsonarotarymixerandallow g. Placetheconjugation efficiency oftheDADEcoupling. of thismaterialbyPAGE or HPLCwillalsoestablishthe conjugates. during analysisoftheoligonucleotide Analysis touseasacontrol of thismaterialshouldbedeprotected this scale. The 15thwillbekeptasaretainer. A smallaliquot from support. Upto14reactionscanbedonesimultaneously f. the to thevialscontaining conjugars Add thedissolved aqueous reactions. to 10% volumeoffreshlyprepared0.5Msodiumbicarbonate are used,and necessary whenlargeexcessesofconjugar this maynotbe to organicreactions,although triethylamine amine contaminants), andwater. We add10%volumeof DMSO, (methylene chloride), highqualityDMF(freeof in acetonitrile,dichloromethane carried outconjugations of solution). As topreferredsolvents, we havesuccessfully the reagent.We commonlywilluse100foldexcesses(200µL you touse largeexcessesof allowing therefore recoverable, M. The conjugarwillnotbeaffected bythereactionandis 0.05 ofapproximately solvents ofchoicetoaconcentration e. Dissolvetheaminestoseparatelyin be conjugated will becarriedout. vials inwhichtheconjugations d. Placethesupportin15separate2mL glassscrew-cap product. full-length ofisolatable give theequivalentof0.3-0.5µmole 1 µmoleeach.Formostsyntheses,thiswill approximately c. Drythesupport anddivideinto15equalaliquots, 100 µmolesofreagent. with thereagent. supplied This scaleofsynthesiswillrequire usingtheprocedures DADEtotheoligonucleotide b. Couple standard. This samplewillbedeprotectedandserveastheunmodified support fromthesynthesiscolumnandresealcolumn. sequence ofinterest.Removeaverysmallamount a. Preparea15µmolescalesynthesisoftheoligonucleotide chosen one. ifyoualreadyhave optimize theformationofaconjugate also beusedtoscreenanumberofdifferentto conditions using avariationofconcept. the following This methodcould A canberapidlyprepared conjugates bearing numberofamine Conjugates of Screening Throughput High 2. overall theoreticalyieldfromthestartingscaleof5µmoles. The finalyieldwas435OD spectroscopy andfoundto have thecorrectmass(6585au). Synthesis andReagentsDNA 260 units;2.25µmoles;45%of www.trilinkbiotech.com Reagent throughGlenResearch. to oldproblems. TriLink nowsellstheDADEDNA Synthesis DADE offersto discovernewsolutions manyopportunities desalting. purify asusual. The productcanalsobeusedcrudeafter through thedesaltingprocessofyourchoice,andthen by first passingthereaction d. Isolatetheoligonucleotide NaOH solutionifyouareuncertain.) using the0.4M for specificdeprotectionrequirements used. Refertothemanufacturerofyourphosphoramidites (The time can bereducediffast deprotecting monomersare hours withthefollowingreagent: c. Deprotectatroomtemperaturefor48 the oligonucleotide phosphoramidites. of anyDNA linkersor5'biotin synthesizerlike5'amino reagent. DADEcanbeaddedtothemodifiedreagentport with the using theproceduressupplied oligonucleotide theappropriateamountof b. Couple DADElinkertothe control. sample willbedeprotectedandserveastheunmodified from thesynthesiscolumnandresealcolumn. This scale desired.Removeaverysmallamountofsupport a. Preparethesequenceofinterestonsolidsupportin oligonucleotides. the useof DADE todirectlyprepare5'COOH linkerson scheme allows deprotection Merely usingthefollowing Acid Linkers 4. stepsg,handifromsection2. e. Follow DADE coupling. PAGE or HPLCwillalsoestablishtheefficiency ofthe conjugates. oligonucleotide Analysis ofthismaterialby be deprotectedtouseasacontrolduringanalysisofthe and thereactionmix. A smallaliquotofthismaterialshould can besealedandisbigenoughtocontainthesupport inavialthat d. Placethesupportboundoligonucleotide triethylamine.) solution willresultin1.5mL ofsolution(0.15mL ofwhichis 0.1 M. solution atapproximately your conjugation A 0.1M reactions. toaqueous sodium bicarbonate Attempt to make are used,and10%volumeoffreshlyprepared0.5M this maynotbenecessarywhenlargeexcessesofconjugar 10% volumeoftriethylaminetoorganicreactions,although solvent choice. See Example1forideasregarding Add in thesolventofchoice. for thisexample)tobeconjugated c. Dissolve100foldexcessoftheamine(1500µmoles synthesis willrequire100µmolesofreagent. withthereagent. supplied procedures This scaleof usingthe DADEtotheoligonucleotide b. Couple control. will bedeprotectedandserveastheunmodified the synthesiscolumnandresealcolumn. This sample synthesis. Removeaverysmallamountofsupportfrom For thisexamplewewillworkwitha15µmolescale a. Synthesizethesequenceofinterestonsolidsupport. Preparation of Oligonucleotides with 5' Carboxylic 5' with Oligonucleotides of Preparation 0.4 MNaOHinMethanol/H 2 O (4:1) [email protected] [email protected] our estimation,the highertheconcentrationbetter. We (approx. 50mg/mL) and0.1Msolutionsoftheamidites. In 4. Concentration: acceptable limits. to standovernight. allowed This willdropthewatercontentto layer ofsievestothebottom vial. The vialissealed and solvent and thenadda single the amiditeinappropriate purchased ormanufacturedbyus, commercially istodissolve used forseveralbottles. work wellherealso. The largepackscanbecutopenand sieves (3Å)justpriortouse. quality molecular The Trap-paks The solutionto this istodrythedissolvedamiditewithhigh draw wateroutoftheatmosphereevenduringfast transfers. tend toclingwater, or arehygroscopicenoughtorapidly remove thebase. be aproblem.Weextensively to co-evaporateyourcompound the synthesis.Ifthenexcessbasewillnot this isdonecarefully process andisremovedusingextensivedryingattheendof your compound. The excessbasecomesfromthepurification NMR orHPLCanalysisof is detectableinthephosphorus of yoursynthesis;excessbaseandwater. Neither contaminant arising fromtheamiditeitselfthatcangreatlyaffect the quality 3. Moreabout water: Therearetwocommoncontaminates are unsureofthequalityyoursupply. supply highlydriedsolventsoversievesforyouramiditeifyou device totest the watercontentof your solvents. TriLink can for thispurpose.)We stronglyrecommendyouuseatitration from PerseptiveBiosystemsarethebestqualitysievestouse at least 24 hoursbeforeuse.(We foundthatthe Trap-paks it tostand sieves and allowing molecular acetonitrile, adding solvent,suchasDNAby usinghighquality synthesisgrade 30 ppmwater, preferably 10ppmor less. This canbeprepared on asynthesizer.decide touseyourcompound problems ifyou ifthereareanycompatibility your manufacturer there areproblemswithflowratechangesandvolatility. Ask canbeusedinmostsynthesizers, Although dichloromethane amidites, suchasfatty acid amidites, require dichloromethane. common solventforDNA synthesis.However, more lipophilic in acetonitrile, whichisthe are soluble phosphoramidites 2. Solventselectionand dryness: Mostnucleoside other parameters. issues byoptimizing help overcomesomeminorcontamination costs. low yieldsandhigh The methodssuggestedbelowcan in anamiditesynthesis.Multiplepurificationscanleadtovery Purificationisusuallythemostcostlystep research question. are veryexperimentalormerelyameanstoanswer of development. true ofcompoundsthat This isparticularly compound asitstands,thantocontinuepayformethods At some pointitbecomesmorecosteffective toacceptthe protocols maystillremaininyourcompound. using established of thecontaminantsthatareremovedfromstandardamidites us onlyonce(andperhapsforthefirst time byanyone),some recommend proceduresthat should workinmostcases. compounds. based onourexperiencewithsimilar And wecan do havesomesuggestionsonhowyouramiditemaybehave likely verylittleisknownaboutyourcustomcompound.We knowledge: scientific of envelope the Pushing 1. few minutesto read thesewordsofadvice. we suggestyoutakea phosphoramidite, custom synthesized Use ofCustomSynthesizedPhosphoramiditeReagents Our procedure with any expensive amidite, whether Our procedurewithanyexpensive Water eliminated.Somecustomamidites islesseasily It is alsoimportanttousesolventthatcontains lessthan Further, since thisparticularamiditewassynthesizedby Before yougrababottleofacetonitriletodissolveyour Most instrumentsusebetween0.05 M Most deprotection conditions usingconc.NH deprotection conditions compounds, suchasdyes,arenotstabletothestandard Deprotection method: 7. analysis ofyourfirstisrecommended. oligonucleotide mustbemadeandtested. an oligonucleotide A massspec will answersomeof the questions.However, in theend, stability experimentsthat can helpyoudeveloppreliminary or tothesynthesisscheme. either thecompound TriLink any oneof these reagents,thenchangesmust bemadeto acid, oxidizerorcappingreagents?If the answer isnoto cycle chemistries.Isstableto coupling yourcompound is ordered,thestabilityofreagenttorest one thatshouldbeaddressedbeforeacustomsynthesis synthesis process: and Another majorconsideration, DNAthe of rest the to reagent your of Stability 6. to 95%orbetter.coupling We willdescribethisprocessbelow. efficiency,coupling this isagoodwaytoboostan80% If atallabout an importantnucleoside.youareconcerned istodoubleoreventriplecouple Another recommendation for it isknowntooccur(methylphosphonates, example). times, although that couplesworsewithextendedcoupling time. It the couplingisveryraretohaveanamidite double time. use thatcoupling Actually, in mostcasesitisbestto amidites. non-nucleosidyl everything else,including and15minutesfor minutes foramodified2'deoxynucleoside will provideananswer. Wetimes of5 usecoupling generally time. Sometimesonlytrialanderror the requiredcoupling of Coupling time:Thesterichindrance theamiditedictates 5. and manualsynthesesusingmodifiedreagents. automated of 0.1Mforboth a concentration using recommend you succeedwith yourresearch. done fromthebeginningofprogram. Ourgoalistohelp for you.Manyclientscontracttohave this oligonucleotide experiments describedaboveand prepareyour fails… else all If 10. as we shouldget Aristotle explained, extremely close. 20%), etc. Theoretically, although wewillnevergetto100% will get youto96%(80%oftheremaining second coupling before oxidationasillustratedbelow. step oneortwotimesinthecycle repeating thecoupling may bebest to domultiplecouplings. This isdonebysimply really wanttomakesureyouramiditecoupleswell,thenit triple coupling: double or Single couplingvs 9. amounts ofmaterial. and littletonowastage. This canbecriticalifyouhavelimited can bedonewithjusttherightamountofmaterial coupling uses morereagentsbecauseof priming needs, etc. A manual time. quantity ofreagentsusedandcoupling An instrument It our modifiednucleosides.usgivesprecisecontroloverthe byhand. to dothecouplings This ishowwecouplemostof the mostefficientlearned waytousepreciousamiditesis 8. Coupling method - automatedvsmanual: again, trialanderrormaybenecessarytooptimizethisstep. hours. Once There aremanyalternativemethodsavailable. If there isareportedreagentthatsimilartoyours, If at80%efficiency,your reagentiscoupling then the 800-863-6801 5. Cap 4. Oxidize step#2as manytimesasneeded 3. Repeat #1(addamidite andactivatortocolumn) 2. Couple 1. Deblock DNA Synthesis andReagentsDNA TriLink canconductallofthe This isanotherstabilityissue.Many 4 OH at55ºCfor15 We have

If you 119 Information Technical 120 Technical Information cell linereduction, etc.). This informationisoftenusedtohelp tain genetargetwillgivedesired biological results(i.e.tumor a cer- in cellculturetodetermineifdownregulating nucleotides agent. Target referstotheuseofantisenseoligo- validation as anactualtherapeutic and theuseofanoligonucleotide tions ofantisense,twoaremost prominent: targetvalidation your interestinantisenselies.Of the manypotentialapplica Target Validation versus Antisense DrugDevelopment (Tablephosphorothioates 1). trials atthistimeare throughclinical compounds progressing and Iversen, 1995).Despitetheissues,mostof (Srinivasan properties invivo severalunexpected compounds exhibited easy.road hasnotbeen Itdiscoveredthatthese was rapidly dancy ofthiscompoundasanantisensedrug.However, the with therelativeeaseofsynthesis,haveledtoascen- Furdon, al., 1988; that thismodificationisalsoasubstrateforRNaseH(Stein,et long known(MatzuraandEckstein,1968).It was laterlearned was of oligonucleotides fication toretardnucleasedegradation linkage. a phosphorothioate producing The abilityofthismodi- oxygen onthephosphatebackbonewithsulfur,non-bridging is toreplacea tions thatcanbemadetoanoligonucleotide cessful todate(Matsukura, tosynthesize,havebeenthemostsuc- easiest modifications oligonucleotide. As itturnedout,phosphorothioates,oneofthe changes toeitherthephosphateorsugarportionof subtle aswell asnottoosubtle included These modifications radation (Blake,etal., 1985; andGoodchild,1987). Agrawal fied backboneswereintroducedthatresistednucleasedeg- andendonucleases. both exonucleases To remedythis,modi- in the blood andincellsby of theoligonucleotide degradation one ofthatneededtobeovercomewastherapid the hurdles became apparentthat, in orderto develop anantisensedrug, Walder, 1988). and Hunt,1986;Dash, H (Minshull in fact, is enzymaticcleavageoftheRNA strandbyRNase region. through thehybridized The truemechanismofaction, are indeedmoreliketrainsandwillquicklyread polymerases 'hybrid arrest'. Infact, it waslaterlearnedthatribosomesand the codeandfalloff,not continuereading thus effecting mRNAwould (orpolymerase) (orgeneticDNA),theribosome to asinglestranded was annealed a syntheticoligonucleotide (Stephenson, 1978). The earlytheoryheldbymanywasthatif first theuseofantisenseoligonucleotides paper describing errors thatnearlycausedtheprematuredeathoffield. antisense technology, as welllearnwaystoavoidcostly the useof regarding afewkeyprinciples novice understand acknowledged. universally This essaywillhopefullyhelpthe is moreorless by antisenseoligonucleotides gene expression of oftheinhibition sion ofinterestnowthatthephenomenon FDA approval. an explo- The antisensefieldisexperiencing the first antisense drug(Isis's Fomivirsen) recentlyreceived and resurrected asapowerfultoolforthemolecularbiologist period ofdoubt(Gura,1995;Stein,1995),antisensehasbeen event, suchastranscription,translationorsplicing. After a (DNAa targetsequence orRNA)designedtohaltabiological of DNA thatarecomplimentary, (oranalogs) or antisense,to (an adaptation of this article was published in By RichardHogrefe, Ph.D.; TriLink BioTechnologies OligonucleotidePrimerAn Antisense Oligonucleotide Applications The designofyourexperimentsispredicatedonwhere One ofthemostsimpleandstraightforwardmodifica- Even beforetheroleofRNaseHwasdiscovered,it the attributed withpublishing Paul Zamecnikisgenerally areshort,syntheticstrands Antisense oligonucleotides et al., 1989). These properties,combined ., 1987; Stein, et al.,1987; et al. 1987; Walder and Antisense and Nucleic Acid DrugDevelopment, 9, 351-357 1999. Table 1 updated 2007.) et al.,1988). www.trilinkbiotech.com - order tobesuccessfulinvitro. The potentialcostofdevelop systemwasneededin when itwasdeterminedthatadelivery almostcollapsed whole fieldofantisensedrugdevelopment as salinesolutionswithoutdeliveryvehicles. administered The inclinicaltrialsare supported sincealloftheoligonucleotides - butiswell is veryboldandsimplistic-evencontroversial vivo deliverydoesnotappeartobeaproblem. That statement is thatintocellsinvitroappearstobedifficult,while delivery in tocomeoutinthelast 15yearsinthisfield findings surprising work iscellularuptakeanddelivery. Perhaps oneofthemost constructs thatwillalsobediscussedbelow.generation" fromthenewer"second have moreflexibilityandcanchoose you an antisenseinvitroassayfortargetvalidation designing rothioates thatwillbediscussedinmoredetailbelow. When RNase Hactivity. However, there areproblemswithphospho resistance andretentionof for anantisensedrug-nuclease of expensive theconstructsthat have thecorrect properties with noothermodification. oligonucleotides They aretheleast most oftheconstructsinclinicaltrialsarephosphorothioate of theconstructcost. for areasonable This isthereasonthat ing concernsneedstobetheabilityscaleupsynthesis struct. drug,oneoftheoverrid- Whendesigninganantisense con- ofoligonucleotide drugischoice assay oratherapeutic course. scale up,toxicity, delivery, andtheFDA, pharmacokinetics, of an antisensedrugmustconcernthemselveswith developing uptake. attention totheissueofcellular Those interestedin with cells,whichrequireslessmaterial,butmore exclusively using antisenseasatooltostudygeneswillmostlikelywork several significantdifferences doexist. Those interestedin as well.However,companies now antisensetargetvalidation are ments, whichiswhysomanyantisensedrugcompanies experi- is similartowhatneededconducttargetvalidation to developatherapeuticagent workneeded of theearlyphase drugs. Fortunately,more classicsmallmolecule develop much Ramasamy, bonus(Stein,1995; an added sense shouldbeconsidered effectspositive therapeutic that cannot beprovedtoanti- and Iverson,1995). McKay 1994;Desjardins The occasional mechanism(Deanand due toanantisense were convincingly to discoverthatinsomeinstancesresultswereobtained animalsanywayonly a bravefewwentaheadandinjected a greatdealofmoneyandwaslosingpatience.Fortunately, vivo frightenedaninvestmentcommunitythatalreadyspent of oligonucleotides forsystemicdelivery ing suchavehicle spective. Makesurethat others canreproduceyourwork. synthesis fromacommercialandscale upper- oligonucleotide chemistry, with seek thecounselofsomeoneexperienced concern. If you havenovelchemistry, or verycomplicated to alargeextent. precede thebiology This shouldbeaprimary uct, and if it isfeasibleinlargescale. The chemistrymust drug, youmustconsiderthecosttosynthesizefinalprod- depends onyourfinalgoal.Ifanantisense you aredesigning Oligonucleotide Construct an Choosing with uptake. cells incultureandthereforeyouwillhavetobeconcerned or aninvitroassay, your initialstudieswillmostlikely bewith anantisensedrug of whether youaredeveloping regardless The othermajordifference betweeninvivoandvitro One veryimportantdifferencean invitro indeveloping As statedearlier, the choiceofbackboneconstruct , 1996, Boiziau, et al.,1996, et al., 1997). Inanycase, in - - T compound. of asingle The overallmixturehasalower number of phosphorothioate internucleotidelinkages)instead arrest and depend on extreme hybridization enhancement on extremehybridization arrest anddepend concept of hybrid These constructsareareturnto the original system. ase resistance. You havetoseewhatworksbestwithyour may reducetoxicity,linkages however italsoreducesnucle ate linkagesforbest results. Removingphosphorothioate al., 1994). The arms mayornot contain phosphorothio Metelev, linkages (Monia,etal.,1993; ate internucleotide withphosphorothio a coreofsix2'-deoxyribosenucleosides has six2'-OMe nucleotides atboththe5'and3'ends,leaving in lengththat nucleotides eighteen to useanoligonucleotide commercially.of choicesareavailable A goodstartingpointis for aparticularconstruct, but forresearchpurposesanumber seek counselofagoodpatentattorneyandobtainlicense specific constructsasproprietary. Intheendyoumayhaveto your bestchoiceforsuchwork. active when usedinanvitroassayandare reproducibly Agrawal, 1998). These allcombinetoyieldacompoundmore stability(PengHo, nuclease increased reduction intoxicity,general increased hybridstability, and offeredenhancements bythisclassofcompoundarea H mechanism(Hogrefe,etal.,1990). significant most The principles oftheRNase to helpunderstandtheunderlying 2'-OMe armswereused containing Chimeric oligonucleotides mixtures ofchemistryarecalledchimericoligonucleotides. 1987, Gilesand Tidds,that contain 1992).Oligonucleotides H activityof(AgrawalandGoodchild, the oligonucleotide core that rothioate modifieddeoxyriboseretainstheRNase that surroundaphospho- 2'-O-methyl (OMe)nucleosides) mon designistohavenucleaseresistantarms(suchas are stillcompletelymodifiedwithphosphorothioates. A com- linkages, andmany them includesomephosphorothioate atthesmallerscales. Infact, most of ate oligonucleotides than phosphorothio- ones arenotmuchmoreexpensive and thelesscomplex commercially constructs areavailable do exist. sibilities These "secondgeneration"oligonucleotide an antisenseinvitroassayasendpoint,otherpos- develop succeed. right tousethiscompoundasatherapeuticagentonceyou BepreparedtopayUncleSamforthe antisense applications. for is thatoligonucleotides the NIHpatentedphosphorothioate preparations. early oligonucleotide Another problemforsome in or duetocontamination a sequencespecificphenomenon that toxic (SrinivasanandIversen,1995),althoughmaybe also haveareputationforbeing 1994). Phosphorothioates the affinityshow forproteins(Brown, phosphorothioates Someofthoseeffectsand undesirable. desirable are dueto propertiesinvivo,both have beenknowntoexhibitunusual (LaPlanche, [email protected] [email protected] a racemicmixtureof2 ate oligonucleotides. The backboneischiral,resultingin often beendifficult toachieve. successinasciencewherehas have demonstrated oligonucleotides program. Besidesprice,phosphorothioate are probablyagoodchoiceifyouwishtorapidlydevelop as obtainFDA oligonucleotides approval.Phosphorothioate to entertheclinic,aswell oligonucleotides phosphorothioate compounds. stream of They obviouslyexpectacontinual the synthesisofthese budget toimproving their development kilogram scales. Antisense firmsstillcommitagreatdealof prices andin otides canbeobtainedforfairlyreasonable otides continuestobephosphorothioates. These oligonucle m than its corresponding phosphodiester oligonucleotide phosphodiester thanitscorresponding "Third generation" compoundsarealsoindevelopment. "Third generation" Since 1987,variousgroupshavestakedoutseveral For thosewhowant to havealternatives,orwish to However, there aresomeproblemswithphosphorothio for antisenseoligonucle The mostpopularmodification , 1986). Phosphorothioate oligonucleotides et al.,oligonucleotides 1986). Phosphorothioate n oligonucleotide species(wheren= oligonucleotide et al., 1998; Zhouand et al., et ------mRNAprocess screening The orproteininvitrothanvivo. look fortrueantisenseindicatorssuchasreductionoftarget an antisense drug.In developing general,itiseasierto ing aninvitroassay, but itisalsohighlyrecommended when in culture. This, ofcourse,isyourendpointwhendevelop vitro assay, the initialexperimentswillmost likely bewithcells Successful a Developing effectthe (Boggs, with5-methylcytidinewhichwillinhibit dines 5'toguanosines on yoursystem, a simpleexperiment istoreplace allthecyti- 1998). Ifyouareconcernedabouttheeffect a CpGmayhave effect for itstherapeuticvalue(Klinman,1998;Millan, this against bacteria.Severalgroupsareactuallyexploiting genomic materialasadefense response tonon-methylated ylated. Apparently, an immune mammals havedeveloped is methylatedatthe5position.Bacterialcytosinenotmeth- tion isthat in mammaliancells,mostoftheexposed cytosine stimulation oftheimmunesystem(Krieg,1998). The explana- CpG causeafairlyprofound es thatcontainthedinucleotide referred toastheCpGeffect. sequenc- Someoligonucleotide rates. have even bettersuccess of thechimericoligonucleotides chance thatoneofwillbeactive.Some those tensequences or G-Carmsthatwillformstronghairpins. There isagood sequences withregionsofpolyguanosine tion whileavoiding sequences alongtheregion,tryingtomaximizehybridiza (Sierakowaka, the mRNAmechanism asopposedtothemessage processing popular inordertoinhibit ing siteshasbecomeincreasingly popular, bysitemutations.Recently,followed targeting splic- the start surrounding codon(AUG)siteisprobablythemost et al.,1994). 1992; BryschandSchlingensiepen, The region advicedoesexist(Cohen,1989;Woolf,nucleotide, although particular geneisthemostactivesiteforanantisenseoligo- work. The onecaveat isthattherenouniversalcationic et al.,havebecomethe standardfor 1995), which (Capaccioli, effective deliverysystemhasturnedouttobecationiclipids of tion totheproblem nomenon. for this phe- does notappeartobeasatisfactory explanation a goodtarget. sans carrieronceyou'vediscovered There Conversely, you cangoforwardwithyourinvivoexperiments that tobesuccessful is is alsofarmoreeconomical.Whatmustberemembered Choice of Sequence of Choice increase. requirements fication is stillaneedtobeattentivepurity, asthemodi- particularly there hasbeensignificantimprovementovertheyears, of thepastwereduetocontamination. conclusions Although of thecompound.Manyfailedexperimentsandfalse whatever theconstructyouchoose,becertainofintegrity unless youhaveextensivein-houseexperience. it isprobablybestnottoexplorethesetypesofcompounds such assolubility, delivery, or costofsynthesis. At this time, work tosomeextent, all haveatleastonesignificantproblem, Swayze, 1997),andothers.Whilemostoftheseconstructs MMIs (Morvan, nates (Reynoldsetal.,1996), PNA's (Hanvey, etal.,puremethylphospho 1992), chirally (Gryaznov andChen,1994;MignetGryaznov, 1998), 2'-MOEs include (Monia,1997),N3'-P5'phosphoramidates using highlymodifiedoligonucleotides. These modifications Whether you are developing an antisensedrugorin Whether youaredeveloping Youwith whatiscommonly dohavetobeconcerned For everysiteofinterest, design uptotendifferent Fortunately, solu- we doseemtohaveareasonable There isnosurewaytodetermine itself, As afinalwordregardingtheoligonucleotide 800-863-6801 ., 1993; Lappalainen, et al.,1993; Oligonucleotide Applications et al., 1996). ., 1997). et al.,1997). in vitro, you mustuseadeliverysystem. cellular uptake. in vitrocellular The most In Vitro Experiment ., 1994; Quattrone, et al.,1994; a prioriwhereon et al.,1996; in vitro et al.,

