DOCSLIB.ORG

Of a Short-Chain Oligonucleotide Without Inert Atmosphere

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Of a Short-Chain Oligonucleotide Without Inert Atmosphere applied sciences Article A Simple and Efficient Microfluidic System for Reverse Chemical Synthesis (50-30) of a Short-Chain Oligonucleotide Without Inert Atmosphere Rahul Bhardwaj *, Phan T. Tue, Manish Biyani and Yuzuru Takamura * Department of Bioscience and Biotechnology, Japan Advanced Institute of Science and Technology (JAIST), 1-1 Asahidai, Nomi city, Ishikawa 923-1211, Japan; [email protected] (P.T.T.), [email protected] (M.B.) * Correspondence: [email protected] (R.B.); [email protected] (Y.T.); Tel.: +81-761-51-1661 Received: 7 February 2019; Accepted: 28 March 2019; Published: 31 March 2019 Abstract: Reverse DNA synthesis (50-30) plays diverse functional roles in cellular biology, biotechnology, and nanotechnology. However, current microfluidic systems for synthesizing single-stranded DNAs at a laboratory scale are limited. In this work, we develop a simple and efficient polydimethylsiloxane- (PDMS-) based microfluidic system for the reverse chemical synthesis of short-chain oligonucleotides (in the 50-30 direction) under ambient conditions. The use of a microfluidics device and anhydrous conditions effectively surpass the problem of moisture sensitivity during oligonucleotide synthesis. With optimized microfluidic synthesis conditions, the system is able to synthesize up to 21 bases-long oligonucleotides in air atmosphere. The as-synthesized oligonucleotides, without further purification, are characterized using matrix-assisted laser desorption ionization–time of flight (MALDI-TOF/TOF) mass spectroscopy (MS) supported by the denatured polyacrylamide gel electrophoresis (PAGE) analysis. This developed system is highly promising for producing the desired sequence at the nanomolar scale on-chip and on-demand in the near future. Keywords: reverse oligonucleotide synthesis (50-30), PDMS chip; on-chip and on-demand; air atmosphere 1. Introduction The past two decades have witnessed significant advancement in research efforts to incorporate synthetic short-chain ssDNAs into a variety of applications and miniaturized devices, such as oligonucleotide-Chip technology [1–5]. In the miniaturization of devices made of nucleic acids, single-stranded DNAs are the preferred choice, which are largely synthesized autonomously or non-autonomously via a staged process that uses conventional phosphoramidite chemistry [6]. The rapid growth in the use of synthetic oligonucleotides made chemical synthesis (30-50 direction) the standard protocol for preparing oligos using a commercial DNA synthesizer. This occurred because of the easy accessibility of the corresponding protected monomeric building blocks [7]. On the other hand, reverse chemical synthesis (50-30 direction) has been limited to nuclease resistance and hairpin loop applications, where the terminal linkage of natural 50-30 antisense oligonucleotides was modified via the construction of alternating 30-30 or 50-50 internucleotide linkages (terminal capping), and hence has not been matured much yet [8–11]. However, addressed micro-arrays of surface-bound ssDNAs (50-30) with a free 30 terminal group recently found multiple biotechnology applications in genetics, for example, in enzymatic extension via DNA or RNA polymerase at the 30 hydroxyl group, and transcriptomics/genomic analysis of cells using 30-poly T tail-based primers (50-30) containing short-chain oligos as a barcode address-tag (<12 bases) [12,13]. In later applications, researchers have Appl. Sci. 2019, 9, 1357; doi:10.3390/app9071357 www.mdpi.com/journal/applsci Appl. Sci. 2019, 9, 1357 2 of 13 been preparing random sequences for barcode-tags using mix and pool synthesis, which limits the application. Direct reverse synthesis of a specific sequence as an address-tag is attractive to overcome this limitation. Our work presents an application of reverse synthesis which centers upon on-chip and on-demand synthesis of a barcode as an address-tag which can be used for applications like positional information-based transcriptomics and proteomics analysis of single cells. To develop a system for the reverse synthesis of oligonucleotides, microfluidic devices were the favored choice because of their advantages like precise and reproducibly actuate fluids, low reagent consumption, and plentiful quantity production [14]. Most of the microfluidic systems have been limited to