- - - 121 Information Technical 122 Technical Information Table 1: Idera Geron ..JmsCn.Hs.GI24 MyeloidLeukemia I GTI2040 A.C. JamesCanc.Hosp. mrn(se ersine)E11I Myastheniagravis II Antisense Pharma EN101 Amarin (EsterNeurosciences) Alnylam Aegera Aegera Cachexia Aegera Aegera II Aegera Aegera AVR118 Aegera Advanced Viral Research aionaCne os T 00I Breastcancer II GTI2040 California CancerCons. AVI Biopharma AVI Biopharma AVI Biopharma AVI Biopharma AVI Biopharma AVI Biopharma Archemix Genta Incorporated Genta Incorporated Antisense/Isis Genta Incorporated Genta Dynavax Dynavax Antisense Therapeutics no hraetcl,Ic Z-98ILymphoma Genta I Genta EZN-2968 EORTC Enzon Pharmaceuticals,Inc. Enzon Therapeutic Dynavax TechnologiesCorp Dynavax TechnologiesCorp Dynavax Coley Pharmaceuticals Antisense Thera./Teva Antisense Pharma Geron Gentium/Dana-Farber Genta Incorporated Genta Incorporated Immune Response Idera Idera Company Oligonucleotide Applications Sampling of Oligonucleotides in Clinical TrialsClinical in Oligonucleotides of Sampling R13 oi uo ainnistelomerase Renalcellcarcinoma Solidtumormalignancies II I IMO-2055 GRN163L E316II ua amr acnm lP(aps niio)AtsnePopoise C0584 Ongoing Ongoing NCT00558545 NCT00558922 Glioblastoma Phosphodiester Ongoing Phosphodiester II NCT00363974 Resp.syncytialvirus Antisense AP 12009 Antisense PancreaticCancer XlAP (caspaseinhibitor) Phosphodiester II XlAP (caspaseinhibitor) HumanMammaryCarcinoma I/II ALN-RSV01 Non-smallcelllungcancer I/II Antisense AEG35156 Solidtumors XlAP (caspaseinhibitor) I/II AEG35156 Advancedtumors I AEG35156 Acutemyeloidleukemia I AEG35156 Advancedcancers I/II AEG35156 I AEG35156 AEG35156 V-68 II Musculardystrophy I/Ib AVI-4658 V-57II Drugmetabolism I/II AVI-4557 V-05I HepatitisC II AVI-4065 V-16ICABG I AVI-5126 etnM IRestenosis II Resten-MP etnN IIIRestenosis II/III Resten-NG R17 IVnWlern ies Platelets Von WillebrandDisease II ARC1779 eaes I/II Melanoma II/III Genasense Genasense Multiplesclerosis II ATL1102 eaes IIINnsalcl ugcne Bcl-2 Melanoma(Skin) III Non-smallcelllungcancer II/III Genasense Genasense ISS(Heplisav) 1018 ISS(Tolamba) 1018 Asthma I ATL1102 eaes I ct yli ekmaBcl-2 Prostatecancer Acutemyeloidleukemia II Prostatecancer TLR9 III Genasense II Genasense HIV ColorectalNeoplasms Genasense I/II TLR9 HepatitisB I Non-Hodgkin'sLymphoma HGTV-43 I 1018 ISS II 1018 ISS 1018 ISS Cancer II ProMune Multiplesclerosis IIa III ATL1102 AP 12009 R13 /ICrnclmhct ekmatelomerase Chroniclymphocyte leukemia I/II GRN163L I Defibrotide Genasense III Genasense M-05INnsalcl ugcne TLR9 HIV Non-smallcell lungcancer II HepatitisC I Amplivax I IMO-2055 IMO-2125 Compound I HepatitisB III Ragweedallergy III I VOD III Phase Anaplastic Astrocytoma Cell Neoplasm Multiple MyelomaandPlasma Chronic Lymphocytic Leukemia Solid Tumors Disease www.trilinkbiotech.com Unknown lP(aps niio)AtsnePopoise C0579 Ongoing NCT00557596 TGF-beta2 Phosphodiester Ongoing NCT00357747 Ongoing viral gene Antisense NCT00372736 XlAP (caspaseinhibitor) Phosphodiester Phosphodiester Antisense XlAP (caspaseinhibitor) Phosphodiester Antisense XlAP (caspaseinhibitor) Antisense XlAP (caspaseinhibitor) ctlhln seaeAtsneMixedChemistry Antisense acetylcholine esterase Cytoprotective dystrophin CYP3A4 S HVpoes)AtsneNugene Antisense NS3 (HCVprotease) c-myc c-myc c-myc Bcl-2 VLA-4 Bcl-2 VLA-4 TLR9 TLR9 Bcl-2 DNA Bcl-2 Antisense viral replicationgenes TLR9 TLR9 VLA-4 TGF-beta2 HIF-1α Bcl-2 Bcl-2 Ribonucleotide Reductase Bcl-2 Unknown TLR9 TLR9 TLR9 Target (tumor factor) (tumor factor) Antisense siRNA Ongoing NCT00070551 Phosphorothioate Antisense mueAtv PNA Immune-Active nies Phosphorothioate Antisense nies Nugene Antisense nies Nugene Antisense nies Nugene Antisense nies Nugene Antisense Aptamer nies hshrtiaeNT0425Ongoing NCT00543205 Phosphorothioate Antisense MixedChemistry Antisense nies hshrtiaeNT0123Ongoing NCT00016263 Phosphorothioate Antisense Phosphorothioate Antisense nies hshrtiaeNT0361Ongoing NCT00030641 Phosphorothioate Antisense nies hshrtiaeNT0828Ongoing Phosphorothioate NCT00085228 Phosphorothioate Antisense Antisense Ongoing NCT00511095 Phosphorothioate Ongoing Immune-Active NCT00435812 Phosphorothioate Ongoing Immune-Active NCT00068588 Phosphorothioate Phosphorothioate Immune-Active Phosphorothioate Immune-Active Antisense Ongoing NCT00761280 Ongoing Phosphorothioate NCT00431561 Phosphorothioate Phosphorothioate Antisense Antisense Antisense nies hshrtiaeNT0240Ongoing NCT00024440 Phosphorothioate Ongoing Antisense NCT00403052 Phosphorothioate Ongoing NCT00251394 Unknown Phosphorothioate Immune-Active mueAtv LNA Immune-Active nies hshrtiaeNT0355Ongoing NCT00636545 Ongoing Phosphorothioate NCT00017602 Ongoing NCT00078234 Phosphorothioate Antisense Phosphorothioate Antisense Antisense Antisense nnw admmxueNT0551Ongoing NCT00358501 Randommixture Unknown mueAtv Phosphodiester Ongoing DNA Immune-Active NCT00729053 Unknown Phosphodiester Immune-Active mueAtv hshdetrNT0359Ongoing NCT00633529 Phosphodiester Immune-Active Mode of Action Thiophosphoramidate Thiophosphoramidate RNA Unknown Chemistry C0309 Ongoing NCT00310895 Ongoing NCT00124189 C0688 Ongoing NCT00658086 C0171 Ongoing NCT00127517 C0648 Ongoing NCT00694785 C0468 Ongoing NCT00466583 C0783 Ongoing NCT00728936 Trial Number Terminated Ongoing Ongoing Completed Completed Terminated Completed Completed Ongoing Ongoing Completed Terminated Completed Ongoing Ongoing Completed Outcome [email protected] [email protected] VasGene UPMC CancerCenters rnesMrae optlGI24 Prostatecancer I GTI2040 Princess MargaretHospital OSI Pharm(Eyetech) OSI Pharm(Eyetech) OSI Pharm(Eyetech) Bevasiranib Opko Health(formerly Acuity) Bevasiranib Opko Health(formerly Acuity) OncoGenex Technologies BreastCancer Oncogenex Kaposi'ssarcoma II Center Norris ComprehensiveCancer I/II OGX-011 NeoPharm VEGF-AS Melanoma National CancerInstitute I National CancerInstitute MethylGene CpG7909 MethylGene Ludwig InstforCancerRes Lorus Isis/Lilly Isis Pharmaceuticals Isis Isis Isis Isis Isis AVI-4658 Inst. forDrugDev. Imperial CollegeLondon nvriyo enyvnabsla I Leukemia II University ofPittsburgh Prostatecancer busulfan University ofPennsylvania II University ofPennsylvania Univ. OGX-011 ofPennsylvania Lymphoma University ofChicago University ofBritishCol. II Genasense Topigen Southwest OncologyGroup Southwest OncologyGroup in frel ioye IN-2 Maculardegeneration I SIRNA-027 Sirna (formerlyRibozyme) Santaris Company vaccine dendritic cell suppressive Diabetes- elnI Cancer II Melanoma I Veglin CpG 7909 aue IRtnlVi clso VEGF VEGF RetinalVein Occlusion II DiabeticMacularEndema II/III Macugen N/A Macugen Macugen Tumors (Cand5) I (Cand5) Cancer I/II OGX-427 Cancer OGX-011 (VEGF-AS) I Veglin™ LErafAON Hematological cancer Solidtumors I I/II Renalcellcarcinoma MGCD0103 MGCD0103 Cancer II Crohn'sDisease II GTI-2040 III LY2181308 HighCholesterol ISIS 2302 II Diabetes ISIS 104838 II Ulcerativecolitis ISIS 301012 II CMVretinitis II ISIS 113715 N/A Alicaforsen I Vitravene GTI 2040 (PMO) ODN c-myb AS LungCancer I/II c-myb AS Allergicasthma Genasense II TPI-ASM8 EGFR AS P29 /ICrnclmhct ekmaBcl-2 Chroniclymphocyteleukemia I/II SPC2996 Compound Type 1Diabetes I II III Mesothelioma I/II Duchennemuscular I/II eaooi ainnisc-myb HematologicMalignancies I Cancer II Cancer III Phase Neovascular AMD Neovascular AMD Neovascular AMD Rheumatoid Arthritis Solid Tumors dystrophy Disease D0 D0adC8 nies hshrtiaeNT0493Ongoing NCT00445913 Phosphorothioate Antisense CD40, CD80andCD86 VEGF isoforms CD8+ T-cell angiogenesis Phosphorothioate clusterin secretory protein Antisense vascular endothelialGF Melan-A VEGF VEGF VEGF Hsp27 clusterin secretory protein c-raf Phosphorothioate HCAC Antisense HCAC R2 compofRNR survivin ICAM-1 TNF-alpha apoB-100 PTP-1B ICAM1 IE2 dystrophin c-myb c-myb Unknown Bcl-2 EGFR bcl-2 GM-CSF CCR3, ILreceptor3&5, VEGFR-1 Ribonucleotide Reductase Target 800-863-6801 Oligonucleotide Applications nies Phosphorothioate Antisense Unknown Immune-Active nies hshrtiaeNT0649Ongoing NCT00668499 Phosphorothioate Ongoing NCT00258375 Antisense MixedChemistry Antisense Unknown Immune-Active Aptamer Aptamer Phosphorothioate Antisense Aptamer Ongoing siRNA Unknown NCT00327340 siRNA MixedChemistry Antisense Antisense Unknown Ongoing Antisense NCT00511576 Ongoing NCT00372437 MixedChemistry MixedChemistry Ongoing Antisense NCT00642018 Ongoing Antisense NCT00048295 MixedChemistry Ongoing Phosphorothioate NCT00362180 Ongoing MixedChemistry Antisense NCT00455598 MixedChemistry Antisense MixedChemistry Antisense Phosphorothioate Antisense Phosphorothioate Antisense Ongoing Antisense NCT00159250 Antisense Phosphorothioate Antisense nies Unknown Ongoing NCT00002592 Unknown Antisense Ongoing Phosphorothioate NCT00138918 Antisense Antisense Mixed Chemistry Ongoing NCT00080847 Ongoing Antisense NCT00049543 Phosphorothioate Phosphorothioate Antisense Antisense ntes hshrtiaeNT0002Ongoing NCT00005032 Phosphorothioate Ongoing NCT00550797 Anitsense Phosphorothioate Antisense siRNA nies hshrtiaeNT0813Ongoing NCT00285103 Phosphorothioate Antisense nies Phosphorothioate Antisense Mode of Action Phosphorothioate Phosphorothioate hshrtiaeNT0545Ongoing NCT00354445 Phosphorothioate RNA RNA RNA Chemistry C0417 Ongoing NCT00471471 C0122 Ongoing NCT00112229 C0499 Ongoing NCT00499590 Ongoing NCT00487786 Ongoing NCT00100672 C0705 Ongoing NCT00780052 Ongoing NCT00002592 Trial Number Completed Completed Completed Completed Completed Completed Completed Completed Completed Completed Completed Completed

Outcome 123 Information Technical 124 Technical Information dard phosphorothioate oligonucleotides, orone ofthemore oligonucleotides, dard phosphorothioate ments willrequiregramsofmaterial. If you areusingstan- go through50mgmuchmorerapidly.obviously Laterexperi to micethatnormallyweigh0.02 kg. However, a ratstudywill At a common doseof5mg/kg,canbemade 500 inoculations rodent studycanbeconductedwith 50mgofoligonucleotide. requirements willnotbeextensive. A fairlycomprehensive Initially,a reasonablequantityofyouroligonucleotide. your new setof concerns. First, you mustbeassuredofobtaining Moving to case activitychanges,whichisnotuncommon. sample toin compare withnewbatchesofoligonucleotide the construct. The extramaterialwillalsoallowyoutoretain on depending erally yield1to2mgofpurifiedoligonucleotide, a1 extensive experimentation, a 200nmolescalesynthesisismorethanenough.For 5 to10ODs(~150-300 is required.Usually mentation ishowmucholigonucleotide in somemannerthatchangesuptakeproperties. to makesurethat gonucleotide thecelllinehasn'ttransformed test periodically yourdeliverysystemusingafluorescentoli- atactivity.you aretrulylooking It is actuallyagoodideato can testyourantisensesequenceswiththeconfidencethat ments withoutdeliverysystems(Shoji,etal.,1991). fate of most usedinthe earlyexperi of theoligonucleotides which wasthe sequestering, endoplasmic merely observing is visibleinthecytoplasminstead,thenyouare fluorescence) tive of proper delivery. If a punctatepattern(isolatedspotsof cells shouldhavefluorescenceinthenuclei. This isindica is uptakeintothenucleusofcells. A fairproportionofthe fixed, thenviewedunderthemicroscope. The desiredeffect as 1to3micromolarsolutions. The cellsareharvestedand the manufacturer'sinstructions. to thecells These areapplied cleotide ismixedwiththecationiclipidmixture(s)accordingto 1991; Sasaki,etal., 1995). oligonu The fluorescentlylabeled isfairlystraightforward (Shoji,etal., oligonucleotide labeled for thispurposefromcommercialsources. are available with mixed basecompositions oligonucleotides cently labeled compound isrequired. fluores- inexpensive As analternative, the oligonucleotide. of your Therefore aseparatepreparation mustbeintroducedduringthesynthesisof orescent molecule will servewell.Pleasenotethattheflu- cein, thatfluorophore with filtersforfluores- fluorescent microscopescomeequipped added, thisdoesnotappeartoaffect uptake. Sincemost you intendto use. must be Although afluorescentmolecule that mattersis the constructionissameaswhat 1995). isrelativelyunimportanthere. The sequence All Sasaski, to observeuptake(Noonberg,etal.,1992; and fluorescencemicroscopy oligonucleotide cently labeled is occurringwithyourdeliverysystembyusingafluores- uptake if goodcellular assay istodetermineconclusively ous lipidmixturesforthispurpose. system. To makeit easier, thatcontainvari- kits areavailable tofindtherightdelivery to havedosomeexperimentation construct, own celllineoroligonucleotidethenyouaregoing can befoundthatworks.However, if your you areexploring highly likelythatamongstthematleastoneuptakesystem construct. cationic lipidmixturetofit your celllineandoligonucleotide particular cellline. The fact is thatyouhavetohandtailorthe to optimizedeliveryintoyour isrequired cific concentrations yet, sometimes amixtureofdifferent cationiclipidsatspe- lipid thatworksforallcelllinesandwithconstructs. Worse Oligonucleotide Applications When youstart your invivostudiesyouhaveawhole One lastquestiontoanswerinregardvitroexperi- Once youhavefoundasatisfactorylipidmixture, The experimenttostudytheuptakeofyourfluorescently a successful The bestwaytobegindeveloping cationic lipidsexist.It availableis Many commercially In Vivo Experiments mg) whichisreadilyobtainedfrom mmole scalesynthesiswillgen- www.trilinkbiotech.com in vitro et al., - - - - to answeryourquestions. out therewilling that thereareplentyofpeople the knowledge your chanceswillbegoodinthelongrunand with knowing prefer todoityourself,thengoforward withtheconfidenceof worth thepriceifyourgoalsarecommercial innature.If you more atfirst, but theexpertiseyoupurchaseisusuallywell your antisenseassays.It you developmaycost you helping in existencethat there arecompanies are inthebusinessof an true ifyouaredeveloping you standagoodchanceofsucceeding. This isespecially cleotides, ifyoupayattentiontothosewhohavegonebefore, Final Words ing aboutthesecondandthirddrugsinyourpipeline. cult. However, you shouldnowbewellonyourwayandthink- next stageofinvestigations. rate to followthedegradation ties willbeneeded on thesensitivityofMoresignificantquanti- your equipment. nation, aslittle1to5 circulatory lifetimeandurinefecalelimi study regarding they canalsobeobtainedcommercially. Foraverysimple tritium (hydrogen-3). These canbemadein-house,although et al., 1994). The mostaresulfur-35and common isotopes ., 1991; Cossum, isotope (Agrawal,etal.,1991; with aradioactive test labeled subject withanoligonucleotide studies. of the These animalexperimentsrequireinjection of anantisensetherapeuticistheneedforpharmacokinetic course istojustuseasalinesolution. company. Untilsuchadeliverysystememerges,yourbest technology byInexofCanada,adelivery tic oligonucleotide merging companies, suchasthepurchaseofLynx's therapeu higher successrate. The mergingneeds,infact, have ledto specified tissuescanonlybeadvantageousandleadtoa belief thatagoodmethodofsystemicdeliveryto universal trials usingnodeliverysystem, ongoing thereisstillafairly (Agrawal, enduring. Oraldeliveryhasalsobeenexamined imagine ocular andotherlocationsevenmoredifficultincluding to merely saline. invariousways, The solutionsareinjected for yourcellwork.Most systemyousoarduouslydeveloped throw outthedelivery - truerationaldrugdesign. the HolyGrailofdrugdevelopment for themechanismofactionlaterwithhopediscovering towards adrugproduct. You canalwayscontinuethesearch The practicalcourseistoacceptpositiveresultsandcontinue If the compounddoeswhatitwasmeanttodo,whyargue? by adherentstothe"Sowhat?"schoolofthought. championed mechanism. Onestrongargumentthatisverypersuasive 1997) andisthebestwaytobeconfidentinanantisense mRNA andproteinproduct.Still,ithasbeendone(Monia, is difficult tolocateandquantitatethereductionofbothtarget It This hasbeenatopicofcontroversyfromthebeginning. of yourexperimentsandproveyouhaveanantisensedrug. vendor. source orfromanexternal rial, whetheritisfromanin-house to paygoodmoneyformate- taken. Bepickyandwilling contaminants isveryeasyto avoid ifproperprecautionsare tant tobesureofthequalityyourmaterial. Toxicity dueto fee. manufacture forareasonable to a drugonlytofindoutitisnextimpossible developing rather thanlater. millions Nothingisworsethanspending scaleupissuesearlyintheprogram, be suretoinvestigate cant concern.However, if yourconstruct is fairlycomplicated, supply willnotbeasignifi common chimericoligonucleotides, Regardless of yourintended useforantisenseoligonu Regardless The roadtoFDA approvalfromhereisstilllonganddiffi- Another setofexperimentsuniquetothedevelopment Another concern,orperhapsarelief, is thatyou can A verysignificantconcernishowtointerprettheresults Along withquantitygoesquality. It is evenmoreimpor- et al., 1995). Despitetheapparentsuccessof some mCi permousewillsuffice, depending oligonucleotide solutions are in vivooligonucleotide in vitroassay. If all elsefails, ., 1993; Iverson, et al.,1993; in vivothe - - - - [email protected] [email protected] 26. 25. 24. 23. 22. 21. 20. 19. 18. 17. 16. 15. 14. 13. 12. 11. 10. 9. 8. 7. 6. 5. 4. 3. 2. 1. References Biophysica Acta, 201-208. Biophysica Acta, I., Syrjanen, K., and Syrjanen,S. (1994) Biochemicaand K., Urtti, Lappalainen, E., Jaaskelainene, A., Soderling, (1986) Nucleic Acids Research, 14,9081-9093. B., Stec,W.J.,Uznanski, M.F., Summers, G. Zon, and L.A., James, LaPlanche, T.L.,W.D.,C., Wilson, Powell, Research and 131(Antisense Application), 243-262. Krieg, A. M. Handb.Exp.Pharmacol, (1998) 8, 181-184. Klinman, D.M. (1998) Antisense Nucleic Acid DrugDev., and Development,4,43-52 Antisense Research Iversen, P.L., Mata, J., Tracewell, W.G., & Zon,G. (1994) (1990) JournalofChemistry,Biological 265, 5561-5566. R.I., Walder,H.H., Hogrefe, Hogrefe, R.Y., Walder, J.A. Science, 258,1481-1485. D.A., Boyd, L.E. (1992) Babis, S.A., and A.L., Noble, M.A., Bonham, K.G., Carter,Au, S.G.,Bruckenstein, R., Josey.Cadilla, C.F.,D.J., Hassman, J.A.,Ricca, Hanvey, J.C.,Peffer,J.E., N.J.,Bisis, S.A., Thomson, Gura, T. (1995) Science,70,575-577. Society,Chemical 116,3143-3144. Gryaznov, S., and Chen,J.K.Journalof (1994) American 20, 763-770. Giles, R.V., & Tidd, D.M.(1992) Nucleic Acids Research, Acids Research.,17,9193-9204. Furdon, P.J., Z., Domininski, andKole,R.(1989)Nucleic 1608-1613. and Experimental Pharmacology Therapeutics, 275, J.P.,Desjardins, P.L.Iverson, and J. of (1995) USA, 91,11762-11766. Dean, N.M., and McKay, R (1994)Proc.Natl Acad. Sci. P. (1987) Proc.Natl Acad. Sci.USA,84,7896-7900. Dash, P., Lotan, I., Knapp,M., Kandel E.R. and Goelet, 267, 1181-1190.Therapeutics, and Crooke,S. (1993) JournalofPharm.and Exper. Cummins, L., Owens, S.R., Markham, P.M., Shea, J.P. P.A.,Cossum, Sasmor, H.,Dellinger, D., Truong,L., J.S. (1989) Cohen, 435-437. TIBS, 10, Comm., 197,818-825. and Quattrone, A. (1993) Biochem. And Biophys.Res. E., Mazzei, G.D., Mini, S., Pasquale, Capaccioli, T, Neurobiology,Molecular 557-568. 14, Brysch, W., K.and and Schlingensiepen, (1994) Cellular (1994) J. Biol. Chem., 269, 26801-5. M. I. X., Nerenberg, S., Xu, O., Sullivan, L., Heidenreich, S.H., Gryaznov,D.A., Kang, Brown, S.M.,DeDionisio, 7, 369-380 Toulme, J.-J. (1997) Antisense Nucleic Acid Drug Dev., F.,R., Duconge, E., Mishra, C., Dausse, Boiziau, & Nucleic Acid DrugDev., 7, 461-471. Villiet, P., Bennett, C.F., & Monia,B. P. (1997) Antisense R.T.,Boggs, McGraw, K.,Condon, T., Flournoy, S., 6139-6145. M.P., Ts'o,P.O.P., Miller, & P.S.Biochemistry, (1985) 24, Blake, K., Murakami, A., Spitz, S.A., Glave,S.A., Reddy, Pharmacology, 50, 571-576. Z., Iyer, R.P., Yu, D. and Zhang,R.(1995)Biochemical J.M., Yan,I., Jiang, R.B., Habus, H., Diasio, J., Cai, Z., Zhao,H., X., Lu, S., Zhang, Agrawal, Tamburin, National Acad. Sci., 88, 7595-7599 Agrawal, S., Temsamani, J. & Tang, J.Y.(1991) Proc. 3539-3542. Letters., 28, J. (1987) Goodchild, S., and Agrawal, Tetrahedron Research., 6,3009-3023. Agrawal, K., and Riftina,F. (1979) Nucleic Acids 52. 51. 50. 49. 48. 47. 46. 45. 44. 43. 42. 41. 40. 39. 38. 37. 36. 35. 34. 33. 32. 31. 30. 29. 28. 27. Lett., 8,3269-3274. Zhou, W., and Agrawal, S. (1998) Bioorg.Med.Chem. Proc. Natl Acad. Sci.USA,89,7305-7309. Woolf, T.M.,C.G.B. (1992) Jennings, D.A., and Melton, Sci. USA, 85, 5011-5015. Walder, R.Y.,Walder, and Proc. Natl J.A.(1988) Acad. 16,971-972. Nucleotides, Nucleosides D., Sanghvi, B., Peoc'h, E.E., Bhat, Swayze, Y. S.(1997) Acad. Sci.USA, 75, 1302-1306. Stephenson, M.L.,andZamecnik,P.C. (1978) Proc.Natl Stein, C.A., (1995) NatureMedicine,1,1119-1120. J.S. (1988) Nucleic Acids Research.,16,3209-3221. Stein, C.A., Subasingfe, C., Shinozuka, K., and Cohen, 129-137. 9, Analysis, Laboratory Clinical Srinivasan, S. K., & Iversen,P. L. (1995)Journalof (1996) Proc.Natl. Acad. Sci.U.S. A., 93, 12840-12844. M. J., H., Sambade, Sierakowaka, R. S., &Kole, Agrawal, 5550 Juliano, R.L Nucleic (1991) Acids Research,19,5543- Shoji, Y., Akhtar, S.,Periasamy, B., & A., Herman, 17, 103-4 Kobayashi, M., & Fujita,H.(1995)Photomed., Photobiol., M., Watanabe,Sasaki, M., Kawakubo, Y.,M., Komiyama, L.J.(1996) Nucleic Acids Research.,24,4584-4591. M.M., Beck, T.A.,S.K., Klem,R.E.& Knowles, Arnold, D.A., Riley, T.A.,W.B., Marvin, Daily,W.J., Vaghefi, R.I., Jaeger,M.A., Hogrefe, Reynolds, J.A.,Schwartz, Biochem. andBiophys.Res.Comm.,218,930-933. G., Rebowski, Stec, W.J.R., and Amerakoon, (1996) Ramasamy,K., R., Misiura, R.,Kanagaratnam, (1998) Mol.BrainRes.,62,1-11. S., Livanov,Ho, Peng V.,W., Zhang, J., Lesher, Li, T. Biochemica, 1,25-29. Quattrone, S. (1995) Capaccioli, G.D., and A., Pasquale, Hunt, C. Anthony (1992) Antisense Res.Dev., 2, 303-13. Noonberg, SarahB., Weiss, Tania L., Garovoy, Marvin R., 118, 255-256. Bellon, L., (1996) Journalof Society,American Chemical F.,Morvan, Sanghvi, Y.,Vasseur,M., Perbost, J.-J., 107-123. Therapeutic Agents), as (Oligonucleotides Monia, B.P. (1997) CibaFound.Symp., 209 Freier,D., & 14514-22. Chem., 268, J. Biol. (1993) S.M. J., Kawasaki, C. D., Guinosso, McGee, P.A. M.,Cook, B.P.,Monia, E. Lesnik, W.F., C.,Lima, A., Gonzalez, Miller, P.S.Bio/Technology, (1991) 358-362. 9, 15553-15558 Davis, H.L.(1998)Proc.Natl. Acad. Sci.U.S. A., 95, C.L.B., Weeratna,Millan, R., Krieg, C., A. M.,Siegrist, 14, 6433-6451. Minshull, J., and Hunt, T. (1986) Nucleic Acids Research, Research, 26,431-438. Mignet, N., and Gryaznov,S.M. (1998) Nucleic Acids Med. Chem.Lett., 4, 2929-34. Metelev, V.,J., and Lisziewicz, Bioorg. S. (1994) Agrawal, 448-452. Matzura, H., and Eckstein,F. Eur.(1968) J. Biochem., 3, 7706–7710. virus, Proc.Natl immunodeficiency Acad. Sci.USA,84, and cytopathiceffectsof replication inhibitors of human analogs ofoligodeoxynucleotides: Phosphorothioate J.S., Broder, M., Cohen, H., Reitz, S.(1987) G., Mitsuya, K., Zon, M., Shinozuka, Matsukura, 800-863-6801