continuous flow reactors fabricated in glass or silicon because a variety of organic reagents are needed for chemical syntheses, which limits the workable complexity and reagent savings [15]. In some cases where solvents are mild, it has been possible to use integrated elastomeric material to perform batch syntheses at the nanogram scale [16,17]. Hua et al. developed a chemically resistant microreactor made of a hybrid silicon–elastomer microfluidic system, but with limited chemical characterizations of the products [18], whereas Quake et al. reported a photocurable perfluoropolyether elastomer for oligonucleotide synthesis (50-30)[19,20]. In addition, Quake et al. succeeded in developing mild reagents, which made DNA synthesis feasible on a polydimethylsiloxane- (PDMS-) based microfluidic device [21]. Despite significant advancements, the necessity of an inert atmosphere, skilled operators, and expensive controller units to perform the synthesis is still a significant challenge. Therefore, the development of an efficient system with a simple design and ample product yield under ambient conditions is necessary; this will not only reduce the cost of the process, but also facilitate on-chip and on-demand processing at a laboratory scale. In this paper, we present a simple and low-cost PDMS microfluidic system that enables the synthesis of reverse oligonucleotides (50-30) under ambient conditions suitable for on-chip and on-demand applications. The single-layered microchannel fabrication makes the microfluidic system simple and easy to use at common laboratory scales. We have also optimized the microfluidic conditions, like flow rates and flow times of the synthesis step, to achieve a high yield of the desired sequence. 2. Materials and Methods 2.1. Materials The DNA phosphoramidite monomers and controlled pore glass (CPG) (nominal particle size 110 µm, average pore size = 1000 Å) were purchased from Link Technologies Ltd. (Scotland, UK). Superdehydrated acetonitrile (water < 0.001%, 10 ppm) was purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). A 25–30% NH4OH aqueous solution was purchased from Fluka Analytical (Munich, Germany). The (1S)-(+)-(10-camphorsulfonyl) oxaziridine (CSO) oxidizer, 5-(ethylthio)-1H-tetrazole (ETT activator, 0.25 mol/L solution in anhydrous acetonitrile), 5-(benzylthio)-1H-tetrazole (BTT activator, 0.3 mol/L solution in anhydrous acetonitrile), and anhydrous acetonitrile (MeCN) were purchased from Glen Research (Sterling, VA, USA). The SYBR gold nucleic acid gel stain was purchased from Invitrogen (Carlsbad, CA, USA). Tris-EDTA buffer (pH = 7.5) was purchased from Integrated DNA Technologies (IDT; Coralville, IA, USA). All the DNA oligonucleotides, except the ones that we synthesized from the microfluidic chips, were ordered from IDT and were purified using high-performance liquid chromatography (HPLC). Other chemicals, including tris (hydroxymethyl) aminomethane (Tris), Tris-borate-EDTA (TBE) buffer 10x, poly (vinylpyrrolidone), and TFA, were purchased from Sigma-Aldrich (Tokyo, Japan). 2.2. Fabrication of the PDMS Microfluidic Chip The chip fabrication steps were conducted in a clean room facility at JAIST (see supplementary information, Figure S1) [22]. The PDMS chip with a microchannel was developed from a defined mold structure, made of SU-8 over silicon wafers using photolithography techniques. PTFE tubes Appl. Sci. 2019, 9, 1357 3 of 13 Appl. Sci. 2018, 8, x FOR PEER REVIEW 3 of 13 (ID 1mm, New England Small Tube Co., Litchfield, NH, USA) were inserted into the inlet and outlet holesholes ofof thethe chip,chip, andand sealedsealed thethe gapgap withwith PDMS.PDMS. AnotherAnother endend ofof thethe inletinlet PTFEPTFE tubetube waswas bondedbonded withwith aa needleneedle toto introduceintroduce reagentsreagents fromfrom rubberrubber cappedcapped glass vials (see supplementary information, FigureFigure S2a).S2a). The PDMS chip consisted of a long chamberchamber (W = 2 mm, L = 10 mm, and H = 220 µm),µm), micropillarsmicropillars (W = 120 µm,µm, L = 500 µµm,m, and H = 220 µµm),m), a zigzag channel (W = 400 µµm,m, L = 60 mm, andand HH = 220 µm)µm) near the outlet, and a through-hole (2(2mm) mm) close to inlet (Figure 1 1).). TheseThese micropillarsmicropillars areare designeddesigned (the(the gapgap betweenbetween twotwo pillarspillars waswas setset toto bebe 100 µµm)m) toto traptrap thethe CPGCPG particlesparticles ofof aa 110110 µμm size.size. For CPG loading,loading, 2020 µµlL of of freshly freshly prepared CPGs slurry (60 mg, loading capacitycapacity 25–4025–40 µµmol/g)mol/g) inin 11 mLmL anhydrous anhydrous acetonitrile acetonitrile was was introduced introduced slowly slowly from from the the through-hole through-hole using using a micropipette, a micropipette, and onceand once the micro-chamber the micro-chamber was filled, was filled, CPGs wereCPGs washed were washed with fresh with anhydrous fresh anhydrous acetonitrile. acetonitrile. A column A ofcolumn porous of CPG porous particles CPG wasparticles therefore was packedtherefore inside packed the inside reaction the chamber reaction in chamber a defined in mannera defined of particlesmanner of monolayer particles monolayer (see supplementary (see supplementary information,
Load more

Recommended publications
  • Self-Catalyzed Site-Specific Depurination of Guanine Residues Within Gene Sequences
    Self-catalyzed site-specific depurination of guanine residues within gene sequences Olga Amosova*, Richard Coulter, and Jacques R. Fresco* Department of Molecular Biology, Princeton University, Princeton, NJ 08544 Edited by Jack W. Szostak, Massachusetts General Hospital, Boston, MA, and approved January 19, 2006 (received for review September 28, 2005) A self-catalyzed, site-specific guanine-depurination activity has been found to occur in short gene sequences with a potential to form a stem-loop structure. The critical features of that catalytic intermediate are a 5؅-G-T-G-G-3؅ loop and an adjacent 5؅-T⅐A-3؅ base pair of a short duplex stem stable enough to fix the loop structure required for depurination of its 5؅-G residue. That residue is uniquely depurinated with a rate some 5 orders of magnitude faster than that of random ‘‘spontaneous’’ depurination. In con- trast, all other purine residues in the sequence depurinate at the spontaneous background rate. The reaction requires no divalent cations or other cofactors and occurs under essentially physiolog- ical conditions. Such stem-loops can form in duplex DNA under superhelical stress, and their critical sequence features have been found at numerous sites in the human genome. Self-catalyzed stem-loop-mediated depurination leading to flexible apurinic sites may therefore serve some important biological role, e.g., in nu- cleosome positioning, genetic recombination, or chromosome su- perfolding. ͉ ͉ DNA self-catalysis guanine depurination stem-loop structure Fig. 1. Localization of the cleavage site. A 32P-labeled 8-nt oligomer com- plementary to the 3Ј end of D-1 was used as a primer in a Sanger sequencing epurination in DNA has been recognized as a spontaneous reaction.
    [Show full text]
  • Role of Sam68 in Dna Damage Responses and Tumorigenesis
    ROLE OF SAM68 IN DNA DAMAGE RESPONSES AND TUMORIGENESIS by Xin Sun A dissertation submitted to Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, Maryland Dec 2016 © 2016 Xin Sun All Rights Reserved Abstract The ability to recognize and repair DNA damage through rapid and appropriate DNA damage responses is pivotal to safeguard genomic information, which is persistently challenged by internal and environmental offenses. DNA lesion initiated poly(ADP- ribosyl)ation (PARylation), catalyzed primarily by poly(ADP-ribose) polymerase 1 (PARP1), is one of the earliest post-translational modifications to orchestrate downstream DNA damage response (DDR) signaling. However, the precise mechanisms through which PARP1 is activated and poly(ADP-ribose) (PAR) is robustly synthesized are not fully understood. Converging evidence support the emerging role of RNA- binding proteins (RBPs) in promoting DNA damage repair at different stages of DDR. We discovered Src-associated substrate during mitosis of 68 kDa (Sam68) as a novel- signaling molecule in DDR. In the absence of Sam68, DNA damage-triggered PARylation and PAR-dependent DNA repair signaling were dramatically diminished. With serial cellular and biochemical assays, we revealed that Sam68 is recruited to and significantly overlaps with PARP1 at DNA lesions and that the interaction between Sam68 and PARP1 is crucial for DNA damage-initiated and PARP1-conferred PARylation. Utilizing cell lines and knockout mice, we demonstrated that Sam68- deleted cells and animals are hypersensitive to genotoxicity caused by DNA-damaging agents. In addition, loss of Sam68 delays basal cell carcinoma (BCC) onset and progression in a mouse model for basal cell carcinoma (BCC) of the skin, suggesting Sam68 is required for BCC tumorigenesis, likely through promoting tumor cell survival.