Oligonucleotide Applications 125 Information Technical 126 Technical Information there areB A SyntheticChallenge as possible,andarereproducible aspossiblefromlottolot. all theseapplicationsisthattheoligonucleotides areasrandom needed todeterminethesequence. A commonrequirementof random inselectsites,reducingthe numberofcompounds diagnostic implications. chromosomes canbedetected,theresultsofwhichhave to mapdifferences inchromosomes.Suchdifferences inthe assays may, forexample,usedyelabeledrandomprimers randomers intheirmicroarrayassaysystems(1). These the twoplotsdeviatedfromeachother(3,4,5,6). polymorphism wasdetectedifthefluorescenceintensityfrom were analyzedwithamicroarray, yieldingaplotofdata. The randomers, butwithdifferent dyes. The labeledamplicons sample andaWT controlwerebothamplifiedusingthe system coupledwiththeirmicroarraydetectionsystem. The polymorphism usingatwodyenonamerrandomerprobe developed anassaythatcoulddetectasinglebasemismatch for short.OnesuchassaywasdesignedbyIncyte. They oligonucleotides containingdegeneratesites,or“randomers” of commercialdiagnosticassaysweredevelopedusing of degeneratesitescontainingallfournucleosides. A number amplification isdonewitholigonucleotidescontaininganumber generate apoolofalltheavailablesequences. This random sites istherandomamplificationofagenetictargetto has thecodeforproteinofinterest. mixtures ofallpossiblebases,inordertoidentifyagenethat Therefore theprobemustalsohavedegeneratesites,or potential codonsthatresultinthecorrectaminoacidsequence. for thegeneisnotknown,thenitnecessarytoprobeall a degeneratesiteinthegeneticcode.Ifspecificsequence still correctlyencodefortheaminoaciddesired. This iscalled of whichcanoftenconsistseveraldifferent nucleosidesand trinucleotide codon,thethirdandsometimessecondbase gene ofaparticularprotein.Eachaminoacidisencodedby probe studiesusingsyntheticoligonucleotidestohuntforthe oligonucleotide chemists. The needoriginatedfromearly “degenerate” sites,havelongbeenasynthetictargetfor base. These sites,knownaseither“random”,“wobble”or in oligonucleotidescontainingsiteswithmorethanone Introduction Oligonucleotide Applications bases (4iftotally degenerate)andn=thenumber ofsitesthat By RichardHogrefe, Ph.D., Terry Beck, NatashaPaul,Ph.D.and Alexandre Lebedev, Ph.D.; TriLink BioTechnologies Randomer Oligonucleotides The syntheticproblemsstemmainly fromthefactthat In anotherapplicationofrandomersthesequenceisonly Other companieshavealsoemployeddyelabeled Another applicationforoligonucleotideswithdegenerate There hasbeenarecentresurgenceintheinterest n sequences in the mixture, where B = the number of B =thenumber where the mixture, in sequences 5’ 5’ 5’ . . Reverse transcriptase or transcriptase Reverse Random PrimerLabeling DNA polymerase DNA Random Primer Labeling primer) random nonamer labeled (Cy3/Cy5 www.trilinkbiotech.com mixture inthisexperiment.) ratios ofthebaseswerenotoptimizedtosynthesizea1:1:1:1 different ratiosofeachbaseat1and15μmolescales.(The same mixofreagentsinthemachinegaveconsiderably column, liketheExpedite8909.Incaseshownbelow, the instruments thatuseasingle,gasdrivenflowthroughthe the different molecularweightsofeachbase. ratio youdesireinasinglebottle.Besuretotakeintoaccount ratio ofbases.Itismuchbettertopre-mixthebasesin caused byrestrictionscancausemajorchangesintheresulting sequentially. Any deviationinflowratebetween reagentbottles of therequirednucleosidephosphoramiditestocolumn ill-advised touseit.Mostmachinesapplypulsesofeach sites consistingofmixes2,3orall4bases,youwouldbe synthesizers thatwillallowforthesynthesisofdegenerate The SynthesisStep HPLC toyieldtheratioofeachbaseinoligonucleotide(2). yielding thefreenucleosideswhicharequantifiedusingRP- venom phosphodiesterase(SVPD)andalkalinephosphatase, digesting theoligonucleotideusingacombinationofsnake Determining BaseComposition manufacture areproducible,highqualityproduct. changed ateachstep,requiringextremecareinorderto processing. The randomnessofthesequenceispotentially followed byconjugationtothedye,repurificationandthenfinal synthesis oftheoligonucleotide,preliminarypurification, throughout theprocesswhichincludesstep-wisechemical maintaining areproduciblerandommixtureofsequences fragments, chemicalmodifications,andextradye,whilestill randomer oligonucleotidethatishighlypurifiedofshorter homopolymers, willhavevastlydifferent characteristics. properties duringmanufacturing.Some,however, likethe bases inlength.Manyofthesequenceswillhavesimilar sequences; 262,144different individualoligonucleotides,nine are random. Thus anonamerrandomerisactually4 Scale playsabiggerroleindeterminingtheratio Although thereisasubroutineonmostcommercialDNA Determining basecompositionisaccomplishedby The challengeliesinmanufacturingadyemodified

Composition HPLC Analysis ofSVPD/CIAP Digestion 0.85 0.95 1.05 1.15 0.8 0.9 1.1 1.2 H H 1 P P L L 0 C C Cd TdA dT dG dC A A ddAdA dA ddAdA dC dG dT dA n n a a l l y y s s i Time (min) i s s o o f f S S 5 10 V V P P D D / / C C I I A A P P D D i i g g e e s s t t i i 15 umole 1 umole o o n n 9 different another fractionalpurificationas described above. conjugation ispurificationtoremove theexcessdye,whichis there isnopreference.However, thestepimmediately after reaction wentfrom20%to100% completion, suggestingthat composition oftheoligonucleotide astheefficiencyof product changed.We foundverylittledifference inthebase to seeifthebasecompositionof the resultingdyelabeled oligonucleotide atvariouspointsoftheconjugationreaction reactive thantheothers. the ratioofbases,especiallyifoneormorebasesare nonamer. We wereconcernedthattheconjugationwouldskew a succinimidylactivateddyeisattachedtoanaminolabeled inadvertently changedisduringtheconjugationstepwhere The ConjugationStep product. good tominimizesideproductsandallowmaximalrecoveryof purifying byHPLC. To doso,thesynthesismust bevery guanosine ratiothroughtheelutionprofile. throughout thefractions,equallysharingchangein adenosine, cytosineandthymidineratioswereessentiallyeven peak showedalackofthesequencesbearingguanosine. The guanosine nucleosidewasenriched,whilethebackof fractionated byRP-HPLCasshowninthefigurebelow. yielded anoverallbasemixturecloseto1:1:1:1was across anHPLCpeakanonamerrandomerthatoriginally of thedesiredproductistoberetained. contaminant impossibleifcompleteintegrityoftherandomness side-product elutionprofilemakingabsoluteremovalofthat often theelutionprofileofproductoverlapsthatn-1 much broaderthanadiscreetoligonucleotideproduct.Infact, different sequences,theelutionprofileofdesiredproductis Unfortunately, inthecaseofrandomers,whichcontainmany necessitating tightcutsinordertoobtainhighqualitymaterial. These contaminantsofteneluteclosetothedesiredproduct, groups stillintact,andinadvertentlymodifiedoligonucleotides. incomplete synthesis,oligonucleotideswithsomeprotecting most commoncontaminantsincludeshorterfragmentsfrom to thedegreerequiredachievedesiredquality. The is purifiedundergoesselectioninordertoremoveimpurities come fromthepurificationstep.Eacholigonucleotidethat The PurificationStep best determinedusingempiricaldata. supports neededtoprepareaperfect1:1:1:1ratioofbasesis ensure uniformitythroughoutthebatch. The propermixtureof 1:1:1:1 aspossible,andthatthesupportitselfiswellmixedto the solidsupport.Itisimportantthatmixtureascloseto [email protected] [email protected] We testedthatconceptbyremovingaliquotsof The nextstepinwhichtheratioofbasescanbe It isveryimportanttoincludetheentirepeakwhen The resultsshowthatinthebeginningofpeak To determineiftherandomness ofthemixturechanges A muchlargerpotentialimpactonlottovariabilitycan Another considerationisthestartingratioofbaseson f f f 1 7 1 f f 2 2 f 3 f Tim 3 f 4 f 4 f 5 f e 5 f 6 f 6 f 7 . Nucleic Acids Research.(2001)29:e41. 6. JournalofBiology. (2006)5:3. 5. GenomeBiology. (2004)5:R19. 4. TheJournalofNeuroscience.(2004)24:2866. 3. Wiley, JohnandSons.CurrentProtocols.Nucleic Acid 2. ProceedingsoftheNational Academy ofScience.(2006) 1. References oligonucleotide synthesis. can workonyournextexperiment,insteadof oligonucleotides from TriLink, ofcourse. That wayyou 6. Purifyallofthefulllengthmaterialwithoutcuttingout 5. EnsureyourDNA synthesizerisrunningverywellto 4. Prepareyouramiditemixturesinonebottleandtestthe 3. Prepareyoursupportbymixingitverywell,andtest 2. Bringanassayin-housesuchasthedescribedenzymatic 1. oligonucleotide, itisnecessarytotakethefollowingsteps: Summary Chemistry. (2000)10.6.1-10.6.6. 103: 4534. A moresimplesolutionwouldbetobuyyourrandomer to removeexcessdyeandmaximizeyield. Ensure acleanconjugation,purification,desaltingprocess synthesis. side fractions,hencetherequirementforanearperfect ensure highcouplingefficiency. overall variationinbaseratio. coupling ofthepreparedmixturestoensurelessthan10% Adjust accordingly. random aliquotstoassessbothmixingandthebaseratio. digestion assaytotestratios. 800-863-6801 In ordertoprepareasuccessfulrandomer

Oligonucleotide Applications 127 Information Technical 128 Technical Information on the companion fluorescent dyechosen. on thecompanion modified atthe5position. There aresomelimitationsbased or 5'terminusinternallyat any deoxybase,which canbe oligonucleotide. These quencherscanbeplacedatthe3' we alsooffer awideselectionforplacementonthe however, if yourexperimentsrequireoneortheother. chemist thantotheenduser. We doallowyoutospecify, of theconjugateandismoreconcernto solubility listed in Table 2. application. The more popular dye/quenchercombinationsare quencherforyour youdeterminetheappropriate 1 willhelp - atlittleornoaddedcost. quenchers more specialized Table one cannowchoosefrom publications, Beacon Molecular synonymous withtheterm“quencher”becauseofearly dyes suchasCy5.5. Although DABCYL hasnearlybecome blue dyessuchasPacificBluetotheextremelyred-shifted quenchers areanalogsoffluorescein. fordifferentspecific quenchers emissionranges. The QSY series andtheBlackHoleQuenchers(BHQ) may notbere-sold,distributed orre-packaged. for R&Dusebythepurchaser.under agreementwith Biosearchandtheseproductsaresoldexclusively or diagnosticpurposesand they They maynotbe used forclinical “Black HoleQuencher”, “BHQ-1”,“BHQ-2”and“BHQ-3”areregistered trademarksofBiosearch Technologies,Inc, Novato,CA. The BHQtechnologyislicensedand sold to were developedovercomethoselimitations,theQSY with thered-shifteddyes. Two differentsystems quenching became apparent. These quencherswerenotveryefficient and TAMRAof multiplexsystemsquickly forthedevelopment quenchers overthelastdecade. The limitationsofDABCYL Quenchers Technical Information Not onlydoes TriLink offer eachofthesequenchers, The difference betweenQSY-7 andQSY-9 iswater There isnowaspecificquencherforeverythingfrom Great advanceshavebeenmadeintheareaof

® . Both offer. Both www.trilinkbiotech.com ® Figure 1: Quencher Structures BHQ-3 QSY-21 BHQ-2 QSY-9 QSY-7 BHQ-1 QSY-35 DABCYL Table 1:Spectral Data of N O O H TAMRA O O O 672 nmn/a 660 nm89,000 579 nmn/a 562 nm85,000 560 nm92,000 534 nmn/a 472 nm23,500 Range Quenching 453 nm32,000 Coef. Ext. max Abs H N + O O H DABCY N N L Quenchers N 620-730 nm 590-720 nm 550-650 nm 500-600 nm 500-600 nm 480-580 nm 410-500 nm 380-530 nm N QS O Y- 7 TM SO 2 N N + H O O [email protected] [email protected] Table 2: This dye selection chart provides a quick reference to the dyes we provide, spectral ranges and offers a starting point for quencher-dye pairing. must beem * There isnoguaranteeaquencherwillbeeffectiveinyourcons truct. Quencher-dyeefficiencies Ore C C Texas-Red- TAMRA-X BODIPY 558 Carbox HEX 2' Ore dT-FAM 7-Aminocoumarin- BODIPY-564 6-FAM DTAF Cascade Blue BODIPY-530 6-JOE BODIPY 493 BODIPY-581 C A A A A A A A A A A A A A A 7-Methox P130 Carbox Carbox P BODIPY-TR- C BODIPY-576 Rhodamine Red-X TE Dans Dimeth Marina Blue Pacific Blue NBD- Rhodamine Green-X Ore Rhodol Green Dyes y y y y y lexa Fluor®750 lexa Fluor®700 lexa Fluor®680 lexa Fluor®660 lexa Fluor®647 lexa Fluor®633 lexa Fluor®594 lexa Fluor®568 lexa Fluor®546 lexa Fluor®555 lexa Fluor®532 lexa Fluor®430 lexa Fluor®488 lexa Fluor®350 , 4' T MPO 5.5 5 3.5 3 g g g on Green514 on Green488 on Green500 , y X 5' l-X ( p y y y y , Fluorescein na -X-Rhodamine rhodamine 6G iricall laminocoumarin 7-erbooufnfursen54m529nm 544nm 7'-Tetrabromosulfonefluorescein / y dT-TAMR p coumarin X hthofluorescein X / / / / / / y 568 503 591 589 570 550 determinedin X ) A ( ROX ) y our ex p erimental conditions Em 5n 535nm 556nm 492nm 520nm 1n 492nm 516nm 4n 520nm 548nm 7n 415nm 570nm 7n 649nm 670nm 550nm 570nm 1n 588nm 616nm 580nm 605nm 583nm 603nm 546nm 576nm 521nm 536nm 466nm 535nm 496nm 516nm 416nm 451nm 353nm 442nm 1n 335nm 518nm 6n 559nm 568nm 500nm 509nm 1n 396nm 410nm 358nm 410nm 2n 496nm 523nm 9n 581nm 591nm 576nm 589nm 564nm 570nm 534nm 551nm 5n 524nm 557nm 7n 340nm 376nm 7n 602nm 672nm 9n 675nm 694nm 588nm 604nm 8n 560nm 580nm 7n 749nm 775nm 702nm 723nm 679nm 702nm 663nm 690nm 650nm 665nm 632nm 647nm 590nm 617nm 578nm 603nm 556nm 573nm 555nm 565nm 532nm 554nm 434nm 541nm 495nm 519nm 346nm 442nm 2n 503nm 528nm 506nm 526nm 495nm 521nm 499nm 519nm 6n 376nm 468nm 5n 362nm 459nm max 800-863-6801 Abs . max Abs BHQ-1 579nm 534nm Abs BHQ-2 Quencher Guidelines max max : : Technical Information Abs QSY-35 661nm 560nm Abs QSY-9 QSY-7 & 475nm Abs QSY-21 max max max : : : 479nm Abs DABCYL max

* : 129 Information Technical 130 Technical Information polymerase replication, nA isselectivelyincorporatedopposite natural nucleobase withhighfidelity. Forexample,inDNA triphosphate shouldbereadilyincorporated oppositeits goal, thecorrespondingpseudo-complementary nucleoside replication ofthecorrespondingtemplate. To accomplish this nucleoside triphosphatesduringpolymerase-mediated incorporation ofthecorresponding pseudo-complementary Advancements towardsthisultimate goalhavefocusedon representative hybridizationtotilingmicroarraysispredicted. diminished forsequencescomposedofpc-nucleicacids,more As thedegreeofsecondarystructureshouldbesignificantly hybridization studies,suchasthosewhichemploymicroarrays. Figure 2depictstheadvantageofutilizingpc-nucleicacidsin be hinderedbysignificantsecondarystructure(12,13,14,15). a nucleicacidsequencetoitscorrespondingcomplementcan has foundutilityinanumberofapplications. property ofpc-nucleicacidsisadesirablecharacteristic,which between nA and T(6,7,8,9,10,11). This dynamichybridization presumably becausethreehydrogenbondscannowbeformed A:T basepair, andthenA:T pairismorestablethan A:T, base pairingstrengthofthe A:sT pairissimilartothatofan sulfur atomofsT. While the nA:sT basepairisunstable, the between theexocyclicamineofnA andthelargesizeof between nA andsT isunstablebecauseofthestericclash Crick basepairbetweenadenine(A)andthymine(T),the DNA, RNA,andPNA(1,3,4,5).IncomparisontotheWatson- extensively inanumberofpolymersystems,whichinclude (sT) (Figure1). This familyofbaseanalogshasbeenstudied is theonebetween2-aminoadenine(nA)and2-thiothymine DNA. be utilizedinmicroarraystudiesandthetargetingofduplex property ofpc-nucleicacidsisanattractivefeaturewhichcan to unmodifiednucleic-acids. Thedifferential hybridization intermolecular secondarystructuresandcanreadilyhybridize pc-nucleic acidshavediminishedintramolecularand strong basepairswithstandardbases(1,2).Consequently, analogs thatformweakbasepairswithoneanotherbut Technical Information By NatashaPaul,Ph.D.; TriLink BioTechnologies Nucleic Acids Pseudo-complementary represented by the dashed arcs. represented by arcs. the dashed are withinrepresented bold.Steric in clashes pair abase are complementary andthenatural corresponding nucleobases Differencescomplementary between Nucleobases. the pseudo- Figure 1: Proposed BasePairs Between Natural andPseudo- Base Pairs Between NaturalBase Between Pairs and Pseudo-complementary Nucleobases N N Adenine 2-Thiothymine : N N H H Watson-Crick Base Pair Literature precedencehasdescribedhowhybridizationof One exampleofapseudo-complementarybasepair Pseudo-complementary (pc)nucleicacidscontainbase (Stabilizing pair)(Stabilizing N N Adenine Thymine : H H (Stabilizing pair)(Stabilizing N N N N O O H H N N O S N N Pseudo-complementary Base Pair N N 2-Aminoadenine Thymine : N N H H 2-Aminoadenine 2-Thiothymine : N N (Stabilizing pair)(Stabilizing H H N N (Destabilizing pair) N N O O H H N N N N H H H H O S N N a) c) b) www.trilinkbiotech.com stranded DNA Figure 3. The of pseudo use technology. us ifyouareinterestedinlearning moreaboutthisexciting STTR granttostudypc-nucleicacids(25,26). Pleasecontact Gamper of Thomas Jefferson Universityandwasfundedbya hybridization propertiesofnucleic acids. development ofnovelapplications thatrelyonthedifferential their naturalcomplement,thereisagreatpotentialforthe sequences withoneanotherandstrongercomplexes formation. As pseudo-complementarysequencesformunstable detection andnon-standardnucleicacid-basedstructure number ofpotentialapplicationsrangingfromnucleicacid in pc-nucleicacidsisanattractivecharacteristicwithalarge duplex DNA(24). endonuclease activity(22,23),andsite-specifichydrolysisof inhibition oftranscriptioninitiation(4),modificationrestriction has beenutilizedinanumberofapplications,whichinclude affinity. Theabilityofpc-PNA toperformdoubleduplexinvasion and thetwoPNA strandswouldbindtooneanotherwithhigh would notbeveryefficient,asthedsDNA wouldremainintact natural nucleobasesattachedwereused,theduplexinvasion a stabledoubleduplexinvasionstructure.IfPNAswiththe to targetduplexDNA (Figure3)(4). The resultantcomplexis of PNAsinvolvestheusepseudo-complementary complementary DNA strand(21).Oneinterestingapplication polyamide sequencesthatformverystableduplexeswitha nucleic acidhybridization. obtained, allowingforfurtheradvancestowardsmoreuniform et al.from Agilent(20). Overall,encouragingresultshavebeen as wellwithinarecentpatentapplicationfiledbySampson recorded inanumberofpublishedmanuscripts(3,16,17,18,19) replication(16,17,18). Work withpc-nucleicacidshasbeen establishing thefidelityofrecognitioninDNA polymerase thymine andsT isselectivelyincorporatedoppositeadenine, nucleic acids nucleic prepared for with sequences moreuniformsignal much pc- microarray to atiling acids reveals a Crick andpc-nucleic Expectedstructure. c) hybridization performance of Watson- displaysprepared reducedsecondary withacids pc-nucleic degreehigh of secondary structure. b) The sequence, same exhibiting sequence a hybridization. a) Natural Watson-Crick Figure 2. Effect of secondaryacid structure onnucleic TriLink iscurrentlycollaboratingwithProfessorHoward Overall, thesignificantreductionofsecondarystructure Peptide Nucleic Acids (PNAs)arenoncharged c b d a f e - complementa i

Hybridization signal j g 100 h 20 40 60 80 0 bdfh j abcdefghi Position on sequence on Position ry (PNA) PeptideAcid Nucleic to target double sequences a bcdefghij Complementary Psuedo- Watson-Crick 6 Nucleic Acids Research(2008)36,6999-7008. 26. Nucleic Acids Research(2008)36,3409-3419. 25. Nucleic Acids Research(2004)32,e153. 24. Nucleic Acids Research(2003)31,3929-3935. 23. EMBOReports(2002)3,956-961. 22. Science(1991)254,1497-1500. 21. USPatent Application NumberUS2004/0086880 A1 (May 20. RNA (2005)11, 1441-1447. 19. Journalofthe American ChemicalSociety(2006)128, 18. Nucleic Acids Research(2005)33,5640-5646. 17. Nucleic Acids Research(1995)23,885-892. 16. Biochemistry(1991)30,10606-10613. 15. Nucleic Acids Research(1998)26,4975-4982. 14. Journalofthe American ChemicalSociety(2001)123, 13. JournalofMolecularBiology(1987)197,349-362. 12. 11. BiochemicalBiophysicalResearchCommunications 10. Nucleic Acids Research(1988)16,305-317. 9. Biochemistry(1984)23,6723-6732. 8. Biochemistry(1983)22,597-600. 7. FEBSLetters(1990)274,103-106. 6. RNA (2005)11, 1441-1447. 5. ProceedingsoftheNational Academy ofSciences(1999) 4. Biochemistry(2004)43,10224-10236. 3. Nucleic Acids Research(1996)15,2470-2475. 2. Biochemistry(1996)35,11170-11176. 1. References [email protected] [email protected] Biochemistry(1996)35,11170-11176. 6, 2004). 396-397. 10805-10813. (1984) 118, 848-853. 96 11804-11808. 800-863-6801