    [Show full text]
  • DNA REPLICATION, REPAIR, and RECOMBINATION Figure 5–1 Different Proteins Evolve at Very Different Rates
    5 THE MAINTENANCE OF DNA DNA REPLICATION, SEQUENCES DNA REPLICATION MECHANISMS REPAIR, AND THE INITIATION AND COMPLETION OF DNA REPLICATION IN RECOMBINATION CHROMOSOMES DNA REPAIR GENERAL RECOMBINATION SITE-SPECIFIC RECOMBINATION The ability of cells to maintain a high degree of order in a chaotic universe depends upon the accurate duplication of vast quantities of genetic information carried in chemical form as DNA. This process, called DNA replication, must occur before a cell can produce two genetically identical daughter cells. Main- taining order also requires the continued surveillance and repair of this genetic information because DNA inside cells is repeatedly damaged by chemicals and radiation from the environment, as well as by thermal accidents and reactive molecules. In this chapter we describe the protein machines that replicate and repair the cell’s DNA. These machines catalyze some of the most rapid and accu- rate processes that take place within cells, and their mechanisms clearly demon- strate the elegance and efficiency of cellular chemistry. While the short-term survival of a cell can depend on preventing changes in its DNA, the long-term survival of a species requires that DNA sequences be changeable over many generations. Despite the great efforts that cells make to protect their DNA, occasional changes in DNA sequences do occur. Over time, these changes provide the genetic variation upon which selection pressures act during the evolution of organisms. We begin this chapter with a brief discussion of the changes that occur in DNA as it is passed down from generation to generation. Next, we discuss the cellular mechanisms—DNA replication and DNA repair—that are responsible for keeping these changes to a minimum.
    [Show full text]
  • Syllabus 2020
    Amrita Center For Nanosciences and Molecular Medicine Program in the Bachelor of Science (B.Sc.) Molecular Medicine 2020 Amrita Vishwa Vidyapeetham 1 2 B.Sc-Molecular Medicine Syllabus 20BIC102 Biophysics and Bioenergetics. Preamble Connecting Physics to biology is an essential factor which eventually determines quantifying primary factors in a physical process that occur at cell level. Physics serves as a nanoscopic visualization tool to dissect such processes to reveal the information to students. This course introduces applications of Physics into Biology. Unit 1 (5 lectures) Physics: general overview, Does Physics treats disease? Why Physics for Biology and Molecular Medicine? Concepts in Physics to apply in Biology, Methods in Physics to elucidate Biology. Unit 2 (10 lectures) Bioenergetics: Laws of thermodynamics. Concept of state functions, free energy change, equilibrium constant, coupled reactions, energy charge, ATP cycle, phosphorylation potential, and phosphoryl group transfers. Electron transport in membrane for oxidative phosphorylation, Chemical basis of hydrolysis of ATP and thioesters. Redox reactions, standard redox potentials and Nernst equation. Unit 3 (10lectures) Biophysics in Medicine: Applications of physical principles in biology and its significance in the development of various biophysical methods for analysing the complexity of biological system. Principles of Medical-imaging, Image analysis, Instrumentation and Working principles, Medical applications of X-ray: Imaging, Introduction to Fluoroscopy. Unit 4 (10 lectures) Nuclear medicine and Radiotherapy: Pros and cons, Nano-bioelectronics: Monitoring and recording bioelectric signals, Transducers in physiology, Diagnostic and Therapeutic Techniques: Cardiac pace makers, Blood flow monitors, Pulmonary function analyzers, Hemodialysis machines, Defibrillators, Short/ wave diathermy, Electrically stimulated pain management, Laser: operating principles, types, Biomedical applications in surgery.