Technical Information 131 Information Technical 132 Technical Information Technical Information to ayieldof0.571 μmole(100/175). the smallscalesynthesesareconventionallyreportedinOD reported inunitsofmass,milligramsorgrams. The yieldsof from alllargerscalesyntheses(100μmoleandabove)are required. The yieldreportedasOD all reactionsareexpressedintermsofmolesreagents representation oftheyield,itisbyitselfnotveryuseful. Almost Conversions likewise reportedinunitsofmassforsimilarreasons. simply becauseitistheconvention. The yieldsontheCOA are and thereforeisnotanaccuraterepresentation. uses anapproximation,aswillbediscussedinthenextsection, accurate representationoftheyield. The conversiontomass number isreportedastheyieldonCOA asitisthemost and theabsorbanceat260nmdetermined. This absorbance the weight. The oligonucleotideisdissolvedinwaterorbuffer water andotherresidualsofthesynthesisthatwouldaffect to measuretheyieldofoligonucleotidessinceitignoressalt, not possible.UVspectroscopyisamuchmoreaccurateway product, obtaininganaccurateweightonroutinesynthesesis orders areplacedatscalesyielding0.05-10milligramof scale, aremeasuredforyieldusingabsorbance.Sincemost units. in opticaldensityunitsmeasuredat260nm(OD in oneoftwoways.Itwillbereportedeitherunitsmassor Measuring Up process canaffect thefinalyieldofaproduct. of howyieldismeasuredandreportedeventhat discussed shortly, butfirstitmayhelptohaveabriefoverview as reagentqualityandevenweather. These topicswillbe attached modificationsthatcanaffect yieldaswell,such yield (whichiswhat TriLink andmostofourcompetitorsdo). to ensurefulldelivery, orsendthecustomerentireresulting can eitherguaranteeonlyasmallamountfromanysynthesis labor neededtopreparethatsynthesisbasedonscale.We the oligonucleotideisbasedonamountofmaterialand material willresultfromanyparticularsynthesis. The priceof as Grichsequences.Itisnoteasytopredicthowmuch of asynthesiscanbeaffected greatlybythesequence,such properties basedonsequenceandmodifications. Thequality oligonucleotide isauniquemolecule,withsynthetic lower, withnoguaranteeoffinalyield. Thisisbecauseevery of thequantity, particularlythesmallerscalesof15µmoleand concentration are(μmole)(mL) required. number ofμmolesproductdirectly, withnofurther math already normalizedto1mL,thefinal calculationyieldsthe in OD product withanε of175(OD which canbederivedto:[concentration]= Absorbance /ε Law: Beer’s Absorbance =[concentration]ε every substance. the extinctioncoefficient(ε),whichisaconstantuniqueto to concentrationusinganotherfigurefoundon TriLink’s COA, Law thatrelatesabsorbance converted toµmolesusingBeer’s By RichardHogrefe, Ph.D.; TriLink BioTechnologies Understanding OligonucleotideSyntheticYields Although theabsorbanceismostaccurate Larger scalesaregenerallyorderedbymass,notyield, All oligonucleotides,eventhosemadeatthemulti-gram The yieldonyourcertificateofanalysis(COA)isreported There aremanyfactorsotherthansequenceandthe Most oligonucleotidesareorderedbythescaleinstead For example,areported yieldof100OD The unitsofεare(OD 260 units.Sincetheabsorbancereadings wereportare 260 260 units)(mL)(μmole) -1 units)(mL)(μmole) , andabsorbanceisexpressed 260 unitsontheCOA canbe 260 unitsfora 260 -1 , theunitsof -1 ). The yields ). The calculates www.trilinkbiotech.com 260 following formula: easily demonstratedbycalculatingthetheoreticalyieldwith few percentagepointsontheyieldcanbedevastating. This is preparation. The resultsofadropincouplingefficiencyjust modified programmingandpayingspecialattentiontoreagent At TriLink wedoourbesttooptimizethisefficiencyusing The couplingefficiencyofthesynthesisisveryimportant. synthesis itself,generallydoneonanautomatedsynthesizer. any particularsynthesisisdeterminedbythequalityof Synthesis What affectstheyieldofyouroligonucleotide? as theamountdeliveredmakesitimpractical. samples. This isnotreasonableforscalesof1μmoleandless, to accountforthesaltformifyouintendactuallyweigh per μmole). This numberiscalculatedforthefreeacid.Besure on theCOA ingramspermole,orperhapsmoreusefully, μg multiply thenumberofmolesbymolecularweight(found as 10%inordertoobtainperfectresults. vary theconcentrationofoligonucleotidereagentsbyasmuch reasonable approximationofthetruefigure.Bepreparedto than manyvendors,theεoffered onourCOA isstillonlya the spectrumofsequences. oligonucleotides thatmayornotextrapolatewellacross of somedyeswesellexperimentally, butthosewereonunique of themodificationonabsorbance. We havedeterminedtheε can onlyuseaneducatedguessastowhatwillbetheaffect and othercompoundsareaddedtoanoligonucleotide.We fairly complicatedcalculationisonlyaccuratetowithin10%. nucleoside, whichcanbeconsiderable.However, eventhis on theabsorbanceofeachnucleosidebyneighboring nearest neighbormodel.We takeintoaccounttheaffect TriLink doesthenextbestthinginusingwhatiscalled experimentally, butthisisaverylonganddifficultprocess. into account. increases asmoreofthecomplexitiessystemaretaken formulations existforthiscalculation,theaccuracyofeach for eachandeveryoligonucleotidesoldby TriLink. Different sometimes withsomeguessworkaddedin. The εiscalculated on anumberthatisitselfobtainedbycalculation,and theoretically yields75%ofproduct(0.99 29 couplings)withanaveragecouplingefficiencyof99% couplings. where xistheaveragecouplingefficiencyandynumberof techniques. impossible, despite usingrigorousanhydrouschemistry of synthesisbymaking nearcompletewaterremoval almost well. Extremelyhumiddayswilladversely affect thequality and phosphoramiditequality. The weatherplaysarole,as be perfect,suchasthemoisture content intheacetonitrile finely tunedtooperateat99%+, but otherfactorshaveto to thecontrary. Notonlydoes theinstrumenthavetobe at anoperationalefficiencyof99% orgreater, despiteclaims 25% yield. could hopeforis50%yield. At 98%itbecomesanabysmal the difficultyofmakinga70mer. Evenat99%,thebestone 55%. That onepercentcostsnearlyhalfthematerial.Consider synthesis at98%efficiencywillhaveamaximumyieldofonly The amountofproducttheoreticallypossiblefrom If youwishtodeterminethemassofoligonucleotide, Therefore, despiteusingamorecomplicatedformula The guessworkoccurswhensomemodifiednucleosides The mostaccuratemethodistogeneratetheε Unfortunately, thisveryconvenientcalculationispremised For example,asynthesisof30mer(whichrequires It isverydifficulttomaintaineverymachineday x y 29 ). That same ). That µmole. manipulation isnotunusual.Our yield isnowat44%,or0.44 to chemicalmodifications,incomplete deprotectionsandmanual the loss.Overallalossofanother 25%ofpotentialproductdue involves transfersandfiltrations that invariablyresultinmostof and inthiscase,somedegradation ofthequencher. Italso small amountofundeprotectedbases, undesiredmodifications, average of99%fortheother26couplingsiscalculatedthusly: example. The aminolinkercoupleswithanefficiencyof95%. three modifiedbases,eachofwhichcoupleat93%inthis further chemistry. The oligonucleotideisfurthermodifiedwith bound reagentfromwhichthesynthesisbegins,requiringno labeled oligonucleotide. The 3'quencherinthiscaseisasupport therefore requiringpost-synthesisconjugationtoa5 This hypotheticaldyeisonlyavailableasasuccinimidylester, above, butaddthefollowing:ithasa5 affected. We willreturntotheexampleof30merdescribed oligonucleotide order? What shouldIexpectfrommy1μmolescalemodified and 95%canbeseveralfold. The difference indeliveredproductbetweenafinalpurityof90% unprotected primaryaminemodifiedoligonucleotides. product toco-elutewithshorterfragments,whichisthecase cases, themodificationsonanoligonucleotidewillcause properties withtheproducttomakepurificationdifficult.Insome containing degradedordamageddyesoftenshareenough variety ofreasons. of thetheoreticalyieldwillbeconsumedinmanyprepsforawide overall processofpurificationiscostlytoyield.Upwards50% acceptable purity. Regardlessofthequalitysynthesis, crowd intotheproductpeak,requiringatightercuttoobtainan syntheses willhavemorecontaminatingfragmentsthat allowing alargercutoftheproductpeak.Moderateandpoor will haveonlyamoderateamountofimpuritiestoremove, be thestepwheremostyieldislost. A highqualitysynthesis Purification compounds. These sidereactionsallresultinlossofyield. occurs tothebasesthemselves,butparticularlydyelabeled bases arenotfullydeprotected.Inmostcases,somedamage modifications requiredeprotectingconditionssomildthatthe long asthedeprotectingreagentsarefresh.Howeverparticular base. Withsimpleoligonucleotidesthispresentslittledifficulty, as Deprotection poorly timetoduesequenceeffects. personnel. Despiteourbestefforts, somereactionswillstillgo well asoperatewithestablishedSOPsandtrained the qualityofallreagentsthroughourISO/GMP program,as affect onfinalproductyield. At TriLink wedoourbesttocontrol quantitative. A mediocreconjugationcanhaveaconsiderable quality ofthebuffers. Reactionscanrangefrom10%tonearly and ageofreagent,thesequenceoligonucleotide reaction isaffected bymanyfactorsaswell,includingthequality conjugation toanamine,thiolorcarboxylfunctionality. This coupling efficiencyaslow90%. for avarietyofreasons.Itisnotunusualreagenttohave [email protected] [email protected] The deprotectionoftheoligonucleotidealwaysresultsina The theoreticalmaximumsyntheticyieldbasedonan Here isahypotheticalsynthesistoillustratehowyield The requestedpuritymakesaconsiderableimpactonyield. Oligonucleotides thatarepartiallydeprotectedandthose Depending onthequalityofsynthesis,purificationcan After synthesis,theoligonucleotideisdeprotectedwith Some modifiedoligonucleotidesarepreparedby Modified reagentsoftenhavepoorcouplingefficiencies (0.99 26 )(0.93 3 )(0.95) =0.59 ' dyeanda3quencher. ' amino modified compoundlikethis. Yields of20-40OD mg, ofproduct. product, reducingthefinalyieldto0.15μmoles. appreciated, resultinginanoveralllossofanother20%the Unfortunately, thisseriesofstepsismorecostlythan and thenremovealiquotsforfinalanalysispriortodelivery. the sample,convertittopropersaltandprecipitateit, the productto0.19μmole. dyes, resultinginafurtherlossof20%theproduct,reducing material, freedye,andanysideproductsduetodamaged efficiency of80%. Theyieldisnowat0.24μmole. unless thereisasequenceorreagentissue.Letusassumean 0.30 μmoleofintermediate. aforementioned undesiredmodifiedmaterials. We arenowat at least25%oftheproductduetocontaminationwith work-up, loweringouryieldto0.40μmole. 10% ofthisgrouppriortopurificationduringdeprotectionand elute withthefailuresequencesonHPLC.Itiscommontolose conjugation. Withoutit,theaminolabeledoligonucleotidewill the purificationofaminolabeledintermediatepriorto The protectinggroupontheaminoislefttoimprove final yieldwouldcorrespondtoadeliveryof52OD and amolecularweightof10,500g/m,or10.5mg/μmole,this pages 17and18. A Table ofGeneralExpectations best possibleunderthecurrentcircumstances. requested. Itisbesttoorderbythe scaleandtrustustodothe we mayhavetopreparealargerscaleensuretheyieldyou This willincreaseyourcostconsiderablyinsomecasessince produce theallottednumberofkits,evenatsmallquantities. guaranteed amountsofmaterialdeliveredsothattheycan amounts maymakesense.Manydiagnosticfirmsrequire the properstartingscale. customers order10mgsamples,dependingonustodecide are orderedbysetquantity, usuallyinunitsofmass.Some When shouldIrequestasetquantity? do withwhatwecanobtain. qualitymaterial.If for high thatfalterswemustmake dependent many ofourreagentshaveasinglesource,uponwhomweare the synthesiscanhavegraveconsequencesonyield.Also, the precedingparagraphs.Subtlechangesinoneaspectof of theexactsameoligonucleotide.Muchanswerisin obtain different quantitiesofmaterialforsubsequentbatches exactly thesame? Why ismyyielddifferentthistimealthoughtheorder coupling efficiencyjustonepercentagepoint. of theproductrightfromonsetsynthesisbydropping with higherthanusualhumiditycanresultinthelossofathird structure thatmakespurificationdifficult. Also,recallthataday cause thelossofatleasthalfmaterialifithassecondary maintenance). A compoundwithhighguanosinecontentcan issues) andsomewithinourcontrol(leveloftraining,routine some completelyoutofourcontrol(mostlyconstruct/sequence described caneasilybecompoundedbymanydifferent factors, compounds ofthissortaremuchmorecommon. The losses We wouldconsiderthisanexcellentyieldforahighly Given a30merwithanεof345(OD This isfollowedbyaseriesofmanipulationstodesalt This materialisnowre-purifiedtoremoveunconjugated The conjugationofadyegenerallygoesat70-90% This productispurified,resultinginanotherlossof For ageneralguideline,seetables ofexpectedyieldson There arecaseswhenorderingasetquantityforsmaller Almost allmid-scalesyntheses(50mgandlarger) We understandhowannoyingitmustbeforacustomerto 800-863-6801 Technical Information 260 units)(mL)(μmole) 260 260 unitsfor units,1.6

-1 133 Information Technical 134 Technical Information are chemically synthesized asamixtureof2 are chemically oligonucleotides (Sp andRp),standardphosphorothioate phosphorus phosphorothioate chirality oftheinternucleotide or antisenseexperiments(6).Duetothe in targetvalidation with modifiedbases. to moreexpensivenucleosides ofaduplexconsiderably,the thermostability without resorting will enhance of 2'-fluoroandOMenucleosides combination duplexes (5). destabilizes the 2'-aminomodification A of theDNA-DNA duplexesby1.3°Cperinsertion(4),while with 2'-fluoroenhancesthethermostability 2'-deoxyribose enhances thermostabilityoftheduplexes.Modification in DNApresence of2'-OMenucleosides orRNA strands derivatives. The are 2'-OMe,-fluoroand-amino DNA target(3).) by RNaseHofaDNA/RNA/DNAto chimerahybridizeda features sufficientconformational and cleavage forrecognition RNA possesses stretchasfewthreeribonucleotides A-form intheduplex.(Aand ratioofdeoxyribo-ribonucleotides extent of A-form characteristicsdepends onthecomposition For theduplexesof DNA withDNA-RNA-DNA chimeras,the of characteristics withapredominance A-form features(2). have aconformationwithboth A-form andB-form conformation ofsugar(1). The DNA:RNA hybridduplexes of DNA:DNA duplexwhichischaracterizedby2'-endo two RNAand ismorestablethantheB-form molecules conformation ofthesugar(1).It is normallyformedby DNA:DNA >Increaseof Tm >>MostStable are representedbysinglestrandform. molecules are inadoublestrandedform,theotherhalfof while temperature (Tm) at whichhalfoftheDNA orRNA molecules by melting is characterized oftheduplexes The thermostability with thesamesequence). thermostability (foroligonucleotides listedbyincreasing setofduplexes shown inthefollowing and sugarmodifiedoligonucleotides. The simpleansweris of theduplexesDNA andRNA withvariousbackbone By RichardHogrefe, Ph.D.and Alexandre Lebedev, Ph.D.; TriLink BioTechnologies of ModifiedOligonucleotideDuplexes Thermostability Technical Information phosphorothioate oligonucleotidesare very expensive to (6). However,and Rpphosphorothioates the chirallypure and RNA or mixedSp comparedto Rp diastereomers with DNAto enhancethethermalstabilityof theduplexes havebeenshown oligonucleotides Sp-phosphorothioate pure were synthesizedandinvestigated (6).Chirally oligonucleotides diastereomers ofphosphorothioate gave a analog phosphorothioate Tm of 57.7°C (4). RNA strandgavea Tm of 62.8°C,whereasthe2'-OMe For theduplexwithDNAoligonucleotides. thecontrol2'-OMe- for2'OMe-RNAreduced considerably phosphorothioate effect of the2'-OMegroup,difference between Tm is respectively, conditions.Duetothestabilization at described RNA15mer oligonucleotide had Tm’s of33.9°Cand45.1° of the samesequencewithacomplementary phosphodiester and acontrol oligonucleotide of a15merphosphorothioate DNA:RNA andDNA:DNA duplexes.Forexample, acomplex destabilize oligonucleotide forms inthephosphorothioate of theSpandRpchiral linkages). Overall,thecombination phosphorothioate (where nisthenumberofinternucleotide Phosphorothioate oligonucleotides are commonly used oligonucleotides Phosphorothioate sugar modifiedoligonucleotides The commonlyavailable The by3'-endo A-form ofaduplexischaracterized We areoftenaskedabouttherelativethermostabilities In several publications individual stereo defined individual In severalpublications n diastereomers www.trilinkbiotech.com 8. 7. 6. 5. 4. 3. 2. 1. References on acasebybasis. permission with Genta'sexplicitwritten of synthesizingthesemolecules throughGenta, can onlybeobtained Inc. TriLink iscapable commercially,are notavailable methylphosphonates and than thecontrolhybrid(7).Unfortunately, the chirallypure hada same oligonucleotide Tm of 55.1° C, justslightlyless had a Tm of40.6°C,whiletheRpchirallypureversion linkages and phosphodiester Rp-mixed methylphosphonate had a Tm of60.8°C. with alternatingSp/ An oligonucleotide had a control Tm of34.3°C,whereasthephosphodiester 15mer oligonucleotide an Rp/Sp-mixedall-methylphosphonate RNAwith For instance, oligonucleotide. phosphodiester target, with aDNA (8)comparedto targetisgreatlyenhanced complex withanRNA target; while the stabilityofcomplex the destabilizes analog, ontheotherhandonlyslightly oligonucleotides. stronger thanphosphorothioate on thecomplexeswithDNAoligonucleotides orRNA is duplexes. effectThe destabilizing of methylphosphonate DNA-DNAthan similarphosphodester andDNA-RNA thermostability ofthesecomplexesissignificantlylower DNAsandRNAs.However,with complementary the which isallthatproducecomplexes is readilyavailable, oligonucleotides, Rp mixedversionofmethylphosphonate linkages). internucleotide of methylphosphonate The Sp/ diastereomers (2 are alsopreparedasamixtureofSpandRp analogs oligonucleotide linkages. These methylphosphonate by TriLinkneutral containing isoligonucleotides phosphorothioate oligonucleotides. for thesynthesisofchirallypure the addedexpenserequired and donot affect the thermostabilityadverselyastowarrant formostapplications especially 2'OMeanalogs,areadequate oligonucleotides, make. SofarmixedSp/Rpphosphorothioate Nucl.Acids Res.22,2401-2405. J.W.,Engels, Lebedev, Wickstrom,E.,and A.V. (1994) Vyazovkina,E.V., E.V., Savchenko, Lokhov, S.G., Reynolds, etal.(1996)Nucl. Acids Res.24,4584-4591. 722. Stec, et al.(1994) Angew. Chem. Int. Ed. Engl.33, 709- Nucleic Aurup, etal.(1994) Acids Res. 22, 20-24. Kawasaki, etal.(1993)J. Med Chem. 36, 831-841. Hogrefe, etal.(1990)J. Biol. Chem.265,5561-5566. Biochemistry32, Lesnik, etal.(1993) 7832-7838. (Springer-Verlag, New York, 1984). Cantor, Ed.,Springer Advanced TextsChemistry in W. Saenger, ofNucleic Principles Acid Structure,C.R. Aoligonucleotide chirallypureRpmethylphosphonate Another commonbackbonemodificationoffered n diastereomers,wherenisthenumber

analysis ofoligonucleotides inboththeUVandvisible ranges absorbance ofa solution ofthesampleat260nm. Spectral Spectral Analysis analyzed byPAGE atnoadditional charge. demanding applications.Everyoligonucleotide soldat TriLink is adequate asthesolefinalQCanalysis forallbutthemost of basicprimersandresearchcompounds itismorethan showing thebasicqualityofanoligonucleotide.Incase costly andtimeconsumingforcommercialproduction. oligonucleotides areradiolabeledpriortoanalysis,butthisis narrow linearrange.Betterquantitationcanbeobtainedifthe However, it isaverypoorquantitativetoolandhasfairly very effective forseparatingoligonucleotidesbasedonlength. oligonucleotides. analysis) isthemostcommonanalyticaltoolforanalysisof any doublestrandstoseparateintosingleduringthe Denaturing PAGE (thegelhasahighureacontenttoforce absorb theUVlight,leavingashadowonbackground. which isonafluorescentbackground. Theoligonucleotides backshadowing, whereaUVlampisshinedonthegel through thematrix. A commonmethodofdetectionisUV matrix. The negativelychargedoligonucleotidesaredrawn charge ratiosbypassinganelectricalacrossagel Polyacrylamide GelElectrophoresis(PAGE) Common Oligonucleotide Analytical Methods method(s) ofanalysis,whichisdeterminedbytheapplication. important totheapplication. Therefore, purityisdefinedbythe analytical methodchosengivesthepuritybasedonfactors by massifexcesssaltistakenintoaccount.Generally, the measured atanalternativewavelength,oronly60%pure the samecompoundcouldbeonly70%pureiffreedyeis exchange (AX-)HPLCatthesamewavelength.Furthermore, at awavelengthof260nmmayonlybe80%purebyanion samples. is veryhardtodeterminetheamountofsaltinsub-milligram purification matrix(especiallyin PAGE purifiedsamples).It that donotabsorblightat260nm,suchasexcesssaltsand are manyothercontaminantsthatcanbeinaproductsample even shorterfragmentsfromthemainproductpeak. Also, there (RP-) HPLCcanoftenbeverypooratseparatingn-1,n-2and that donotseparatefromtheproductpeak.Reversephase for manyreasons.Forexample,contaminantsprobablyexist analysis ofthefinalproduct. Thisnumbercanbeerroneous Analyses (COA)foranoligonucleotideistheresultofHPLC How isPurityDefined? some generaldecisionsbeforeyouplaceacallforadvice. are severalguidelineswecanoffer thatwillhelpyoumake answers youneedforyourspecificproblem.However, there end, your TriLink ProductSpecialistisyourbestwaytogetthe highly modifieditis;whatisitsapplication;andsoon.Inthe novel compoundoronewemadeseveraltimesalready;how it isaresearchscaleorcommercialsynthesis;if be complicated. The decisionwillvarydependingonwhether ask for? The answerispredicatedonmanyfactorsandcan clients. Howdoyouknowwhatpurityisneededandto [email protected] [email protected] By RichardHogrefe, Ph.D.; TriLink BioTechnologies PureWhen ismyOligonucleotide Enough? Everyoligonucleotideisquantitated bymeasuringthe PAGE analysisisinexpensiveandveryadequatefor PAGE isgoodforqualitativelyexaminingaproduct.It PAGE separatesoligonucleotidesonthebasisofmass/ A samplethatis90%purebyRP-HPLCanalysisobserved Usually, thepercent purityseenonmostCertificateof This isaverycommonquestionwereceivefromour oligonucleotides: T12mer,oligonucleotides: - T22mer. T18mer Figure 2: AX-HPLC analysisofamixture of6polythymidine 1). Inmorerecentyearsithasbecomereproducible. from thefulllengthproduct(Figure2,ascomparedtoFigure oligonucleotide andovercomethemassdifference. the amineispolarenoughtochangelipophilicityof will oftenelutealongwiththefailuresequencesbecause contains aprimaryaminofunctionalityforconjugationpurposes longer thanthefulllengthproduct.Forexample,aproductthat to theextentthatinafewcasesshorterfragmentwillretain alone cangreatlyaffect theretentionofanoligonucleotide, longer thanapproximatelya12mer(Figure1).Sequence being itsinabilitytoseparateoligonucleotidesofsimilarlength, over 50yearsofcommercialdevelopmentbehindthem. reproducibility issues.Ontheotherhand,RP matriceshave in theearlydaysbecauseofmanufacturingproblemsand and stillis. AX chromatographywasverydifficulttoperform available tooligonucleotidechemistshistoricallywasRP, diethylaminoethyl (DEAE)oraquarternaryamine. a coatingofcationicmolecules,mostcommonlyform most commonbeingoctadecyl,orC-18. An AX matrixhas particle coatedcovalentlywithalongchainhydrocarbon,the charge ratio. exchange (AX),whichseparatesonthebasisofanionicmass/ separates onthebasisofmass/lipophilicityratio,andanion in oligonucleotidechemistryarereversephase(RP),which different classesofHPLC. The twomostcommonlyused of manyclassescompounds. There arealsomany commonly usedanalyticaltoolfordeterminingthepurity High PerformanceLiquidChromatography(HPLC) oligonucleotide asafreeservice. of thefinalanalysisperformedby TriLink oneverydye-labeled remains inthesample. This isaveryimportantassayandpart dye atitsabsorbancemaximumtodetermineifanyfree of theoligonucleotideat260nmandabsorbancea can alsobeveryuseful.We usetheratioofabsorbance oligonucleotides: T12mer,oligonucleotides: - T22mer. T18mer Figure 1:RP-HPLCanalysisofamixture6polythymidine AX-HPLC isverygoodatseparatingshorterfragments However, RP-HPLChasmanyproblems,themostglaring The mostwidelyusedandleastexpensivematrix A RP matrixgenerallyconsistsofasilica,glassorpolymer Due toitsversatility, HPLChaslongbeenthemost 800-863-6801

Technical Information 135 Information Technical 136 Technical Information some oftheseassays areveryhighforwhatthey return. NMR andmetalcontent byatomicabsorption. The costsof hybrid meltingtemperature,water content,elementalanalysis, capillary electrophoresis,basecomposition sequenceanalysis, include theaforementionedendotoxin assay, bioburdenassay, an endotoxinassayinthelattercase. Specifically, theseassays pre-clinical therapeuticsyntheses, withperhapstheadditionof sections areallthatneededfor allGMP diagnosticand applications. Infact,thebasicassays listedintheabove beingusedinspecial orforoligonucleotides samples for clinical their costintime,moneyandmaterial.Mostareonlyneeded analytical chemist.Inmostcasestheirvalueisnowherenear Special Assays circumstances whereESwillnotwork. error canbeasmuchawholenucleotide. large, hidingmanymodifications.Inlongeroligonucleotidesthe thrown outofcalibration.Further, theleveloferrorcanbe tolerant ofsamplequality, particularlysaltcontent,butiseasily desalting andhandlingofthesamples.MALDIismuchmore of thesample,particularlyexcesssalts,necessitatingstringent calculated figure.However, itiseasilyaffected bythequality very accurate,usuallygivingmasseswithin1or2amuofthe of oligonucleotides,electrospray(ES)andMALDI.ESis differentiate betweenthetwo. has vastlydifferent properties.InthiscasePAGE willeasily the self-annealingsequenceCCCCAAAATTTTGGGG, which a veryinnocuouslooking16merthathasthesamemassas oligonucleotide withthesequence ACTCGCTTGACAGAGT is calculated doesnotconfirmastructure.Forexample,an of themrathersignificant. contaminants. However, thismethodalsohaslimitations,some way tohelpverifyitsidentityanddetermineunknown products. Determiningthemassofacompoundisanexcellent become agreattoolfortheanalysisofproductsandside- from oligonucleotidefirms,including TriLink. MSanalysishas Mass Spectroscopy(MS) bases inlength. must beconsideredunusableforanythingovertentotwelve oligonucleotides, andwithoutcarefulmethodsdevelopment much lesseffective fortheseparationofstandardunmodified in length,andthatistheabsolutebestcaseswith AX. RP is not effective atseparatingfragmentslongerthan50-60bases of oursmallscalemodifiedcompounds. greater areanalyzedby AX-HPLC freeofcharge,asaremany contaminants usingthismethod. All synthesesof50mgsor in general,analysisby AX ispreferable.We oftenseemore AX andRP-HPLCisbetterthaneitheronealone;however, contaminants. Havingananalysisofyourcompoundbyboth two methodsarebetteratseparatingdifferent typesof to answerdefinitively. Itwasalreadypointedoutthatthe approximation ofthepurity. especially whenitistheonlyanalyticalmethodused,an be understoodthatthepuritynumberobtainedfromHPLC, of pharmaceuticalgradeoligonucleotides. Therefore itmust development ofHPLCanalyticalmethodsfortheanalysis as analyticalreferences. This iscommonlydoneforthe of interestwithoutsynthesizingthesuspectedcontaminants certainty theabilityofanymethodtoseparatecontaminants as RP. separate somechemicalmodifications,suchasdyes,well Analytical columnsaremuchmoreexpensiveanddonot Technical Information There aremanyassaysavailabletotheoligonucleotide We useESunlessMALDIisrequested,orinspecial There aretwoMSmethodstypicallyusedfortheanalysis For instance,havingafoundmassidenticaltothat Recently, thisassayhasbecomeroutinelyavailable One significantproblemthatplaguesHPLCisit Which methodisbest, AX orRP-HPLC? That isdifficult It shouldbekeptinmindthatitisdifficulttopredictwith www.trilinkbiotech.com purified, theband shouldbesharpandthecontamination Assays torequestandresults expect: purification. of sequence,modificationsand dye thatdonotallowforgood with mostassays.Occasionally, therearesome combinations present inalabeledoligonucleotide samplecanwreakhavoc respect toaspecificcontaminant. Forexample,anyfreedye applications wherehigherpuritymaybenecessary, usuallyin 90% purityoneverycompound.However, therearesome than adequate.Onceagain,weattempttoachieveatleast biological systematacellularlevel,80%willoftenbemore properties ofmodifiedoligonucleotidesorhowtheyaffect a modified oligonucleotides,suchasstudyingthephysical cannot achievegreaterthan70%-80%. achieve ascloseto90%possible,althoughsometimeswe limited bythenumberandtypeofmodifications. We attemptto twice atmostwithanyparticularsequence.Purityisoften oligonucleotides: 2. Generalbiologicalresearchgradeprobesandmodified a largen-1band;thisisoftenindicativeofotherproblems. contaminants arefaintifobservableatall. There shouldnotbe Make surethattheproductbandsarestrongand Assays torequestandresultsexpect: interfere withenzymes. or precipitationattheleasttoremoveexcesssaltsthatmay desalted tolessthan20%saltbyweighteithercartridge possible shorterfragments. All oligonucleotidesshouldbe impurities shouldbespreadoutfairlyevenlyamongthe In mostcasesapurityof70%ismorethansufficient. The These compoundsareoftenpureenoughascrudedesalts. primers usedforsimpleamplificationorextensionexperiments. oligonucleotides usedforbasicbiologicalresearch,suchas daily basisfallintothiscategory. These consistofthe 1. Generalbiologicalresearchgrade,non-diagnosticprimers: general classesofoligonucleotidesorderedfrom TriLink. the bestwecandoisoffer basicguidelinesforthefollowing dye labeledoligonucleotides.Sinceeverysituationisunique, the developmentofveryexactingmicroarrayassaysusing to theassaycominglaterinprocess. This isoftentruein just toallowdevelopment,withtheintroductionofrobustness some assaysrequirehighqualitymaterialatthebeginning progresses towardcommercialization.Ontheotherhand, identified andarereproduciblefromlottolot. sequences, aslongtheindividualcomponentsarepositively a therapeuticdrugcantoleratesmallpercentageofother with particularsequences,especiallytargetwhile instance, someassaysrequireabsolutelynocontamination than aprimersoldinkit. That isnottrueinallcases.For injected intohumanshasamuchhigherpurityrequirement obvious. Forinstance,youmayexpectthatacompoundbeing the application,purityrequiredbyeachapplicationisnot Decision Driven AnApplication for theproduct. TriLink willsuggestitinthecourseofdevelopingspecifications to thecompositionofcompound,inwhichcasemostlikely FDA requeststhemorifneededinyouruniquesituationdue should onlybeconsideredifyourregulatorydepartmentorthe expensive mammalssuchaspigs,dogsorprimates. The rest stage pre-clinicalsamples,particularlyformaterialgoinginto In manycasesPAGE issufficient. Ifthematerialis For mostapplicationsingeneralresearchthatuse These compoundsaregenerallyonlypreparedonceor PAGE ismorethansufficientforthesecompounds. Many ofthecompoundsorderedworldwideona Purity requirementsalsonormallychangeastheproduct Although itmaybeobviousthatpurityisdeterminedby The endotoxinandbioburdenassaysareusefulforlate profile foryourproduct’s purity asitrunsitscoursethrough possible atalltimes. Infact,werecommendasort ofconcave definitely notinyourbestinterest toobtainashighaquality are quiteuniqueinmanyways. This isonecasewhereit oligonucleotides: 4. Therapeutic assays areunnecessaryfordiagnostic products. degradation oftheoligonucleotide.Mostotherspecial composition assay. This specialservicerequiresenzymatic oligonucleotides bearingdegeneratepositionsusingabase We dohavethecapabilitiesoftestingrandomness performance intheassay. specifications aresubordinatedtotheproduct’s required and reproducibilityofthesyntheticprotocol.Ofcourse,all turn setbythevalidationruns,whichestablishcapabilities results aresetbytheproductspecifications,whichwerein mandatory. Eachrelevantassaymustberun. The desired assays thatmayhavebeenoptionalpreviouslyarenow category ofproducts. The onlyconsiderationhereisthat general biologicalresearchgradeproductsapplyforthis Assays torequestandresultsexpect: manufacturing offluorescentoligonucleotides. high purity. We areoftenlimitedbyimpuredyesinthe completely aspossible. Some contaminants,suchasfreedye,mustberemoved that yieldwillbegreatlyaffected ashigherpurityisdesired. or greater. This isactuallyquiterare.Itmustbeunderstood Some specificapplicationsmayrequirehigherpurity, 95% to theremovaloffreedyesorotherinterferingcontaminants. usually goodenoughwiththesamecarefulattentiongiven at thelowestpricepossible.Inthiscase,80-90%isalso The reasonisthatyouwanttheassaytoberobust,but higher thanwhatisneededfortheassaytoworkroutinely. 3. Commercialdiagnosticgradeprimersandprobes: appropriate productinacomplexmixture. process control,givingusanexcellenttooltoidentifythe to aquantitative,method.Itisalsoextremelyvaluablefor of charge.Itisconsideredmoreaqualitative,asopposed oligonucleotides androutinelyonall50mgorlargerordersfree future releasesofaproduct. representative ifyouintendtousethisasaspecificationfor is hardtointerpretandshouldbediscussedwitha TriLink compared topreviouslotsensurereproducibility. This data extinction coefficients;however, repeatsynthesesare oligonucleotide. This isanapproximationbasedoncalculated dye ratioiscorrectforthenumberofdyesattachedto during synthesis.We alsocheckthattheoligonucleotide/ spectral qualitiesanddidnotundergochemicaldegradation spectroscopy toensurethatthedyestillhasdesired overall mixture. eachnomorethanafewpercentofthe with thecontaminations HPLC analysesshouldshowstrongproductbandsorpeaks include ananalysisbyRP-HPLCifneeded.BothPAGE and not. We alsoanalyzethecompoundby AX-HPLC. We will since itdoesquicklyshowusproblemsthatothermethodsdo quality ofthefinalcompound. Thesuitestillincludes PAGE, or more,agreaternumberofassaysareusedtoascertainthe the dataappearsasitdoes. explanation shouldbeaccompanyingtheCOA detailingwhy PAGE, howeveritshouldbeveryclosetoasingleband,oran it isveryhardtoobtainproductsthataresinglebandson very minimal.Withsomemodificationsandsequences [email protected] [email protected] The purityrequirementsforatherapeutic candidate There aretimeswhenadditionalanalysisisrequired. All theassaysdiscussedinprecedingsectionon Some sequencesand/ormodificationsdonotallow The specificationsfortheseproductsshouldbesetno Mass spectroscopyisalsorunonmanysmallscale All dyelabeledcompoundsareanalyzedbyUV/Visible For highlymodifiedcompounds,orforordersof50mg more guidanceifneeded.We arehappytohelp. oligonucleotide. Pleaseaskyour TriLink representativefor that willhelpyoumakedecisions regardingthepurityofyour Summary manufacturing. harmonized, itwillbeeasyforyoutotransitionclinicalscale taking yourprogramtothenextlevel.Sinceourmethodsare with assaydevelopmentassoonyouarereadytodiscuss for youatthisstage. Avecia willtakeanactiveroleinhelping of oligonucleotidedrugsfortheclinic,isawonderfulresource are consideredstandard. described aboveifthemanufacturingprotocolsbeingfollowed our experience,theFDA willnotrequiremuchbeyondthat your regulatorystaff andtheirinteractionswiththeFDA.In may ornotberequired,dependingontheviewpointof critical, usingeitherclassicalornewerMSmethodology. order animaltoxicitystudies.Sequenceanalysisbecomes necessary forthebatchesmadefinalpre-clinicalhigher that testthebioburdenandendotoxincontaminationbecome oligonucleotide andtheassaysrequiredtofileanIND. Assays regulatory departmentwillbeexploringthespecificationsofthe up aswell. As yougetclosertoaPhaseIclinicalstudyyour number andcomplexityoftheassaysautomaticallyratchet your researchcompounds. As yourprogramprogresses,the quality youhavecometoexpectfrom TriLink willsufficefor oligonucleotides. Inyourearlyresearchthenormalhigh with researchgradeoligonucleotideswillbeusedforthese Assays torequestandresultsexpect: research. for youtotrustourproductsallearlystagepharmaceutical early stageresearch,butourexpertiseinthisareaissufficient assays, andtheonesrequiredareusuallyimpracticalduring for someofthesmallmoleculecontaminantsusingstandard in cellularoranimalexperiments.Itisnotpossibletoassay other contaminantsfromoligonucleotidesthataretobeused nm profileonHPLC. We alwaysusecaretoremovesaltsand organics andsalts,thanitistohaveanextremelyclean260 contaminants fromthebeginningofprogram,suchas and 90-95%forPhaseII/IIIstudies. pre-clinical animalstudies,85-90%forPhaseItoxicitystudies work withthenewermaterial–averycostlymistake. later clinicalphases,youmayhavetorepeatallyourearlier If thatpurityisnotmetwhenthecompoundscaledupfor pre-clinical animalstudiesandevenearlyphaseclinicaltrials. the drugisbeingpreparedatarelativelysmallscaleforusein important tonotsetextremelyhighpurityspecificationswhen development ofthecommercialmanufacturingprotocol.Itis or betteryet,getsincreasinglypurerduringeachstageofthe consideration isthatitcriticalthedrugstaysaspure, must evolveregardingthepurityofproduct. The main scaled upforanimalstudies,amorecomplicatedstrategy cytotoxicity. types ofcompoundsandunderstandhowtoreduceunwanted providers. At TriLink wespecializeinthesynthesisofthese higher puritythanisnormallyavailablefrommostsmallscale syntheses leadingtocytotoxicity. Therefore, youwantamuch Much timehasbeenwastedasaresultofpoorquality to beworkingwithashighaqualityofmaterialpossible. research andintotheclinic. Hopefully youfoundsomeuseful hintsinthisarticle TriLink’s alliance with Avecia, theleadingmanufacturer The moreesotericassays,suchasmetalcontent,etc., Again, alloftheassaysapplicabledescribedforuse It isactuallymorecriticaltoremovesmallmolecule A goodruleofthumbistouse80-85%purematerialfor Once atargetmoleculeispreparedandgoingtobe At theveryearlystagesofresearchyouwanttobesure 800-863-6801

Technical Information 137 Information Technical 138 Technical Information prior touse. will alsoensurethat reading acompoundisfullysolubilized of anyexcesssaltpresentinthesample. An absorbance spectrophotometric measurementswillbeaccurateregardless is toolowto accurately weighonabalance. In addition, Often, an oligonucleotide. thetotalmassofoligonucleotide method forquantitatingoraliquoting is therecommended absorbance measurements centimeter lightpath.Utilizing ofsolutionusinga1.0 1.0 atinmilliliter 260 nanometers reading of required togiveanabsorbance oligonucleotide unit of measurement thatisdefinedastheamount of concentration, listhepathlength,and OD by on howtousethedataprovided TriLink. which thenumbersarederived,andgivepracticalexamples discuss themeansby ofoligonucleotides, weights molecular will offer oftheextinctioncoefficients anexplanation and stock solutions. concentrations andpreparing This paper molar needed tocalculateimportantdata,suchasdetermining coefficientweight. andthemolecular These numbersare crucial totheuseofextinction an oligonucleotide: Oliva; By JudyNgoandShawna TriLink BioTechnologies Extinction Coefficientsand An Introductionto and TablesOligo Data quantify an oligonucleotide. quantify anoligonucleotide. The OD OD the exactbasesequenceandcompositionisknown, systems. doublebond Because due totheirconjugated strand hasanabsorbanceatornear260nanometers, Eachofthebasesinanucleicacid oligonucleotide. of Beer'sLaw: A = known astheextinctioncoefficient. It is acomponent present isdonemathematicallyusingaphysicalconstant of the individual bases. Because it is dependent on the bases. Becauseitisdependent of theindividual of each bases aswelltheabsorbance the neighboring The extinctioncoefficient takesintoaccounttheeffects of coefficient, aconstantforthematerialbeinganalyzed. Weights of 260 260 Associating theOD The Every TriLink Certificateof Analysis liststwonumbers 5' AGC TAA GTC ACT GCC ATT GA 3' Sample sequence: Oligo UnitConversion µmoles ofoligo= Example 1:Converting to µmoles (Use theµmoles Example 2:Converting to µgrams µgrams of oligo = µmolesxMW µgrams ofoligo unitisaveryaccurateandconvenientmethodto unit,isaspectrophotometricmeasurementofan optical densityunit, or morecommonlythe ε Cl; where A isabsorbance,C = 32.4÷195.5ODunits/µmole = 0.166µmoles = 0.166µmolesx 6101.0g/mole = 1012.8µgrams calculated in Example1) calculated OD (A Oligonucleotides 260 unitwiththeamountofoligo Extinction Coefficient ( Coefficient Extinction Molecular WeightMolecular (MW): OD (A 260 ) ÷ 260 ε 260 ): unitisanormalized ε istheextinction www.trilinkbiotech.com ε )*: 32.4 195.5 ODunits/µmole 6101.0 g/mole Client RelationsDepartment.We willbehappytoassistyou. units. If you haveanyfurtherquestions,please contactour to examples ofconversionssomethemorecommonlyused convert fromOD units, itisimportant tounderstandhowusethisdata equation units,L mmole to thestandard derivedandequivalent are mathematically of Analysis andisexpressedinODs/µmole. These units the extinctioncoefficient ofanoligonucleotide. offersconveniently calculates that automatically aspreadsheet DNA andRNA nucleotidesanddimerpairs. TriLink's website coefficient constantsforboththe aswelltheestablished the extinction The nextpageshowstheformulaforcalculating calculate theextinctioncoefficient ofeacholigonucleotide. coefficients. TriLink modelto usesthis"nearest neighbor" extinction by theindividual bases andmultiplying individual the accuracy thanthemorecommonmethodofmerelyadding oligonucleotide. method givesgreater The "nearestneighbor" several waystodeterminetheextinctioncoefficient ofan coefficient isuniquetoeveryoligonucleotide. There are composition andsequence,theextinction exact nucleotide Since TriLink oftheproductinOD providesthefinalyield oligonucleotide. weight ofanunmodified molecular usedtocalculatethe The nextpageshowstheinformation weight ofthefreeacidform molecular theoligonucleotide. compound ingramspermole(6.02x10 is simplythe massofthe weight ofanoligonucleotide Certificate of weight. Analysis isthemolecular The molecular and maybeoffapproximation by asmuch10%. model to calculate theextinctioncoefficient, itisstilljustan TriLink usestherelativelyaccurate"nearestneighbor" for convertingOD may contain. atoms theoligonucleotide This valueisneeded sum ofeachthecomponentmolecularweightsall ε 195,500 L195,500 mmole convert the The extinctioncoefficient islistedon TriLink's Certificate Another importantpieceofdatafoundon TriLink's *If youareworkinginL mmole L mmole -1 cm ε 260 260 units into other desired units. Below are unitsintootherdesiredunits.Below to ODunits/µmole. -1 unitsintoof mass. TriLink givesthe ÷ 1000= -1 cm -1 cm -1 Molecular ÷ 1000=195.5ODunits/µmole -1 . Please notethatalthough ε ODunits/µmole -1 cm -1 23 units,youwillfirstneedto molecules).It is the 260 [email protected] [email protected] Molecular Weight Calculations Oligonucleotide ε 260DpEpFpGp…KpL =[2(εDpE+ mula withthetableabove: latest version of our molecular weight weight calculator asanExcelspreadsheet.) latest versionofourmolecular To calculatethenumberofOD every dimerpairinthefirst part. the 3'and5'terminalbasesinsecondpart(monomers)ofequation,whereasyouuse Note thatyoudonotinclude Touse theformulabelow.weight ofanoligonucleotide, calculatethemolecular (Or, the go to ourwebsitetodownload = Molecular weight ofoligonucleotide =Molecular + 2for3'and5'terminalhydrogens in sequence) linkages + (#ofphosphorothioate linkages insequence) + (#ofphosphodiester + (#of T insequence) + (#ofdGinsequence) + (#ofdCinsequence) (#ofdA insequence) dCpdC 7.3 dCpdA 10.6 dApT dApdG 12.5 dApdC 10.6 dApdA 13.7 pT pdG pdC pdA 11.4 8.7 11.5 7.4 15.4 ε 260 260 Extinction Coefficient FormulaExtinction Coefficient unitsper ε EpF+ mole of oligonucleotide with the sequence 5' DpEp…KpL, use the following for- with thesequence5'DpEp…KpL,usefollowing mmole ofoligonucleotide FpG + .. + FpG + ε TpT TpdG TpdC TpdA dGpT dGpdG dGpdC dGpdA dCpT dCpdG ε KpL)- ε E- x x x x x x 800-863-6801 F - ε G - .. - G - ε 8.4 9.5 8.1 11.7 10.0 10.8 8.8 12.6 7.6 9.0 ε 260 80.03 63.97 240.23 265.2 225.23 249.24 ε K]

Oligo Data and TablesOligo Data 139 Information Technical 140 Technical Information 2. 1. The SELEXProcess • • • • • • of Aptamers Advantages target molecule. SELEX istofindanaptamerthatbindstheactivesiteof and its target (4). As mentionedabove,theultimategoalof shapes thatallowthelock-and-keyfitbetweenanaptamer but inrealitytheyfoldintospecificconformations.It is these as linearsequences, are oftenrepresented Oligonucleotides ofthebasestoanothernucleicacidstrand. hydrogen bonding mechanism fornucleicacids: to thestandardrecognition as opposed recognition, interest throughconformational 15-60 basesinlength.It generally bindstothetargetof meaning "tofit" (3). ligand, An aptamerisanoligonucleotide group wasgrantedapatentin1993. Joyce attheScrippsInstitute in LaJolla,CA (2). The Colorado Ellington atMassachusettsGeneralHospital,andGerald at theUniversityofBoulder,Colorado Jack Szostakand Andy of thistechnology:LarryGoldandCraig development Tuerk working onthe weresimultaneously separate laboratories library thatbindtothetarget(1). from theoriginal Three goal ofSELEXistoidentifyasmallsubsetaptamers against atargetofinterest,suchasprotein. The main simultaneous screening of1x10 simultaneous aptamers. oligonucleotide The SELEXprocessallowsforthe Enrichment) processofdeveloping Ligands byExponential One exampleistheSELEX(SystematicEvolutionof typicallyusedindrugdiscovery.existing technologies triphosphates offers researchersmanynovelwaystoexplore By StephanieM.Schuitt, TriLink BioTechnologies SELEX ProcessAn Overviewofthe Modified Nucleoside Triphosphates 6. 5. 4. 3. Create a"library"of(~1x10 random oligonucleotides structure.) protein, smallmolecule,orasupramolecular can bea Define targetmolecule.(Themolecule proteins (5) Highly specific;candiscriminatebetweencloselyrelated Useful astherapeuticagents antibodies Greater stabilitythanmonoclonal Favorable toxicityprofiles Easily modifiedtoincreaseresistanceendonucleases Straightforward synthesis The wordaptamercomesfromtheGreek"aptus", TriLink's wideselectionofmodifiednucleoside endonuclease degradation (7). endonuclease the stabilityofaptamerandmake itmoreresistantto modify aptamers. These typesofmodificationsincrease dUTP (TriLink Catalog # N-1010)arecommonlyusedto 2'-Fluoro-dCTP (TriLink Catalog#N-1008) and 2'-Fluoro- triphosphates. Forexample, using modifiednucleoside and refined aptamers are isolated,sequenced Individual from trillions,toasmallnumberthroughthisprocess. The numberofhighaffinitymolecules isreduced binding selectioncycles. and thenputthroughseveraladditional oligonucleotides. binding Those thatbindareamplified areseparatedfromthe oligonucleotides Non-binding aptamers. will bindtothetargetandarethenconsidered molecule. A fewofinthelibrary these oligonucleotides "library" tothetarget Expose theoligonucleotide that bindtothetargetmolecule. efficient waytofindandPCRamplifyoligonucleotides and wobblebasesinbetween(6). This providesan sites at theendofeacholigonucleotide primer binding oligonucleotides) The randompoolofDNAhas generally Nucleoside Triphosphate Applications 15 different oligonucleotides www.trilinkbiotech.com 15 8. 7. References for researchpurposes. triphosphates thatcanbeused variety ofmodifiednucleoside triphosphates canbeusedin. nucleoside TriLink offers awide proteins canbetestedatthesametime(8). photoaptamers areputintoarrayssothatlargenumbersof away, while thosethatremainareamplifiedbyPCR. These target molecule. Those thatdonotcrosslinkarewashed conformation willcovalentlycrosslinktospecificsitesonthe are exposedtoradiationandthosethatinthecorrect tyrosine aminoacidonthetarget. The Br-dUoligonucleotides between theBr-dUresidueofaptameranda bonding addition totheconformationalbinding,thereisalsocovalent # N-2008)residueinsteadofastandard Thymidine. In 2'-deoxyuridine-5'-Triphosphate(Br-dU) (TriLink Catalog whichusea5-Bromo- photoaptamers, technology involves their owntechnology,developed called PhotoSELEX. This inventors, Larry Gold,has founded byoneoftheoriginal United Statesorforeignpatent. with otherproducts,orinanyprocess, combination willnotinfringetheclaimsofa makes norepresentationsorwarrantiesthattheuseofanycompound,alonein from TriLink doesnotconveyrightsforuseinanyparticularapplication. TriLink limited rightstoendusersforresearchpurposes. The purchaseofacompound triphosphates aresoldby Certain nucleoside TriLink underlicensesthatprovide Spiegelmer® isaregisteredtrademarkofNOXXONPharma AG. PhotoSELEX ispatentedbySomaLogic,Inc. SELEX ispatentedbyGilead. 6. 5. 4. 3. 2. 1. Spiegelmer to NOXXON, who isusingitforthedevelopmentoftheir for aptamers. this technology Archemix hassublicensed applications to developpharmaceutical the technology it to technology andhaslicensed Archemix, whoisusing Somalogic http://www.somalogic.comSomalogic (accessedDecember2002) Archemix http://www.archemix.com (accessedDecember2002) SELEX is just one of many applications that SELEX isjustoneofmanyapplications modified SomaLogic (http://www.somalogic.com), a company chem ernie.chem.indiana.edu/~dhburke/SELEX.htm Burke, Donald,Invitroselections:Evolutiona Tube http://bl- Archemix http://www.archemix.com (accessedDecember2002) http://www.somalogic.comSomalogic (accessedDecember2002) 1628-1650. Rival Chemistry 45:9 Antibodies inDiagnostics(1999)Clinical Jayasena, S.D. Aptamers: that An EmergingClassofMolecules tube http://bl-chem-ernie.chem.indiana.edu/~dhburke/SELEX.htm Burke, Donald,SELEXtutorial,Invitroselections:Evolutionina Archemix http://www.archemix.com (accessedDecember2002) Gilead currentlyholdsthepatentrights to theSELEX ® technology(http://www.noxxon.com). [email protected] [email protected] of theirabilitytoserveastoolsforthedifferentof themainclassesNTPDAsandillustration applications. biochemical with areferencetoprimarysourceofinformation. indicated derivatives andtheiranalogs.Whereitisknown,themodeofNTPDAis briefly actionandtheresearchorpracticalapplication(s) enzymatic reactions. in the andmostimportantlytheirbehavior 5'-triphosphates, result inchangestheessentialpropertiesofnucleoside of those.Manythemodifications represent variousmodificationsofthebases,sugars,triphosphatechainandcombinations important detailsofthemechanism reactions catalyzedbyafamilyofNTPenzymes. Different dependent versionsofNTPDAs By Alexandre Lebedev, Ph.D.; TriLink BioTechnologies and TheirAnalogs Nucleoside 5’-Triphosphates ofSelected Enzymatic Activity The data presented in the tables should not be considered as a complete compilation by anymeans.Rather,as acompletecompilation The datapresentedinthetablesshouldnotbeconsidered it isanoverview pages areexamplesofdifferentThe tablesonthefollowing 5'-triphosphate enzymaticactivitiesoftheselectednucleoside many andtheiranalogs(NTPDA)havebeenextremelyhelpfulinexplaining 5'-triphosphate derivatives Nucleoside PFK PK HK CK GC AC HIV RT 2',5'-OAS aa tRNA S tRNA NT TNDT PRPPS Poly(A) Pol PNP RNR RD RP RD DP DD RP DD DP ATP/CTP tRNA NT ASS ATPase AMV RT Abbreviations usedin Tables:

2. 1.