    [Show full text]
  • Role of Depurination in Mutagenesis by Chemical Carcinogens1
    [CANCER RESEARCH 42, 3480-3485, September 1982] 0008-5472/82/0042-0000$02.00 Role of Depurination in Mutagenesis by Chemical Carcinogens1 Roeland M. Schaaper,2 Barry W. Glickman, and Lawrence A. Loeb3 The Joseph Gottstein Memoria/ Cancer Research Laboratory, Department of Pathology, School of Medicine, University of Washington, Seattle, Washington 98195 {R. M. S., L. A. L.], and Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709 [B. W.G.] ABSTRACT significant extent during the presumably modified mode of replication in SOS-induced bacteria. In these experiments, The effect of modifying <f>x"174viral DMA by the chemical apurinic sites were introduced by a combined acid-heat treat carcinogens /?-propiolactone, A/-acetoxyacetylaminofluorene ment. Although a role for other heat-acid-induced lesions can and anf/-benzo[a]pyrene diol-epoxide was investigated by not be rigorously excluded, the mutagenicity of apurinic sites transfecting the modified DMA into Escherichia coli sphero- is strongly supported by the abolition of mutagenesis by alkali plasts. Modification of the DMA in vitro by each of these agents (20) and by purified apurinic endonuclease.4 Apurinic sites was mutagenic for the <(>x174amber mutants am3 and am86. could also be important for mutagenesis by chemical carcino Mutagenicity depended on the induction of the "SOS" re gens. Modification of purines at A/3 and A/7 and of pyrimidines sponse in the host spheroplasts. Heating ß-propiolactone- at O2 positions labilizes the /V-glycosylic bond, leading to treated DNA at neutral pH caused strong inactivation such that sharply increased spontaneous depurination rates (2, 11, 22).
    [Show full text]
  • Depurination of Nucleosides and Induced by 2-Bromopropane At
    Depurination of Nucleosides and Calf Thymus DNA by 2-BP Bull. Korean Chem. Soc. 2009, Vol. 30, No. 10 2309 Depurination of Nucleosides and C이 f Thymus DNA Induced by 2-Bromopropane at the Physiological Condition Jyoti Sherchan, Hoyoung Choi, and Eung-Seok Lee* College of Pharmacy, Yeungnam University, Kyongsan 712-749, Korea. * E-mail: [email protected] Received July 28, 2009, Accepted August 25, 2009 Depurination, the release of purine bases from nucleic acids by hydrolysis of the N-glycosidic bond, gives rise to alterations of the cell genome. Though cells have evolved mechanisms to repair these lesions, unrepaired apurinic sites have been shown to have two biological consequences: lethality and base substitution errors. 2-Bromopropane (2-BP) is used as an intermediate in the synthesis of pharmaceuticals, dyes, and other organics. In addition, 2-BP has been used as a replacement for chloroflurocarbons and 1,1,1-trichloroethane as a cleaning solvent in electronics industry. However, 2-BP was found to cause reproductive and hematopoietic disorders in local workers exposed to it. Owing to the toxicity of 2-BP, there has been a tendency to use 1-BP as an alternative cleaning solvent to 2-BP. However, 1-BP has also been reported to be neurotoxic in rats. Though N-guanine adduct of 2-BP has been reported previously, massive depurination of the nucleosides and calf thymus DNA was observed in this study.腿 incubated the nucleosides (ddG, dG, guanosine, ddA, dA and adenosine) with excess amount 2-BP at the physiological condition (pH 7.4, 37 oC), which were analyzed by HPLC and LC-MS/MS.
    [Show full text]
  • Polymerase Chain Reaction (PCR)
    Polymerase Chain Reaction Secondary article (PCR) Article Contents . Polymerase Chain Reaction: Overall Description Michael L Metzker, Baylor College of Medicine, Houston, Texas, USA . DNA Polymerase Reaction Thomas C Caskey, Cogene Biotech Ventures, Houston, Texas, USA . Sensitivity and Contamination of PCR . PCR Introduces Mutations . PCR is a rapid in vitro DNA synthesis process, which can amplify up to a billion copies of a PCR Length Limitations . given nucleic acid target. It has been extensively applied for the identification, detection Creation of Novel Recombinant Molecules by PCR . and diagnosis of genetic and infectious disease. PCR as a Detection System . Degenerate PCR . Ancient DNA Polymerase Chain Reaction: Overall . Quantitative PCR Description . Related Nucleic Acid Amplification Procedures . Ligation Chain Reaction Few techniques rival the impact that the polymerase chain . Summary reaction (PCR) has made in the age of molecular biology. Cloning and deoxyribonucleic acid (DNA) sequencing are other such techniques that have become embedded into synthesis fidelities, respectively. PCR is an elegant but everyday life on the molecular biologist’s bench. Over 60 simple technique for the in vitro amplification of target books alone (not to mention the tens of thousands of DNA utilizing DNA polymerase and two specific oligo- research articles) have been devoted to the strategies, nucleotide or primer sequences flanking the region of methods and applications of PCR for the identification, interest. PCR is a cyclic process of double-strand separa- detection and diagnosis of genetic and infectious diseases. tion of DNA by heat denaturation, specific hybridization Rightfully so, the inventor of PCR, Kary B. Mullis, was or annealing of short oligonucleotide primers to single- awarded the Nobel Prize in Chemistry for his discovery of stranded DNA, and synthesis by DNA polymerase (Saiki the technique in 1993.