For example: of theenzymaticreactionsthroughvariousmechanisms. NTPDAs maybecomeinhibitors Substrate activityofNTPDAsoftenchangescomparedtothattheparentNTPs(usuallyreduced).

c. b. a. structure (ordisrupttheassociationforceskeepingfunctionalmultiunitenzymes). catalytic action,orconformationstabilityof forsubstraterecognition,thepeptide functions responsible NTPDAs possessingareactivegroup(s)mayalsocausechemicalmodificationofthecrucialenzyme of theenzymaticprocess. for continuation ing thechemicalfunctionrequired a modified productlack- NTPDAs maycauseaterminationoftheenzymaticchainreactionbyproducing sites oftheenzymethuscompetingwithsubstrate,reactionproductoranallostericregulator(s). may physicallyblockthebinding,catalyticorallosteric onthetypeofmodification) NTPDAs (depending Phosphofructose Kinase Pyruvate Kinase Hexokinase Creatine Kinase Guanylate Cyclase Adenylate Cyclase VirusHuman Immunodeficiency Reverse Transcriptase 2',3'-Oligoadenylate Synthesase Aminoacyl tRNA Synthesase tRNANucleotidyl Transferase TerminalNucleotidyl Transferase Phosphoribosylpyrophosphate Synthesase Polyadenylate Polymerase Polynucleotide Phosphorylase DNA Polymerase Ribonucleotide Reductase DNA Polymerase RNA Dependant RNA Polymerase RNA Dependant DNA Polymerase DNA Dependant DNA Dependant ATP/CTPtRNA Dependent Nucleotidyl Transferase Adenylylsuccinate Synthesase Adenosine 5'-triphosphatase AvianVirus Myeloblastosis Reverse Transcriptase 800-863-6801

Nucleoside Triphosphates 141 Information Technical 142 Technical Information Table 1:Sugar modifiednucleoside5'-triphosphateanalogs Nucleoside Triphosphates Modified RNA Modified Synthesis Of Synthesis RNA Sequencing Modified DNA Modified Research Synthesis Of Of Synthesis Enzymatic Ribosomal DNA Sequencing Research plcto Md fAto R of Action Mode Application H O O PO H O O O Substrate P H Substrate O O O P H O Chain Termination Activation Chain Terminator H Modification ofEnzymeOH Substrate Inhibition Substrate Chain Termination H R 2 O Ba R s 1 e NHC(O)CH NCS OMe N OH NH Me NH OH OMe NHC(O)CH OH NH Xylo-OH OH OH NCS F H H H OH CHO H NH OH NH CHO OMe N H NH Me CHO 2',3'-EpoxideA 2',3'-Epoxide 3 3 2 2 2 2 2 2 2 2 2 Br OH Br OH www.trilinkbiotech.com H OH OMe OH Ara-OH A,C OH OMe OMe OH H OH OH OH Arylazido A OH F CHO OH F CHO H OH OH OH CHO OH OH R Ara-OH G OH OH OH OH OH 1

A A A A,C,G,U A,C,G,U A U,T A A,C,G,U A A,C,G,U U,T A,C,G,U A,C,G,U A,C,G,U A,C,G,U A,C,G,U A,C,G,U A A,C,G,T,I A A A,C,G,U A A,G A,C,G,U Base A,G A A A,C,G,U A,C,G,U A A DD RP RNR RNR DD RP RNR DD RP DD RP DD RP aa tRNA S RD RP DD RP DD RP DD RP aa tRNA S ATP-ase DD RP DD DP DD RP DD RP DD DP aa tRNA S DD DP aa tRNA S Poly(A) Pol DD RP DD DP ATP-ase RD RP DDRP DD RP class Enzyme RD RP DDRP RD RP, DD RP tRNA-NT tRNA-NT DD RP DD RP DD RP DD RP 17 10 10 15, 25-27 10 25 11 67 8 3,9 17 14 64 8 12 3-6 13 30,31 7 13 32-34 1,2 8 24 15, 25-27 28 29 22,23 15 References 22 22,23 19-21 19-21 15-18 15-18 17 17 [email protected] [email protected] Table 3:Nucleoside5'-triphosphateanalogswithmodifiedγ-phosphate Table 2:Basemodifiednucleoside5'-triphosphateanalogs H X * Nucleoside5’-Trimetaphosphate Research Enzymatic Substrate Application Mode of Action Mode Application R of Action Mode DNA Sequencing Application Research Ribosomal Research Enzymatic DNA Modified Of Synthesis PCR RNA Modified Of Synthesis O O P O O PO H H O O O O P O O P H H O O O O P Substrate Substrate O O P H H Inhibition Enzyme Modification Enzyme Photo-Modification4-Azidoaniline O O Substrate H H O Ribosomal protiens Photomodification ofOH Modification ofEnzymeH Inhibition Substrate Substrate O O O Ba RO H (H) Ba s e s e 2,4,6-(Me) 4-Azidoanilide 4-Azidoanilide Anilide 4-Azidobenzylamide 4-Azidoaniline 4-Azidoaniline N N-Methyl,N-(4-azidobenzyl)-amide rA 1-(5-sulfonatenapthyl)-amide Cyclic trimetaphosphate* Cyclic trimetaphosphate* amide N-Methyl,N-(4-azidobenzyl)- methylamino)-benzylamide 1-(5-sulfonatenapthyl)-amide 1-(5-sulfonatenapthyl)-amide 4-Azidoanilide Anilide 2,4,6-(Me) methylamino)-benzylamide 4-(N-2-Chloroethyl, N- X 4-Azidoanilide FSO BrCH 4-(N-2-Chloroethyl, N- H H OH OH OH OH H OH OH OH OH H H H H H H H OH OH OH OH OH OH OH H 3 2 -C 2 C(O)NH-C 6 H 4 3 3 -C(O)O- -C -C 6 6 H H 4 4 -C(O)O- -C(O)O- 6 H Base N 7-Deaza (A,G,I) 3-(Azidooxymethylphenyl)-G 5-I-U 9-Purinyl 8-Cl-A 8-Br-A 8-Azido-A 7-Deaza (A,G) Xanthine 4-Thio-U 5-Formyl U(αanomer) 8-Azido-A 5-Br-U Benzimidazole N 7-Deaza (A,G) 5-Me-C Benzimidazole N Formycin A 5-Br-C 2-Thio U 5-Formyl U(βanomer) 5-I-C 5-I-U 5-Br-U Dihydrothymidine 4 4 6 4 -Me-C ,N -Me-C O- 6 -Etheno-2,6-DAP rA, Etheno-rA rA rA rG rA, Etheno-rA rA r(A, C,G) d(C, T) rA rA rA, Etheno-rA rA r(A,C,G,U) rA rA rA dA, dT Nucleoside r(A, C, G) r(A, C, rA rA rA rG dA, dT rA rG 800-863-6801 Enzyme class Enzyme DD DP DD DP DD DP DD DP DD RP aa tRNA S aa tRNA S aa tRNA S aa tRNA S Hum. telomerase ASS DD RP DD RP DD RP DD RP RNR Hum. telomerase DD DP DD DP DD RP DD DP DD RP DD RP tRNA NT DD RP DD RP DD DP DD DP Nucleoside Triphosphates DD RP CK aa tRNA S145-150 aa tRNA S138-140 CK aa tRNA S145-150 DD RP SVPD atN S124,148 aa tRNA S DP,DD HK SVPD atN 124,148 DD RP aa tRNA S166 aa tRNA S DP, HK RD DP, nyecas References class Enzyme DD RP aa tRNA S145-150 DD RP DD RP DD RP RD DP,RD DD Ribosome 159 ATP-ase 155,156 aa tRNA S PK 163-165 References 38-41 46 46 52 49 8 8 8 8 45 61 57-59 53-56 51 36,37 10 45 42-44 35 60 59 59 53-56 50 49 49 48 47

154 151-153 151-153 142 -144 162 162 167, 168 163-165 160, 161 141 142 -144 154 157 158 143 Information Technical 144 Technical Information Table 5:Sugar andBasemodifiednucleoside5'-triphosphateanalogs Table 4:Nucleoside5'-triphosphateanalogswithmodifiedtriphosphate chain Nucleoside Triphosphates H Enzymatic Synthesis Of Synthesis RNA Sequencing Enzymatic Inhibition Research DNA Sequencing DNA Sequencing Research Application Mode of Action Mode Application R Y X of Action Mode Application Modified DNA Modified H O PO R O O PO 1 H X O O P R O O P 2 H Y O Substrate O P R O O P 3 H Substrate O O Substrate H of Enzyme Photo-Modification H CHO Etheno-A CHO Modification ofEnzyme Inhibition Inhibition ofDNA synthesisH Inhibition ofDNA synthesisH Chain Terminator R O 1 O O H (OH) Ba Ba R s s 2 e e O

CF O

CH O NHO O O CH CH NHO O O NH O O CH CH O O S O S O O O O O O O O O O O O O O CH O O O O O 2 2 2 2 2 O O O O O O O O O O O O rlzd H -zd- ATP-ase 8-Azido-A Arylazido OH H O 8AioA DDRP 8-Azido-A OH OH yoO H 5-Butyl-U Xylo-OH OH yoO H 5-Propyl-U Xylo-OH OH yoO H 5-Ethyl-U Xylo-OH OH N yoO H 5-I-U Xylo-OH OH yoO H 5-Br-U Xylo-OH OH yoO H 5-Cl-U Xylo-OH OH yoO H 5-F-U Xylo-OH OH N NH N NH R 2 2 O O 1 3 3 3 HRibavirin H5-(2-Br-vinyl)-U H5-(2-Br-vinyl)-U R 2 2 H 5-(2-Br-vinyl)-U H 5-(2-Br-vinyl)-U H www.trilinkbiotech.com - - 1 ------O O O O S S S S O O O O O O O O O O O O O O O O O O O O O O O O O O O O R OH 5-F-U OH 5-F-C 2 Base - - - - 2 ------O O O O S S S S S O O O O O O O O O O dT Me S BH S S BH dT Me O O S S S S S S S S R ------3 ------

3 3 d(A,C,G,T) 5-(Me,Et,Br,orI) C uloie nyecas References class Enzyme Nucleoside rA rA rA rA rA rA rA, rG rA rA rA rG rA, rG rA dA rA rA rA rA rA rA, rG rA rA r(A,C,G,U) Myosine dA, dT d(A,C,G,T) dA, dT dA dA dA, dT rA rA rA rA rA rA Nitrogenase 65 DD RP DD RP DD RP DD RP DD RP DNA Primase7 DD RP DNA Primase7 DD RP RD RP AMV RT DD DP nyecas References class Enzyme AMV RT DD DP HIV-RT, AMV-RT HIV-RT, AMV-RT DD DP DD DP DD DP,DD TDNT, DD DP,DD TDNT, DD RP,DD CK CK DD DP CK Various enzymes67-69 Various enzymes74-82 AC PFK Various enzymes67-70 DD DR T n T-ss125,130-132 ATP andGTP-ases Kinases DD DP ATP-ase aa tRNA S Kinases 2',5' OAS DD RP PRPPS Kinases DD DP AMV RT UDP GPP, GP UT AS GS RNA ligase tRNA NT PNP DD DP DD DP DD DP 63 62 64 64 64 64 64 64 64 22 35 35 35 35 133,134 133,134 109 135, 136 137 83 71 66 71 73 72 66 126-129 110-111 125 123, 124 86-92, 120-122 84, 85 100, 101 94-99 93 86-92 118 119 107, 108 106 105 102, 103 110-117 110-117 110-117 104 [email protected] [email protected] References 52. 51. 50. 49. 48. 47. 46. 45. 44. 43. 42. 41. 40. 39. 38. 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PentavalentPhosphorus 31 By InnaKoukhareva, Ph.D.; TriLink BioTechnologies cal reactions of mono-, oligo and polynucleotides, their analogs,andderivatives.Many oftheaspects cal reactionsofmono-,oligoandpolynucleotides, and SomeRelatedCompounds* Organophosphorus routine work. data presentedinthetables,purposesand areintendedtohelpourcustomersintheir including arebroughthereforinformational spin-spin decoupling{ in most cases. However,of the toassistyouintheassignmentofallsignalsandcompleteinterpretation we areglad the compounds. accumulated in atom. the substituentsonphosphorusHowever,and theirderivatives dataonnucleotides a hugevolumeoftheexperimental introduction wecannotgointoanydetailsof ppm). As onecandeducefromthetablesbelow, correlation between there isnounique Many ofP(III) derivatives resonateinalowermagneticfield(200ppmto50ppm)comparedP(V)(70-30 were thesubjectof reviews [1],of and systematiccompilation you areorderingourproductsmayrequest of thestructure,assurance quality,serves asoneofthemostimportanttoolsforevaluation and integrityoftheproducts.When O -NH S -OH O O -OAryl O -OAryl O O O -OAlkyl O O O -OAryl O -OH **Denotes rangeof S S O O O 3'-O-Nucleoside O -OH X R A. Phosphatemono-,di-,triestersandtheir mono-anddiamides P Chemical Shifts in NMR Spectra of Nucleotide Derivatives P ChemicalShiftsinNMRSpectraofNucleotide In ourworkonthesynthesisofdifferentcompounds), containing derivatives (aswellasotherphosphorus nucleotide Phosphorus Nuclear Magnetic Resonance ( MagneticResonance Phosphorus Nuclear The Typical range ofthe The theoryof 31 31 chemical shifts P chemical arereferencedto85% H P chemicalshiftchanges[2]. 5'-or 3'-O-Nucleoside-NHAryl 5'-or 3'-O-Nucleoside-NHAryl 5'-or 3'-O-Nucleoside-NH 5'-or 3'-O-Nucleoside-NHAryl -NH Aryl 5'- O-Nucleoside 5'- O-Nucleoside 5'-or 3'-O-Nucleoside-NHAlkyl -NH Alkyl 1 2 31 31 P asimplesetof NMRspectroscopyover50years,hashelpedintroduce rulesthatareusefulintheinterpreta 31 P NMRspectraareusuallyeasytointerpretandsomereferencedatapresentedinthe Tables shouldhelpyou P chemicalshiftsfortwodiastereomers 31 1 H}- P NMRchemicalshiftscomparedto is verycomplexandnotwelldeveloped 31 P havebeenconsidered. asarangeoranaverage number constants arereported The chemicalshiftsandcoupling 31 P shiftsofmostknownnucleotidederivativescoverstheregionfrom200ppmto-30ppm. chemical -OH -OAryl -OAlkyl -OAlkyl -OH -OH -OH -OH Oly -Nt 3'-O-Nucleoside -OAlkyl (-CNEt) -OH -OH -OH -OH R 2 2 3 PO 31 P NMRspectroscopy. Somefacts below about 5'-or 3'-O-Nucleoside ' r3--uloie(r-Akl -7.7 5'- or3'-O-Nucleoside (or -OAlkyl) 5'- or3'-O-Nucleoside -NHAryl 5'- or3'-O-Nucleoside -OAryl 5'- or3'-O-Nucleoside 3'-O-Nucleoside 5'-or 3'-O-Nucleoside 5'- or3'-O-Nucleoside 5'-O-Nucleoside 5'-or 3'-O-Nucleoside 5'-or 3'-O-Nucleoside 5'- or3'-O-Nucleoside 5'-O-Nucleoside 5'- or3'-O-Nucleoside 5'-or 3'-O-Nucleoside R 4 31 and positive values mean downfield shifts of andpositivevaluesmeandownfield 3 31 Pderivatives toberecordedandsentyouwith NMRspectraofthenucleotide Pof structureandchemi- NMR)spectroscopyiswidelyusedintheinvestigation 31 P NMRdataispresentedintheliterature[2]. 800-863-6801 43.5** -(12.0-13.0) Pyridine 2.9 -(0.7-3.0) Pyridine -(2.2-2.8) Pyridine -(5.0-6.0) 1.0 2.6-2.8 -1.0 -2.0-1.0 0.4-1.2 69.0** 55.0-57.0** H 8.4-9.2 5.0-6.5 1.2; 8.8-9.3 10.8-12.1 D 2.8-3.6; , ppm δ, Nucleoside Triphosphates 31 31 P signals.Only P NMRchemicalshiftsandthenatureof 31 H Pyridine Pyridine Pyridine 1,4-dioxane; Pyridine1 H Pyridine Pyridine Pyridine Pyridine H Pyridine Pyridine D H Solvent H Pderivatives, NMRofnucleotide 2 2 2 2 2 2 2 2 O O O O, Pyridine1 O 7.0 O, pH O; 7.0; O, pH 31 3 1 P NMRspectrarecordedwith H NMR.Inthisshort 31 P NMRspectroscopy 1 1 1 1 1 1 1 1 1 1 1 3 1, 3 1 1 3 2 Reference R 1 P X R 31 2 P NMR R

3 31

- P 147 Information Technical 148 Technical Information I. PentavalentPhosphorus Nucleoside Triphosphates 5--uloie O -H -NH -OH -OH 5'-O-Nucleoside O 5--uloie Orl Orl 'ONcesd -19.0 5'-O-Nucleoside -OAryl -OAryl 5'-O-Nucleoside O 5--uloie O -H -OH -OH -OH 5'-O-Nucleoside S O **Denotes range of **Denotes 5--uloie O -Ay -OH -OAryl -OH 5'-O-Nucleoside O 5--uloie NAy -Hrl 'ONcesd -7074 -7074 na yiie 1 Pyridine n/a -(7.0-7.4) -(7.0-7.4) 5'-O-Nucleoside -NHAryl -NHAryl 5'-O-Nucleoside O 5--uloie O -H -OH -OH -OH 5'-O-Nucleoside O Reference S 5--uloie O -H -OH -OH **Denotes rangeof -OH -11.2 5'-O-Nucleoside 5'-O-Nucleoside -NHAryl O S -OH 5'-O-Nucleoside O -OH -OH -OH -OH -OH 5'-O-Nucleoside O 3'-O-Nucleoside O z ovn Reference Solvent Hz J αγ, Hz βγ, J Hz δγαβ, J δβ -23.0 -24.0 -24.6 23.5 23.023.7Pyridine δα 1 3'-O-Nucleoside -13.0 H R R Y X O X S 75.0-76.0** O 17.6-18.5 X **Denotes range of **Denotes F. Nucleoside5'-O-trimetaphosphate E. Nucleoside3',5'-cyclopyrophosphate D. Pyrophosphatederivatives ofnucleosides B. Nucleoside3',5'-cyclophosphate C. Nucleoside2',3'-cyclophosphate , ppm δ, 52.0-55.0** -(1.5-2.0) ppm δ, 1 31 31 31 P chemicalshiftsfortwodiastereomers P chemicalshiftsfortwo diastereomers P chemicalshiftsfortwodiastereomers -13.7 δα, ppm H DMF; Pyridine;MeOH1 Solvent Pyridine D Solvent 2 2 R O O, pH7 2 -15.0 -14.4 δβ, ppm R 3 (continued) R 1 Reference 1 www.trilinkbiotech.com Reference 1 5'-or 3'-O-Nucleoside 4 2 22.0 27.6 Jαβ, Hz -1.-10 -00 / Prdn 1 Pyridine n/a -10.0 -(10.0-11.0) 42.1** -11.2 -10.0 1.* 3.* 31.2H 34.0** -11.7** H 21.4 -(10.0-11.0) -(5.0-6.0) δα, ppm -11.3 H DMF Solvent 2 O, pH 9 O, pH 1. na Pyridine 1 n/a -19.0 63* 30.8H -6.3** 1. 1. Pyridine1 17.0 -16.6 -6.0 -6.8 , ppm Jaß, Hz Solvent Solvent Hz Jaß, ppm δβ, -0.8 20.7 H 75 Pyridine1 17.5 20.0 D 1 Reference 1 HO O 2 2 2 2 2 O O 1 O 1 O, pH101 O 3 O 3 O P P H H X γ O O β O O H O O P O P R P α O H O O 4 α O O O O P β P Y H R O X O 3 O O O R O O P Ba O O H(OH P X O R O Ba s Ba H e 2 Ba αβ H(OH H(OH s s e ) s R e e 1 ) ) 6. 5. 4. 3. [email protected] [email protected] 2. 1. References II. TrivalentCompounds Phosphorus I. PentavalentPhosphorus O O -H O -H 53d -86t -5.3(d) -18.6(t) -5.3(d) -OH -OH -OH -OH O O Reference ***See ref.2forsolventinformation ,3 'ONc 5--u. (081.)d 2.() -1.-12 902. 1.-00 H 19.0-20.0 19.0-20.0 -(10.8-11.2) -22.7(d) -(10.8-11.2)(d) 5'-O-Nuc. 5'-O-Nuc. 2, 3 O 5--u. O -H 'ONc -10d 2.() -10d na / H n/a n/a -11.0(d) -22.6(t) -11.0(d) 5'-O-Nuc. -OH -OH 5'-O-Nuc. O O O 3 5'-O-Nuc.-OH 2 R N R R Z X Y G. Triphosphate derivatives of nucleosides 3'-or 5'-O-Nucleoside-N(Alkyl) 3'-O-Nucleoside -Cl **Denotes rangeof -OAlkyl 'o 'ONcesd 3'-or5'-O-Nucleoside -OAlkyl 3'-or 5'-O-Nucleoside -OAryl -OAryl -OAryl -OAryl -OAlkyl -Cl 1 DMF:Pyridine=1:1 H n/a n/a D n/a n/a 19.5 -19.7 -10.2 21.8 -3.6 H -9.7(d)** -23.2 -24.5 -(23.5-24.0)** -22.0 25.0(d)** 20.0 -11.4 -14.0 -11.8 -NHAryl 20.0 -OH -OH 5'-O-Nuc.-OH -(5.0-7.7)(d) -NHAlkyl -(20.0-23.0)(t) O -OH -(10.0-11.0)(d) -OAryl O O -OH 5'-O-Nuc.-OH -Alkyl -OH O O O 5'-O-Nuc. -OH O 6 5'-O-Nuc. O O -OH O O -OH O -OH 5'-O-Nuc. O O O S 'ONc O -H O -06 2. 3. 1. 2. H H 29.0 19.6 27.3 6.5 35.0 H 19.3-20.5 -6.0** 23.0-31.8 -22.0 -(4.7-9.6)** -(21.1-22.0)** 30.0** 42.0-44.0** -10.6 -11.4** **Denotes rangeof -OH -OH -OH 5'-O-Nuc.-OH -OH -OH S -OH O O 5'-O-Nuc.-OH -OH O S O 5'-O-Nuc. O O S H. PolyphosphatederivativesofNucleosides 1 V.20, N4,783-789. VictorovaD.F.,N.B., Mozzherin L.S., Dyatkina Atkazhev A.M., Crayevsky M.K., Nucl. A.A., Kukhanova 1992, Res., Acids H., Endo Higuchi T., Kaji Biochemistry,A., 1990, 8747-8753. 29, J. Ludwig, F. Eckstein, J. Org. Chem., 1989, V.54, N3,631-635. TriLink Biotechnologies,Inc. data. Publishers, 1967. M.M. Crutchfield, A.V. Lebedev, A.I. Rezvukhin, Nucl. Acids Res.,1984,V.12, N145547-5566. 1 1 R 31 31 P chemicalshiftsfortwodiastereomers P chemicalshiftsfortwo diastereomers R et. al, 2 R -N(Alkyl) -OAryl -N(Alkyl) -Cl -OAryl -Cl -Cl 2 R 2 31 3 2 2 2 Pin NuclearMagneticResonance Topics inPhosphorusChemistry. New York, Intescience R -11.0 (d) δρ 1 , ppm 4 -OAlkyl R -N(Alkyl) -N(Alkyl) -OAlkyl -N(Alkyl) -Cl -Cl -Cl -Cl 3 δα, ppm (continued) 2.() -10.3 -22.7(d) δρ 2 2 2 n , ppm δβ, ppm δn+1, ppmJ 165.0-170.0 , ppm δ, 141.0 131.0 131.0-138.0 149.0-150.0** 140.0-141.0** 176.0-184.0 153.0-157.0 177.0-180.0 215.0-220.0 z Solvent Hz Jβγ, Hz δγ, ppmJαβ 800-863-6801 902. 1.-00 H 19.0-20.0 19.0-20.0 1,2 z (+),H Slet Reference Solvent Hz J(n+1)n, Hz , 22.5 *** Solvent *** *** *** CD CDCl *** *** *** *** 3 CN; CDCl Nucleoside Triphosphates 3 3 H DMSO: Pyridine=1:11 2 2 2 2 2 2 2 2 O O, pH7.0 3 O,pH 7.0 O O, pH 7.0 O, pH 7.0 O, pH 7.0 O, pH O, pH11 2 Reference 1 3 2 2 2 2 2 2 2 2 2 R O, pH 7.0 O, pH O 2 OH O P n+ R 1 4 ( R O P Z 3 γ OH OO P O 1, 3 3 O P Y OH R β 1, 2, 3, 5, 3 1 1 3, 4 1 n= 1 O ) 2,3,... n OH R P P P X R 3 α 2

R R

R 2 1 1 149 Information Technical 150 Technical Information Nucleoside Triphosphate Bibliography To [email protected]. ontheweb.Wefound belowwiththefulllistavailable your citation. areconstantlyupdatingourdatabaseandwouldliketoinclude TriLinkon ourwebsiteatwww.trilinkbiotech.com/refs. maintainsatechnicalreferencedatabaseavailable A selectionofreferencescanbe dUTP 5-Bromo-2'- dCTP 5-Methyl-2'- 2'-dUTP, 2'-dITP, rine-5'-TP, iaminopu- 2'-deoxyd- rine-5'-TP iaminopu- 2'-deoxyd- Microarrayanalysisofnewlysynthesized dGTP 8-Oxo-2'- 8-Oxo-GTP, Microarray N-1025 PCR,cross- 4-Thio-UTP N-1025 4-Thio-UTP 2',3'-ddGTP Me-ITP, ITP, GTP, 2'-O- 2'-O-Me- 3'-dGTP, GTP 2'-O-Me- 3'-dGTP, ATP N6-Methyl- dUTP 2'-Fluoro-2'- 2'-dCTP, 2'-Fluoro- dUTP 2'-Fluoro-2'- Title 2'-dCTP, Application 2'-Fluoro- Catalog# Product Nucleoside Triphosphates -08Reverse N-2008 N-2026 N-2020, N-2012, N-2003, Enzymatic N-2003 N-2034 N-1066, N-4002 N-3002, N-1021, N-1020, N-1017, N-3002 Asymmetricnucleotidetransactionsofthe N-1017, ATP hydrolysis N-1013 N-1010 N-1008, N-1010 N-1008, therapeutic Transcription, PCR acid Glycerol nucleic synthesis, Incorporation linking Effects ofMutagenicandChain- Therapeutic Therapeutic Multivalent4-1BBbindingaptamers Therapeutic SELEX with Primer-Template Template Strand inStableComplexes Transcriptase andtheDownstream Interactions betweenHIV-1 Reverse edited RNAs rule toselectivelyamplifyGC-rich ADAR- Inversing thenaturalhydrogenbonding template duplex formationbetweenproductand nucleic acidtemplateswithoutstable Enzymatic synthesisofDNA onglycerol with electrochemicaldetection high performanceliquidchromatography guanosine nucleotidetriphosphatesby Analysis ofguanosineandoxidized stress: of oxidative Assessing biomarkers RNA incellsandanimals ribosome entrysite in adicistroviralintergenicinternal interacts withtheconservedloopregion Eukaryotic ribosomalproteinRPS25 Strains ofVarious Genotypes Enzymes IsolatedfromHepatitisCVirus Terminating Nucleotide Analogs on Replication at aHotSpotforInhibitionofViral Viral RNA-DependentRNA Polymerase Inhibitor ofPestiviruses That Targets the AG110 IsaNovel,HighlySelective Imidazopyrrolopyridine Analogue The HslUV protease tumor growthinmice costimulate CD8+ T cellsandinhibit AMPA receptors Water solubleRNA basedantagonistof www.trilinkbiotech.com Scott W Meyer P, tinunt W, Rutvisut- R, etal Suspène Szostak Jack W. Chen, and Jingyang Tsai, Hsuan Ching- Pelaez F Cardozo- Bolin C, et al mann M, Kenzel- T, etal Nishiyama et al Heck J, et al shuyse J, Pae- Sauer R Baker T, vich J, Yakama- et al mara J, McNa- et al Du M, uhrJunlYear Journal Author PLoS ONE Research Nucleic Acid America United Statesof of Sciencesthe National Academy Proceedingsofthe Science Applications B: Biomedicaland Chromatography Journal of America United Statesof of Sciencesthe National Academy Proceedings ofthe Research Nucleic Acid and Chemotherapy Antimicrobial Agents 2007 Journal ofVirology Biology Journal ofMolecular Clinical Investigation The Journalof 2008 Neuropharmacology 2008 2008 2007 2007 2007 2007 2008 2008 2008 pp -01TeaetcInteraction ofBacillussubtilisCodY with Therapeutic Nutritional ControlofElongationDNA N-6001 Replication ppGpp N-6001 ppGpp 2',3'-ddTTP 3'-Azido- 2',3'-ddTTP 3'-Azido- 2',3'-ddGTP 3'-Azido- 2',3'-ddCTP, 3'-Azido- 2',3'-ddATP, 3'-Azido- Transcription couplednucleotideexcision 2',3'-ddGTP 3'-Azido- Transcription 2',3'-ddATP, 3'-Azido- Mechanism of Activation ofß-d-2'- N-3005 Therapeutic P-TEFbisCriticalfortheMaturation 3'-dUTP N-3003 Therapeutic 3'-dCTP N-3002 3'-dGTP dGTP 8-Oxo-2'- dGTP 8-Oxo-2'- dGTP 8-Oxo-2'- 2'-dUTP 5-Propynyl- 2'-dCTP, 5-Propynyl- 2'-dUTP, Title dCTP, Application 5-AA- 5-AA-2'- Catalog# Product [email protected] [email protected] -09TeaetcMechanismsbyWhichtheG333D Therapeutic N-4009 Reverse N-4009 N-4014 N-4008, N-4007, N-4007 N-4002, Trace amountsof8-oxo-dGTP in Therapeutic N-2034 Substrate Selectionof Anthrax Toxin Protective N-2034 Therapeutic N-2034 N-2049 N-2048, N-2017, N-2016, therapeutic Transcription, A novelmechanismofselectivityagainst Therapeutic therapeutic Transcription, Reverse specificity PCR GTP Replication by(p)ppGpp and Lamivudine Facilitates DualResistancetoZidovudine Virus TypeReverse Transcriptase1 Mutation inHumanImmunodeficiency Reverse TranscriptaseInhibitors Confers ResistancetoNucleoside Virus TypeReverse Transcriptase1 Mutation inHumanImmunodeficiency Molecular MechanismbyWhichtheK70E polymerase AZT bythehumanmitochondrialDNA human HL60cells nucleosides causetelomereshorteningin activity invitro,andthecorresponding 5'-triphosphates inhibittelomerase 3'-Azido-2',3'-dideoxynucleoside of theβsubunitRNA polymerase by changingthearginineatposition529 repair inEscherichiacolicanbeaffected Polymerase Inhibition ofHepatitisCVirus NS5BRNA Deoxy-2'-Fluoro-2'-C-Methylcytidine and Elongation InVivo of RNA PolymeraseIIintoProductive polymerase ?replicationfidelity mitochondrial dNTP poolsreduceDNA thermophilus HB8 from Thermus Pyrophosphatases Specificities of Two ADP-Ribose Structural BasisforDifferent Substrate Tumor Cells and CMG2 Achieve Targeting of Between theCellularReceptors TEM8 Antigen Variants thatDiscriminate on enzymaticproductionofmodifiedDNA polymerases byPCRandkineticstudies analogs assubstratesforDNA 2'-deoxynucleoside- 5'-triphosphate Systematic characterizationof 800-863-6801 shein A Sonen- Shivers R, Handke L, man A G, Gross- Sanders Wang J, et al Zelina S, et al Cremer N, Sluis- Johnson K Hanes J, Liu X,etal A, etal Ganesan E, etal Murakami Molecular and Ni Z,etal et al Pursell Z, et al matsu T, Waka- et al Chen K, M, etal Kuwahara uhrJunlYear Journal Author Nucleoside Triphosphates Bacteriology Journal of Cell and Chemotherapy Antimicrobial Agents and Chemotherapy Antimicrobial Agents Research Nucleic Acid Research Nucleic Acid 2007 DNA Repair(Amst) and Chemotherapy Antimicrobial Agents Cellular Biology Research Nucleic Acid Bacteriology Journal of Chemistry Journal ofBiological Research Nucleic Acid

2008 2007 2008 2007 2007 2007 2007 2008 2008 2008 2008 2006 151 Information Technical 152 Technical Information orders, regardless of theformatorderis placed. orders, regardless Inc. requires thatallclients indemnify TriLink fromanylegalactionthatmayensuethesynthesisofanorderedcustom product. to all will beapplied This indemnification Because TriLink customsynthesizes manyproducts attherequestofitsclientele,itcannotdeterminepatentstatus eachrequest. Therefore TriLink BioTechnologies, Indemnification System, Polarization which isaregisteredtrademarkofPanVeraBeacons aredistinctfromtheBeaconFluorescence Molecular Corporation,Madison,WI. Panvera Corporation Systems, Specialist at(510)814-2984,RocheMolecular Inc., obtained bycontactingtheLicensing 1145 Atlantic Avenue, 94501. Alameda, California, chaser bythepurchaseofany TriLink BioTechnologies, Inc.product.Purchasersof PCR-related theseproductsmayneedtoobtainalicense.Further informationmaybe require alicense.Nolicensetousethe5'Nuclease to thepur- process isconveyedexpresslyorbyimplication amplification Assay oranyRochepatentedhomogeneous Systems, Reaction ("PCR")ProcessmaybecoveredbypatentsownedRocheMolecular Inc. andF. Hoffmann-La RocheLtd("Roche").Useofthesemethodsmay Inc. and F. Hoffmann-La RocheLtd("Roche"). withPolymeraseChain methods usedinconnection detection assayandotherhomogeneousamplification The 5'Nuclease Systems, those ownedbyRocheMolecular with PolymeraseChainReaction("PCR") Process maybecoveredbypatentsincluding methods usedinconnection Amplification PCR-Related Products: Disclaimer Statements trademarks ofGELimited. Healthcare product orportionsthereofismanufacturedunderlicensefromGEBiosciencesCorpU.S. Healthcare PatentNumbers5,808,044and5,556,959.CyCyDyeare patent applications. and otherpending Mellon UniversityunderPatentNumberDE3943862 This productorportionsthereofismanufacturedunderlicensefromCarnegie This Cy Dyes may notbere-sold,distributedorre-packaged. for R&Dusebythepurchaser.under agreementwithBiosearchandtheseproductsaresoldexclusively purposes andthey or diagnostic They maynotbeusedforclinical “Black HoleQuencher”,“BHQ-1”,“BHQ-2”and“BHQ-3”areregisteredtrademarksofBiosearch Technologies,Inc, Novato,CA. The BHQtechnologyislicensedandsold Black HoleQuenchers The MolecularProbes Probes. Alexa dyescanonlybeusedifyouhaveadirectlicense withMolecular Alexa Dyes Pthrough licensewithMRC. nucleosidesaremadeavailable P Nucleosides through licensewithEpochBioSciences. CPG aremadeavailable Phthalimidyl Phthaliamide with thepurchaseofthisproduct.Neither TriLinkcost foranyreason. norPHRIwarrantsthisproductbeyondreplacement Beacons aresoldunderlicensefromPublicHealthResearchInst. Molecular ofNew York. They maybeusedforresearchpurposesonly. Nolicenseisgrantedorimplied Molecular Beacons Licensed Technologyand Statements in whichtheproductisused. patented applications may notberesold,distributedorre-packaged.Nolicenseisgrantedimpliedwiththepurchaseofthisproduct.It may benecessarytoobtainaseparatelicenseforcertain forR&Dusebythepurchaser.products are soldexclusively Health andHumanServices.CleanAmp™ or diagnosticpurposesandthey They maynotbeusedforclinical from theDepartment of licensed , andUSPatent6762298 US2010003724 WO2009151921, CN101517091, WO2007139723, EP2032714, US2007281308, CA2653841, is atrademarkof CleanAmp™ TriLink BioTechnologies,Inc. productsorportionsthereofarecoveredby CleanAmp™ TriLinkPatent pending Applications: AU2007268075, CleanAmp BioTechnologies,Inc. TriLink useonly. issellingDADEforresearchanddevelopment requires alicensefrom application Nootherlicenseisgrantedorimplied.Commercial TriLink "DADE" T Black HoleQuenchers(BHQ-1,BHQ-2,BHQ-3),CalFluorGold Biosearch Technologies, Inc. TriLink Logo,OligoBuilder TriLink BioTechnologies, Inc. Cy3, Cy3.5,Cy5,Cy5.5 GE Healthcare Trademarks Trademark, Trademark, Patent andLicensingInformation riLink Patents

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153 Information Technical 154 Technical Information Provisional Patent Provisional 61/056,324, May27,2008. Application Lebedev, 5’-TriphosphatesA., I. ModifiedNucleoside Koukhareva Chemically for Thermally Initiated of Nucleic Amplification Acid. Patents andPending Patents Hogrefe, R. An Primer.Antisense Oligonucleotide AntisenseandNucleic Acid DrugDevelopment 40(2): 402-405. Zhang, P., Beck, T., Tan, W. Design ofaMolecularBeaconDNA Probewith Two Fluorophores. approach. Lebedev, A., Koukhareva, I., Beck, T., Vaghefi,using solidsupport 5’-triphosphates M.ofoligodeoxynucleotide Preparation Fluorescence Anisotropy. Fang, X., Cao, Z., Beck, T., Tan, W. Molecular by Growth FactorMonitoring Aptamer forReal-timeOncoproteinlatelet-derived DNABeacon Molecular Recognition. ProbesforBiomolecular Wang,X., Schuster,J., Fang, K., Li, S.,Vicens,M., Kelley, J., Beck, H., Li, S.,Lou, T.,R., Hogrefe, Tan,W. Fluorescent Novel Nucleotides &Nucleic Nucleosides, Acids 2004,23[1-2]:171-181. Timoshchuk, V., Hogrefe, R., Vaghefi, M.Synthesisof Improved andReliable3'- Derivatives. Azido-2',3'-dideoxyguanosine 1680. Koukhareva, I., Lebedev, & Nucleic Nucleotides A. route tothecappedRNAs.Nucleosides, Chemical Acids 2004,23(10):1667- Nucleoside triphosphates: Koukhareva I., Vaghefe M., and Lebedev A. Synthesis andpropertiesofNTP analogs withmodifiedtriphosphatechainsin C6-(1,2,4-Triazol-1-yl)Derivatives. Nucleosides, Timoshchuk, V.Substitution of at C4andC6:C-Nucleophilic and PurineNucleosides ofPyrimidine Functionalization Their C4-and Interfering RNA Delivery. Pirollo, K., Zon, G., Rait, A., Zhou, Q., Yu, W., Hogrefe, R., Chang, E. Tumor-TargetingComplex forShort Nanoimmunoliposome Journal ofPhysicalChemistry A. 2006,110: 4320-4328. Conjugates. in Anthraquinonyl-Linker-Deoxyadenosine Dynamics and Energetics Charge-Transferon State AQ•-/dA•+ Influences Hussein, Y., Anderson, N., Lian, T., Abdou, I., Strekowski, L., Victor A. Timoshchuk, V., Vaghefi, M., Netzel, T. Solvent andLinker Acids 2006,25:889-907. hybrids (siHYBRIDS):deliveryinvitroandvivoforRNAiofHER-2. Nanoimmunoliposome Hogrefe, R., Lebedev, A., Zon, G. Kathleen F. Pirollo K., Rait A., Zhou O., Yu, W., Chang E.modifiedshortinterfering Chemically DNA chainextension. polymerasemediated Nawrot, B., Paul, N., Rebowska, B.,primerin Stec, J. of stereochemistry 3'-terminalphosphorothioate-modified Significance or cytosine. analogs ofguanine with weaklypairing G., Lahoud, Timoshchuk,V., Lebedev, A., Arar, K.,Hou, Y., Gamper,Jr. H., DNApseudo-complementary of Properties substituted Structure-Free DNAResearch Properties. NucleicAcids withPseudo-Complementary G., Lahoud, Timoshchuk,V., Lebedev, Vega,A., De M., M., Salas, Arar, K.,Hou, Y., Gamper, H.,Jr.of Synthesis Enzymatic 10: 259-263. Republic, 5’-Triphosphates with Thermolabile 3’-Protecting Group. Koukhareva I., Huang, H., Yee, J., Paul, N., Hogrefe, R., Lebedev, A. A new Approach toImprovedHotStart PCR: Nucleoside for HotStart Application PCR. Koukhareva I., Huang, H., Yee, J., Shum, J., Paul, N., Hogrefe, R., Lebedev, A. Heat dNTPs: Synthesisand Activatable 3’-Modified 266-272. Shum, J.,modifiedprimersforimprovedmultiplexpolymerasechainreaction. Paul, N.Chemically Nucleotides. Fluorescent andDouble-Headed Timoshchuk, V., Hogrefe, R. as aModifying The “CoreyReagent”,3,5-Di-tert-butyl-1,2-benzoquinone, Agent intheSynthesisof and Real-Time Experimentsin Ashrafi, E., Paul, N. Heat PrimersforHotStart Activatable Primers forHotStart PCR andHotStart One-step RT-PCR -EndPoint Chemistry, Wiley Interscience2009(inpress). Nucleic Acid Lebedev, A. Heat Activatable PrimersforHotStartSynthesisandBasicPCRSet-upinCurrentProtocols PCR: Oligonucleotide PCR. Koukhareva, I., Lebedev, 5’-TriphosphatesA. 3’-Protected 2’-Deoxynucleoside as aNovel Tool forHeat-Triggered Activation of Publications TriLink's TriLink Publications Small InterferingRNA viaa Tumor-TargetingSystem. Nanodelivery K., Rait, Pirollo, of Potential the E. Materializing G., Chang, R., Palchik, G., Hogrefe, J., Zon, S.,Dagata, Q., Hwang, A., Zhou, Solid Support.BioconjugateChemistry2007,18:1530-1536. Lebedev, A., Combs, D., Hogrefe, R.of Preactivated Carboxyl LinkerfortheRapidConjugation on to Oligonucleotides Alkylamines Analytical Chemistry2009,81(inpress). Nucleosides, Nucleotides &Nucleic Nucleotides Nucleosides, Acids 2001,4-7[20]:1403-1409. Publications, Patentsand Presentations Human GeneTherapy2006,17:117-124. American Chemical Society 2001,73(23):5752-5757. American Chemical Chemistry, Biotechnology and Biological Applications Biological Chemistry,and Biotechnology Nucleic Nucleic Acids SymposiumSeries2008,OxfordUniversityPress,No.52:259-260. Current Protocols in Molecular Biology, Current ProtocolsinMolecularWileyInterscience2009(inpress). Molecular Biotechnology Nucleosides, Nucleotidesand Nucleic Acids 2009(inpress). Nucleotides and Nucleic Acids 2005,24:1043-1046. and Nucleotides 2008,36:6999-7008. Research Nucleic Acids www.trilinkbiotech.com Collection Symposium Series2008, Collection Academy ofSciencestheCzech Biomedical Photonics Handbook Biomedical Cancer Research 2008,40(2):119-126. , Taylor andFrancis2005:39-104. 2007,67(7):2938-2943. 2008,36:3409-3419. Nucleosides, Nucleotides& Nucleic 2003,57:1-23. Wiley-VCH Verlag GmbH2001, 1999,9:351-357. Analytical Chemistry2009,388: Timoshchuk, V., Vaghefi. M. Derivatives. Synthesis of3’-Azido-2’,3’-dideoxyguanosine Improved andReliable Timoshchuk, V. Triazol-1-yl) Derivatives. Substitution of at C4andC6:C-Nucleophilic of andPurineNucleosides Functionalization Pyrimidine Their C4-andC6-(1,2,4- Lebedev,I., A. Koukhareva, RNAs. Route totheCapped Chemical Lebedev, A., Hogrefe, R., Paul, N., Timoshchuk, V., Yee, J., Zon, G. PrimersforImprovedHotStart Modified PCR. Chemically Lebedev, N., A., Paul, Yee, J., J., Kellum, Timoshchuk,V., G. R., Zon, J., Hogrefe, Shum, PrimersforHotStart Modified PCR. Chemically Koukhareva, I., Huang, H., Yee, J., Paul, N., Hogrefe, R., Lebedev, A. A NewGeneral Approach toHotStart5'-TriphosphatesPCR: Nucleoside with 3'-Protecting Group. Thermolabile Yee,E., Lebedev,J., Hildalgo-Mendez, G., Le, R., Zon, A., Hogrefe, T.,J. N., Shum, Paul, PCR. PrimersforImproved Multiplexed Modified Chemically Shum, J., Le, T., Yee, J., Huang, H., Timoshchuk V., Paul, N., Hogrefe, R., Koukhareva, I., Lebedev, A. dNTPsforImproved HotStart Modified PCR. Chemically Posters Peptide TechnologyLa Costa,CA,May1-4,2006. andProductDevelopment, Industry.for theDiagnostic Oligonucleotides of SynthesizingRandomer The Challenge Beck, T. TIDES: and Oligonucleotide Based Technologies Meeting, Arlington, VA, June 26-28,2006. PrimersforImprovedHotStart Modified PCR. Paul,N.CambridgeHealthtech Institute’sChemically 14th Annual Nucleic Acid- Unique toHotStart Approaches PCR. Paul, N.QuantitativePCRMeeting,SanDiego,CA, April 22,2008. Symposium onChemistryofNucleic Acid ComponentsConference,CzechRepublic,June8,2008. A NewGeneral Approach toHotStart5'-TriphosphatesPCR: Nucleoside with 3'-Protecting Group.Lebedev,Thermolabile A. XIVth 18-20, 2008. dNTPsforImprovedHotStart Modified PCR. Paul,N.16thNucleic Chemically Acid-Based Technologies,Baltimore, MD,June USA, Millbrae,CA,November10-13,2008. Modified dNTPsforImprovedPCRPerformance– Protecting Groups.Paul,N.qPCRSymposium An InnovativeuseofNucleoside 9-11, 2009. The NextGenerationinHotStartPrimersanddNTPs.Paul,N.qPCREvent2009,Munich,Germany,PCR -CleanAmp™ March The NextGenerationinHotStartdNTPs.Paul,N.QuantitativePCR.SanDiego,CA,March16-18,2009. PCR -CleanAmp™ and Peptide Oligonucleotide Technologyand ProductDevelopment,LasVegas, NV, May 17-20,2009. Modified dNTPsforImprovedPCRPerformance– Protecting Groups.Paul,N. An InnovativeuseofNucleoside TIDES: Presentations Hogrefe, R., Vaghefi, M.Patent6,320,041,November20,2001. Pre-activated carbonyllinkersformodificationofoligonucleotides. December6,2007. WO2007139723, CA2653841, EP2032714,US2007281308, AU2007268075, Zon, G., Lebedev, Primers forNucleic A. ModifiedOligonucleotide Chemically Acid Amplification. Patent Applications [email protected] [email protected] Gordon Research Conference, Salve Regina University,Gordon Research Conference,SalveRegina Newport, RI, July 20-25,2003. and Nucleic Nucleotides XVII on Nucleosides, International Roundtable MN, September12-16,2004. Acids, Minneapolis, and Nucleic Nucleotides on Nucleosides, XVI Roundtable International MN, September 12-16,2004. Acids, Minneapolis, Nucleic Acid Based Technologies, Arlington, VA, June 26,2006. and Nucleic Nucleotides XVII onNucleosides, International Roundtable Acids, Bern,Switzerland, September3-7,2006. Conference, Boston,MA, Discovery 2DiagnosticsSeptember25-27, 2006. Quantitative PCR,SanDiego,CA,March19-21,2007. and Peptide Oligonucleotide Technologyand ProductDevelopment,LasVegas, CA,May21-23,2007. Gordon ResearchConference(Nucleic Acids), Newport, RI, June 3,2007. Newport, RI, & Oligonucleotides),July1,2007. Nucleotides, Gordon ResearchConference(Nucleosides, Quantitative PCR,SanDiego,CA, April 21-23,2008. and Peptide Oligonucleotide Technologyand ProductDevelopment,LasVegas, CA,May19-21,2008. XIVth Symposium onChemistryofNucleic June 8,2008. Acid Components,CzechRepublic, Beyond Genome,SanFrancisco,CA,June8-11, 2008. XIVth Symposium onChemistryofNucleic June 8,2008. Acid Components,CzechRepublic, Discovery,Data-Driven Providence, RI, September 22-25,2008. San Diego,CA,October20-22,2008. 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TriLink Publications 155 Information Technical 156 Index Amino Linkers Aminoallyl Nucleoside TriphosphatesAminoallyl Alexa FluorDyeSeries ...... 75 Adenosine-5'-O-(1-Thiotriphosphate) Acridine...... 32 Abasic ...... 32 ...... 41 5'-Aminothymidine A Amino ModifiedBasesforOligonucleotides Amino ModifiedNucleoside Triphosphates Selective Placement Amino Linkers 5' and3' Amino Linkers 62 5-Aminoallylcytidine-5'-Triphosphate ...... 52, 56 ...... 52, 5-Aminoallyl-2'-deoxyuridine-5'-Triphosphate 62 5-Aminoallyluridine-5'-Triphosphate ...... 52, -mnall2-exctdn-'Tihsht ...... 2 56 5-Aminoallyl-2'-deoxycytidine-5'-Triphosphate ...... 52, Alexa Fluor700,750 ...... 29 Alexa Fluor430, ...... 23 Alexa Fluor350,405 ...... 21 2-Aminopurine-2'-O-methylriboside ...... 44 2-Aminopurine-2'-O-methylriboside ...... 39 2-Aminopurine-2'-deoxyriboside ...... 43 2-Aminopurine Alexa Fluor 660, 680, 633, 647 Alexa Fluor660,680,633,647 ...... 28 Alexa Fluor594,610 ...... 27 Alexa Fluor568 ...... 26 Alexa Fluor546,555 ...... 25 Alexa Fluor514,532 ...... 24 Alexa Fluor488 ...... 22 2'-Amino-2'-deoxyguanosine-5'-Triphosphate ...... 67 2'-Amino-2'-deoxycytidine-5'-Triphosphate ...... 67 2'-Amino-2'-deoxyadenosine-5'-Triphosphate ...... 67 ...... 39 8-Amino-2'-deoxyguanosine ...... 39 8-Amino-2'-deoxyadenosine ...... 21 7-Aminocoumarin-X 2'-Deoxyadenosine-8-C6 Amino Linker 2'-Deoxyadenosine-8-C6 Amino 5' C6andC12 Amino Linkers ...... 34 3' C3,C6andC7 Amino Linkers ...... 34 2'-Deoxyguanosine-8-C6 Amino Linker 2'-Deoxyguanosine-8-C6 Amino Linker 2'-Deoxycytidine-5-C6 Amino Thymidine-5-C2 andC6 Amino Linker ...... 35 C7 Internal Amino Linker ...... 35 ...... 52 ...... 35 ...... 35 ...... 35 www.trilinkbiotech.com Azido ModifiedNucleoside Triphosphates Aza ModifiedNucleoside Triphosphates Ara ModifiedNucleoside Triphosphates ...... 120 Oligonucleotides Antisense Biotin ModifiedNucleoside Triphosphates Benzimidazole-riboside-5'-Triphosphate ...... 63 B 8-Azidoadenosine-5'-Triphosphate ...... 63 ...... 75 3'-Azido-3'-deoxythymidine-5'-O-(1-Thiotriphosphate) 3'-Azido-2',3'-dideoxyuridine-5'-Triphosphate ...... 69 3'-Azido-2',3'-deoxythymidine-5'-Triphosphate ...... 69 3'-Azido-2',3'-dideoxyguanosine-5'-Triphosphate ...... 69 3'-Azido-2',3'-dideoxycytidine-5'-Triphosphate ...... 69 Aracytidine-5'-Triphosphate ...... 68 3'-Azido-2',3'-dideoxyadenosine-5'-Triphosphate ...... 68 2'-Azido-2'-deoxyuridine-5'-Triphosphate ...... 68 2'-Azido-2'-deoxyguanosine-5'-Triphosphate ...... 68 2'-Azido-2'-deoxycytidine-5'-Triphosphate ...... 68 2'-Azido-2'-deoxyadenosine-5'-Triphosphate ...... 68 8-Azaadenosine-5'-Triphosphate ...... 62 6-Azauridine-5'-Triphosphate ...... 62 6-Azacytidine-5'-Triphosphate ...... 62 6-Aza-2'-deoxyuridine-5'-Triphosphate ...... 56 Arauridine-5'-Triphosphate ...... 68 Araadenosine-5'-Triphosphate ...... 68 3'-Amino-3'-deoxythymidine-5'-Triphosphate ...... 67 itn1-mnallCP...... 5, 63 Biotin-16-Aminoallyl-CTP ...... 52, 57 Biotin-16-Aminoallyl-2'-dUTP ...... 52, 56 Biotin-16-Aminoallyl-2'-dCTP ...... 52, Biotin-16-7-Deaza-7-Aminoallyl-2'-dGTP ...... 52 3'-Amino-2',3'-dideoxyguanosine-5'-Triphosphate ...... 67 3'-Amino-2',3'-dideoxycytidine-5'-Triphosphate ...... 67 2-Aminoadenosine-5'-Triphosphate ...... 62 2-Amino-6-chloropurineriboside-5'-Triphosphate ...... 62 2-Amino-6-chloropurine-2'-deoxyriboside-5'-TP...... 56 2'-Amino-2'-deoxyuridine-5'-Triphosphate...... 67 3'-Amino-2',3'-dideoxyadenosine-5'-Triphosphate ...... 67 2-Aminopurine-2'-deoxyriboside-5'-Triphosphate ...... 56 La Jolla,California.©istockphoto.com/JGould ...... 52 Cytidine-5'-O-(1-Thiotriphosphate) ...... 75 Cytidine-5'-O-(1-Thiotriphosphate) Cyanine DyeSeries Custom ChemistryandContractServices...... 7 ...... 20 Oligonucleotides Conjugates, 2',3'-Dideoxy ModifiedBasesfor Oligonucleotides D ( Carboxy Thymidine ...... 24 6G Carboxyrhodamine 6-Chloropurineriboside-5'-Triphosphate ...... 64 6-Chloropurine-2'-deoxyriboside-5'-Triphosphate ...... 57 ...... 41 5-(Carboxy)vinyl-2'-deoxyuridine C BODIPY DyeSeries Black HoleQuenchers ...... 74 Nucleosides Bisphosphate Biotin OligonucleotideConjugates...... 31 CleanAmp Cholesteryl TEG...... 32 ...... 21 Blue Cascade ...... 27 Carboxy-X-Rhodamine Carboxynaphthofluorescein DADELinker Carboxyl Linkers-See CAL FluorDyeSeries C4-(1,2,4-Triazol-1-yl)-2'-deoxyuridine ...... 42 8-Chloro-2'-deoxyadenosine-5'-Triphosphate ...... 57 CAP andmCAP 74 ...... 53, Bromo ModifiedNucleoside Triphosphates Bromo ModifiedBasesforOligonucleotides CleanAmp y, 5.5...... 28 Cy5, Cy3.5 ...... 27 Cy3 ...... 25 ...... 31 Conjugates Non-Fluorescent CleanAmp CleanAmp 2',3'-Dideoxythymidine ...... 45 2',3'-Dideoxythymidine ...... 45 2',3'-Dideoxyguanosine ...... 45 2',3'-Dideoxycytidine ...... 45 2',3'-Dideoxyadenosine N 63 Biotin-16-Aminoallyl-UTP ...... 52, BODIPY-493/503 ...... 22 CAL FluorGold540 ...... 23 BODIPY-530/550 ...... 24 CAL FluorRed610,635 ...... 27 CAL FluorRed590 ...... 26 CAL FluorOrange560 ...... 24 5-Bromouridine-5'-Triphosphate ...... 63 5-Bromocytidine-5'-Triphosphate ...... 63 5-Bromo-2'-deoxyuridine-5'-Triphosphate ...... 57 5-Bromo-2'-deoxycytidine-5'-Triphosphate ...... 57 5-Bromo-2',3'-dideoxyuridine-5'-Triphosphate ...... 69 ...... 39 8-Bromo-2'-deoxyguanosine ...... 39 8-Bromo-2'-deoxyadenosine ...... 44 5-Bromo-2'-O-methyluridine ...... 41 5-Bromo-2'-deoxyuridine ...... 41 5-Bromo-2'-deoxycytidine BODIPY-TR-X ...... 27 ...... 26 581/591 BODIPY-576/589, ...... 25 564/570 BODIPY-558/568, 5-Bromouridine ...... 43 5-Bromouridine 4 Boi-BA2-CP...... 2 57 -Biotin-OBEA-2'-dCTP ...... 52, Turbo vs.PrecisionPrimers...... 87 Procedure forSynthesis ...... 99–101 ...... 93–98 Note Application ...... 90–92 Note Application ...... 89 Syntheses Successful Procedure forSynthesis ...... 99 TM Products...... 82–101 TM TM TM Primers ...... 86–87 dNTPs...... 84–85 Amidites...... 88–89 5-(Carboxy) vinyl-2'-deoxyuridine)...... 41 [email protected] [email protected] ...... 28 14, 30,128 3'-Deoxy ModifiedNucleoside Triphosphates 3'-Deoxy ModifiedBasesforOligonucleotides 2'-Deoxy ModifiedNucleoside Triphosphates 3-Deaza-5-aza-2'-O-methylcytidine ...... 44 3-Deaza-5-aza-2'-O-methylcytidine Dansyl-X ...... 22 Dansyl-X DADE Linker (5' and Internal) DADE Linker(5'andInternal) ...... 36 7-Deaza ModifiedBasesforOligonucleotides 5,6-Dihydrouridine-5'-Triphosphate ...... 64 5,6-Dihydrothymidine...... 41 ...... 41 5,6-Dihydro-2'-deoxyuridine 2'-Deoxy ModifiedBasesforOligonucleotides ...... 44 2,6-Diaminopurine-2'-O-methylriboside ...... 39 2,6-Diaminopurine-2'-deoxyriboside ...... 43 2,6-Diaminopurine 2,4-Difluorotoluyl 2',3'-Dideoxy ModifiedNuceloside Triphosphates DABCYL K-2'-deoxyriboside ...... 46 K-2'-deoxyriboside 7-Deaza ModifiedNucleoside Triphosphates 2'-Deoxy-P-nucleoside-5'-Triphosphate ...... 58 3'-Deoxyadenosine-5'-Triphosphate ...... 70 3'-Deoxy-5-methyluridine-5'-Triphosphate ...... 70 ...... 45 3'-Deoxythymidine ...... 3'-Deoxyguanosine ...... 45 3'-Deoxycytidine ...... 45 3'-Deoxyadenosine 2'-Deoxyzebularine-5'-Triphosphate ...... 59 2'-Deoxyuridine-5'-Triphosphate ...... 59 76 ...... 75, 2'-Deoxythymidine-5'-O-(1-Thiotriphosphate) 2'-Deoxypseudouridine-5'-Triphosphate ...... 59 2'-Deoxy-L-thymidine-5'-Triphosphate ...... 70 2'-Deoxy-L-guanosine-5'-Triphosphate ...... 69 2'-Deoxy-L-cytidine-5'-Triphosphate ...... 69 2'-Deoxy-L-adenosine-5'-Triphosphate ...... 69 2'-Deoxyinosine-5'-Triphosphate ...... 58 ...... 75 2'-Deoxyguanosine-5'-O-(1-Thiotriphosphate) ...... 75 2'-Deoxycytidine-5'-O-(1-Thiotriphosphate) ...... 75 2'-Deoxyadenosine-5'-O-(1-Thiotriphosphate) ...... 75 2'-Deoxycytidine-5'-O-(1-Boranotriphosphate) ...... 41 2'-Deoxyuridine 3'-Deoxyguanosine-5'-Triphosphate ...... 70 3'-Deoxycytidine-5'-Triphosphate ...... 70 ...... 41 2'-Deoxypseudouridine ...... 46 2'-Deoxynebularine ...... 46 2'-Deoxyisoguanosine 2',3'-Dideoxyuridine-5'-Triphosphate ...... 71 ...... 76 2',3'-Dideoxyuridine-5'-O-(1-Thiotriphosphate) 2',3'-Dideoxythymidine-5'-Triphosphate ...... 71 2',3'-Dideoxyinosine-5'-Triphosphate ...... 71 2',3'-Dideoxyguanosine-5'-Triphosphate ...... 71 ...... 76 2',3'-Dideoxycytidine-5'-O-(1-Thiotriphosphate) 2',3'-Dideoxyadenosine-5'-Triphosphate ...... 70 Applications and Methods Applications andMethods ...... SE...... 30 DABCYL DABCYL-dT ...... 30 7-Deazaadenosine-5'-Triphosphate ...... 64 ...... 39 7-Deaza-2'-deoxyadenosine 3'-Deoxyuridine-5'-Triphosphate ...... 70 ...... 76 3'-Deoxythymidine-5'-O-(1-Thiotriphosphate) ...... 46 2'-Deoxyinosine ...... 76 2',3'-Dideoxyguanosine-5'-O-(1-Thiotriphosphate) 2',3'-Dideoxycytidine-5'-Triphosphate ...... 70 ...... 76 2',3'-Dideoxyadenosine-5'-O-(1-Thiotriphosphate) 5-(Dabcyl-3-Aminoallyl)-2'-deoxyuridine-5'-Triphosphate .58 CPG ...... 30 3'-DABCYL 7-Deazaguanosine-5'-Triphosphate ...... 64 7-Deaza-2'-deoxyguanosine-5'-Triphosphate ...... 58 ...... 39 7-Deaza-2'-deoxyguanosine 7-Deaza-2'-deoxyadenosine-5'-Triphosphate ...... 58 ...... 39 7-Deaza-8-aza-2'-deoxyadenosine ...... 39 7-Deaza-2'-deoxyxanthosine ...... 41 800 863-6801 800 ...... 45

117 157 Index 158 Index Iodo ModifiedBasesforOligonucleotides ...... 33 Oligonucleotides Inverted Inosine-5'-Triphosphate ...... 64 ...... 43 Inosine I 5-Hydroxy-2'-deoxycytidine-5'-Triphosphate ...... 59 H Guanosine-5'-Triphosphate-5'-Guanosine (GpppG,CAP) 74 ...... 53, ...... 76 Guanosine-5'-O-(1-Thiotriphosphate) ...... 74 Guanosine-3',5'-Bisphosphate ...... 74 Guanosine-3',5'-Bisdiphosphate Grants ...... 153 Ganciclovir-5'-Triphosphate ...... 72 G ...... 22 (Fluorescein) 6-FAM 2' FluoroRNA Oligonucleotides ...... 19 F N 5-(C2-EDTA)-2'-deoxyuridine ...... 41 E ...... 10 Diagnostics/OEM Deprotection Methods,Phthalimidyl Amino CPG ...... Hydroxy ModifiedBasesforOligonucleotides Extinction Coefficients ...... 39 Etheno-2'-deoxyadenosine Hot StartPCRProducts-SeeCleanAmp HEX ...... 24 5-Hydroxy-2'-deoxyuridine-5'-Triphosphate ...... 59 Flouro ModifiedBasesforOligonucleotides of Duplex, Thermostability dT-TAMRA ...... 26 dT-FAM ...... 23 DTAF ...... 22 ...... 37 dSpacer DNP-X ...... 31 DNP TEG...... 32 DNA, StandardSynthesisOligonucleotides ...... 17 InsideBackCover Distributors-See ...... 22 Dimethylaminocoumarin Dimer,Cis-Syn Thymidine Diaminopurine Ribose(2-Aminoadenosine-5'-Triphosphate) ..62 Fluoro ModifiedNucleoside Triphosphates Fluorescent Dyes...... 20–30 4 -Ethyl-2'-deoxycytidine ...... 41 -Ethyl-2'-deoxycytidine 8-Hydroxy-2'-deoxyguanosine ...... 60 8-Hydroxy-2'-deoxyguanosine 5'-Iodothymidine ...... 45 5'-Iodothymidine ...... 42 5-Iodo-2'-deoxyuridine ...... 42 5-Iodo-2'-deoxycytidine 8-Hydroxy-2'-deoxyadenosine ...... 60 8-Hydroxy-2'-deoxyadenosine ...... 41 5-Hydroxymethyl-2'-deoxyuridine ...... 42 5-Hydroxy-2'-deoxyuridine ...... 41 5-Hydroxy-2'-deoxycytidine 5-Fluoro-2'-O-methyluridine ...... 44 5-Fluoro-2'-O-methyluridine ...... 41 5-Fluoro-2'-deoxyuridine 5-Fluoro-2'-deoxyuridine-5'-Triphosphate ...... 59 2'-Fluoro-thymidine-5'-Triphosphate ...... 71 2'-Fluoro-2'-deoxyuridine-5'-Triphosphate...... 72 2'-Fluoro-2'-deoxyguanosine-5'-Triphosphate ...... 71 2'-Fluoro-2'-deoxycytidine-5'-Triphosphate ...... 71 2'-Fluoro-2'-deoxyadenosine-5'-Triphosphate ...... 71 Dye SelectionChart ...... 129 ...... 44 5-Fluoro-4-O-TMP-2'-O-methyluridine ...... 138, 139 ...... 138, ...... 47 ...... 134 TM Products www.trilinkbiotech.com 116 N 7-Methoxycoumarin ...... 21 7-Methoxycoumarin ...... 44 5-Methyl-2'-O-methylthymidine Monophosphates ...... 77 Monophosphates Molecular Weight Calculation ...... 138–139 Molecular Beacons ...... 14–15 Beacons Molecular Modified BasesforOligonucleotides Mid-Scale OligonucleotideSynthesis ...... 13 Methylphosphonate Oligonucleotides...... 19 Iodo ModifiedNucleoside Triphosphates 5-Nitro-1-indolyl-2'-deoxyribose-5'-Triphosphate ...... 60 N Methyl ModifiedNucleoside Triphosphates M Linkers-See Amino Linkers,Carboxyl Thiol Linkers ...... 153–154 Licensing L 6-JOE ...... 24 J Methyl ModifiedNucleoside Triphosphates Methyl ModifiedBasesforOligonucleotides ...... 21 Blue Marina (7-Me-GppG,mCAP) 74 ...... 53, 7 -Me-Guanosine-5'-Triphosphate-5'-Guanosine 5-Methyluridine-5'-Triphosphate ...... 65 5-Methylcytidine-5'-Triphosphate ...... 64 5-Methyl-2'-deoxycytidine-5'-Triphosphate ...... 60 3'-O-Methyluridine-5'-Triphosphate ...... 73 Sugar Modified Analogs Ribonucleoside Analogs Ribonucleoside Analogs ...... 44 2'-O-Methyl N ...... 45 5'-O-Methylthymidine 3'-O-Methylguanosine-5'-Triphosphate ...... 73 3'-O-Methylcytidine-5'-Triphosphate ...... 73 2'-O-Methyl-5-methyluridine-5'-Triphosphate ...... 72 2'-O-Methyluridine-5'-Triphosphate ...... 73 Wobble andUniversalBases ...... 46 2' Deoxyribonucleoside Analogs -Pyrimidines ...... 41 2' Deoxyribonucleoside Analogs -Purines ...... 39 N N N N 5-Methyluridine ...... 43 5-Methyluridine ...... 43 5-Methylcytidine 5-Iodo-2'-deoxyuridine-5'-Triphosphate ...... 59 5-Iodo-2'-deoxycytidine-5'-Triphosphate ...... 59 ...... 43 5-Iodouridine 3'-O-Methyladenosine-5'-Triphosphate ...... 73 2'-O-Methylpseudouridine-5'-Triphosphate ...... 73 2'-O-Methylinosine-5'-Triphosphate ...... 72 2'-O-Methyladenosine-5'-Triphosphate ...... 72 2'-O-Methyl-2-aminoadenosine-5'-Triphosphate ...... 72 DNA LinkerandSpacerReagents Their Utility ...... N N 5-Methyl-2'-O-methylthymidine ...... 38, 44 ...... 38, 5-Methyl-2'-O-methylthymidine ...... 44 5-Methyl-2'-O-methylcytidine ...... 46 5-Methyl-2'-Deoxyisocytidine ...... 42 5-Methyl-2'-deoxycytidine ...... 44 2'-O-Methylinosine 5-Iodouridine-5'-Triphosphate ...... 64 5-Iodocytidine-5'-Triphosphate ...... 64 6 6 4 2 1 1 6 -Methyladenosine-5'-Triphosphate ...... 65 -Methyl-2'-deoxyadenosine-5'-Triphosphate ...... 60 -Methyl-2'-deoxycytidine-5'-Triphosphate ...... 60 -Methyl-2'-deoxyguanosine-5'-Triphosphate ...... 60 -Methylguanosine-5'-Triphosphate ...... 65 -Methyladenosine-5'-Triphosphate ...... 65 ...... 39 -Methyl-2'-deoxyadenosine ...... 45 ...... 43 ...... 38 112 Oligonucleotides O O O O Nucleotide Kits O 8-Oxoguanosine-5'-Triphosphate ...... 65 8-Oxo-2'-deoxyguanosine-5'-Triphosphate ...... 60 ...... 40 8-Oxo-2'-deoxyguanosine 8-Oxo-2'-deoxyadenosine-5'-Triphosphate ...... 60 ...... 40 8-Oxo-2'-deoxyadenosine O Nucleoside Triphosphates NBD-X ...... 23 NimbleGen, RocheValidated RandomNonamers ...... 6 6 6 6 4 -Phenyl-2'-deoxyinosine ...... 39 -Phenyl-2'-deoxyinosine -Methylguanosine-5'-Triphosphate ...... 65 -Methyl-2'-deoxyguanosine-5'-Triphosphate ...... 60 ...... 39 -Methyl-2'-deoxyguanosine -Methylthymidine...... 42 Therapeutic Services ...... 12 Services Therapeutic Synthesis ...... 9 Stocked RandomerProducts ...... 48 Spacer Modifications Roche NimbleGenValidated RandomNonamers ...... RNA-See RNA ...... 11,Randomers 48 ...... 30 Quenchers Purification Primer GradeDNA ...... 16 Pricing Pricing-See ...... 17 Phosphorothioate Post-Synthetic ModificationReagents Hybridization ModulationKits ...... 80 Phosphodiester ...... 17 Phosphodiester 138–139 Weight ...... Molecular ...... 10 Diagnostics/OEM Methods Common Analytical Clinical Trials (Oligonucleotidesin) ...... 122 CleanAmp Dimer Cis-Syn Thymidine ...... 120 Primers Antisense 5'-5' Modifications 3'-3' Modifications Chain TerminationKits ...... 78 Alpha-Thiol NucleotideKits ...... 78 2' SugarModifiedRNA Kits...... 79 Process ...... 140 SELEX Guide ...... 54–55 Quick Overview ...... 51 Enzymatic Activity...... 141 Custom SynthesisService ...... 51 ...... 84 dNTPs CleanAmp CAP andmCAP ...... 53 Nucleoside TriphosphatesBiotin ...... 150 Bibliography Nucleoside TriphosphatesAminoallyl Molecular Beacons ...... 14–15 Beacons Molecular Modified Bases ...... 13 Synthesis Mid-Scale 36 Linker Modifications Inverted ...... 33 138–139 Extinction Coefficient Dyes-See FluorescentDyes DNA ...... 17 Alpha PhosphateModifiedNucleoside Triphosphates ...... 75–77 Alliance, TriLink-Avecia ...... 12 2' FlouroNucleoside Triphosphates ...... 53 Troubleshooting ...... 109 Short Historyof ...... 103 TM -See Purification Primers ...... 86 ...... 38–49 ...... 33 ...... 33 ...... 34, 35, ...... 34, ...... 37 ...... [email protected] [email protected] ...... 50–80 ...... 47 ...... 135 ...... 52 ...... 52 ...... 79 11 11 Primers Optical DensityUnit(OD A260 unit) ...... 138 Psoralen C2, C6 Psoralen C2,C6 ...... 32 PCR Products-SeeCleanAmp Payment-See InsideFrontCover Patent andLicensingInformation ...... 152 Pacific Blue P130 ...... Pricing...... 9 Phthalimidyl Oregon Green Dye Series Oregon GreenDyeSeries ...... 22–23 InsideFrontCover Ordering-See Purification Publications, TriLink's PatentsandPresentations ...... 154 Pseudouridine-5'-Triphosphate ...... 65 Nucleic Acids ...... 130 Pseudo-complementary Propyl P-2'-deoxyriboside...... 46 C3 (Propyl)Spacer ...... 37 5-Propynyl-2'-deoxyuridine-5'-Triphosphate ...... 61 ...... 42 5-Propynyl-2'-deoxyuridine 5-Propynyl-2'-deoxycytidine-5'-Triphosphate ...... 61 ...... 42 5-Propynyl-2'-deoxycytidine P Phosphorylation Reagents,Useof ...... CustomSynthesis...... 51 Phosphoramidite Pyrrolo-2'-deoxycytidine ...... 42 Pyrrolo-2'-deoxycytidine PyMPO ...... 25 Puromycin-5'-Triphosphate ...... 66 Puromycin...... 43 Purity-See Purification Pyrrolocytidine ...... 43 Pyrrolocytidine Oligonucleotides ...... 17–19 Oligonucleotides Nucleoside Triphosphates NTP QuickGuide ...... 54 Yields, Expected ...... 138 Conversion Unit Synthesis ...... 47 Trimer Thermostability ofDuplexes ...... 134 CleanAmp Standard DNA Oligonucleotide ...... 17 Simple OligonucleotidePricing ...... 9 Primer GradeDNA Oligonucleotides ...... 16 ...... 15 Beacons Molecular CleanAmp 2' O-MethylRNA ...... 18 2' OHRNA ...... 18 2' FluoroRNA ...... 19 Effect on Yield ofConjugatedProduct ...... Methods Deprotection Mid-scale Oligonucleotides ...... 13 Oligonucleotides Mid-scale ...... 19 Methylphosphonate C3 (Propyl) Spacer C3 (Propyl)Spacer ...... 37 Primer GradeDNA Oligonucleotides ...... 16–17 DNA Purification 3', 5'and Terminal Phosphates ...... 33 RNA Purification Oligonucleotide ConjugatesPurification When is my Oligonucleotide Pure Enough? When ismyOligonucleotidePure Enough? ...... 135 Primer Grade Oligonucleotides Primer GradeOligonucleotides ...... 16 DNA SynthesisScales ...... 17 Understanding OligonucleotideSynthetic Yields ...... RNA SynthesisScales ...... 18 CleanAmp CleanAmp CleanAmp ...... 17 ...... 21 TM TM Primers ...... 86 Products TM TM TM Primers...... 87 dNTPs ...... 85 Amidites...... 88 ...... 17 ...... 18–19 800 863-6801 800 ...... 54–80 TM Products ...... 20

132–134 21 114 116 119 159 Index 160 Index Thio ModifiedNucleoside Triphosphate Thio ModifiedBasesforOligonucleotides Thiol Linkers Quasar DyeSeries Quality Assurance Thermostability ofModifiedOligonucleotideDuplexes Texas-Red-X ...... 26 TET ...... 23 Technical InsideFrontCover Support-See TAMRA-X ...... 25 rSpacer ...... 37 ...... 26 Red-X Rhodamine Rhodamine Green-X...... 23 ...... 153 ResearchRewards Randomer Oligonucleotides RNA R QSY QuencherSeries ...... 30 QSR andcGMP Manufacturing 10 ...... 6–7, Q Therapeutic Oligonucleotide Services Therapeutic OligonucleotideServices ...... 12 ...... 42 6-O-(TMP)-5-F-2'-deoxyuridine 2',4',5',7'-Tetrabromosulfonefluorescein T Spacers ...... 37 InsideFrontCover Shipping-See Process...... 140 SELEX S Quenchers ...... 30 Quenchers Spectral Data ...... 128–129 Data Spectral Quote Request-See InsideFrontCover Quote Request-See Synthesis of Oligonucleotides-See Oligonucleotides, Synthesis Synthesis ofOligonucleotides-See 2-Thio-2'-deoxycytidine-5'-Triphosphate ...... 61 ...... 40 6-Thio-2'-deoxyguanosine ...... 43 4-Thiouridine ...... 42 4-Thiothymidine ...... 42 4-Thio-2'-deoxyuridine ...... 42 2-Thiothymidine 5' C6DisulfideLinker 3' C3DisulfideLinker Quasar 570 NHS Quasar 570NHS ...... 25 Technical Articles, DNA SynthesisandReagents ...... 103–121 Stocked Oligonucleotide Products Stocked OligonucleotideProducts ...... 48 Applications andSynthesis ...... 126 2' O-MethylRNA ...... 18 2' OHRNA ...... 18 2' FluoroRNA ...... 19 Quasar 670NHS ...... 28 Spacer 12 ...... 37 12 Spacer Spacer 9(Triethylene glycol;PEG-150) ...... 37 rSpacer ...... 37 Propyl, C3Spacer ...... 37 ...... 37 dSpacer DNA LinkerandSpacerReagents Their Utility ...... 37 Spacer C6 Spectral Data ...... 128–129 Data Spectral ...... 14 Selection Quencher Spacer 18 (Hexaethylene glycol; PEG-282) Spacer 18(Hexaethyleneglycol;PEG-282) ...... 37 CleanAmp S-18 Randomers ...... 48 Randomers S-18 Roche NimbleGenValidated RandomNonamers ...... 6-FAM (Fluorescein) Antisense OligoControls ...... 48 5' HydroxylLabeledNonamers ...... 48 5' Amine LabeledNonamers ...... 48 TM Primers, Procedure for Synthesis Primers,ProcedureforSynthesis ...... 99 ...... 6–7 ...... 36 ...... 36 ...... 24 ...... 134 www.trilinkbiotech.com 11 112 Trimer OligonucleotideSynthesis ...... 47 ...... 42 Glycol Thymidine Thymidine Dimer, Cis-Syn ...... 47 Yields-See Oligonucleotides,Yields Y Xanthosine-5'-Triphosphate ...... 66 X ...... 76 Uridine-5'-O-(1-Thiotriphosphate) U 6-Thio-2'-deoxyguanosine-5'-Triphosphate ...... 61 4-Thiouridine-5'-Triphosphate ...... 66 4-Thiothymidine-5'-Triphosphate ...... 61 2-Thiouridine-5'-Triphosphate ...... 66 2-Thiothymidine-5'-Triphosphate ...... 61 2-Thiocytidine-5'-Triphosphate ...... 66 Distributors

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