    [Show full text]
  • Synthesis of Oligodeoxynucleotides Using Fully Protected
    Molecules 2010, 15, 7509-7531; doi:10.3390/molecules15117509 OPEN ACCESS molecules ISSN 1420-3049 www.mdpi.com/journal/molecules Article Synthesis of Oligodeoxynucleotides Using Fully Protected Deoxynucleoside 3′-Phosphoramidite Building Blocks and Base Recognition of Oligodeoxynucleotides Incorporating N3-Cyano-Ethylthymine Hirosuke Tsunoda, Tomomi Kudo, Akihiro Ohkubo, Kohji Seio and Mitsuo Sekine * Department of Life Science, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama, Japan; E-Mails: [email protected] (H.T.); [email protected] (T.K.); [email protected] (A.O.); [email protected] (K.S.) * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +81-45-924-5706; Fax: +81-45-924-5772. Received: 27 August 2010; in revised form: 8 October 2010 / Accepted: 14 October 2010 / Published: 27 October 2010 Abstract: Oligodeoxynucleotide (ODN) synthesis, which avoids the formation of side products, is of great importance to biochemistry-based technology development. One side reaction of ODN synthesis is the cyanoethylation of the nucleobases. We suppressed this reaction by synthesizing ODNs using fully protected deoxynucleoside 3′-phosphoramidite building blocks, where the remaining reactive nucleobase residues were completely protected with acyl-, diacyl-, and acyl-oxyethylene-type groups. The detailed analysis of cyanoethylation at the nucleobase site showed that N3-protection of the thymine base efficiently suppressed the Michael addition of acrylonitrile. An ODN incorporating N3-cyanoethylthymine was synthesized using the phosphoramidite method, and primer extension reactions involving this ODN template were examined. As a result, the modified thymine produced has been proven to serve as a chain terminator.
    [Show full text]
  • Development of a Quantitative RT-PCR Assay to Examine The
    Downloaded from rnajournal.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press METHOD Development of a quantitative RT-PCR assay to examine the kinetics of ribosome depurination by ribosome inactivating proteins using Saccharomyces cerevisiae as a model MICHAEL PIERCE, JENNIFER NIELSEN KAHN, JIACHI CHIOU, and NILGUN E. TUMER Department of Plant Biology and Pathology, School of Environmental and Biological Sciences, Rutgers University, New Brunswick, New Jersey 08901-8520, USA ABSTRACT Ricin produced by the castor bean plant and Shiga toxins produced by pathogenic Escherichia coli (STEC) and Shigella dysenteriae are type II ribosome inactivating proteins (RIPs), containing an enzymatically active A subunit that inhibits protein synthesis by removing an adenine from the a-sarcin/ricin loop (SRL) of the 28S rRNA. There are currently no known antidotes to Shiga toxin or ricin, and the ability to screen large chemical libraries for inhibitors has been hindered by lack of quantitative assays for catalytic activity that can be adapted to a high throughput format. Here, we describe the development of a robust and quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay that can directly measure the toxins’ catalytic activity on ribosomes and can be used to examine the kinetics of depurination in vivo. The qRT-PCR assay exhibited a much wider dynamic range than the previously used primer extension assay (500-fold vs. 16-fold) and increased sensitivity (60 pM vs. 0.57 nM). Using this assay, a 400-fold increase in ribosome depurination was observed in yeast expressing ricin A chain (RTA) relative to uninduced cells.
    [Show full text]
  • Avoiding Depurination During Trityl-On Purification Author: Greg Scott
    Application Note: TN-0008 Avoiding Depurination During Trityl-on Purification Author: Greg Scott Introduction epurination in DNA and RNA sequences whether in nature or through fabrication is an alteration of the sugar- Dphosphate backbone in which hydrolysis of a purine base (adenine or guanine) occurs. In the typical mammalian cell, depurination is a rather common event as nearly one thousand purines are lost daily due to endogenous chemical reactions. [1] Systemic mechanisms, notably enzymes and amines, are in place that can effectively cleave voided fragments to prevent errant and potentially damaging transcriptions. [2] As in nature, synthetic oligonucleotides suffer from similar errors during the assembly process resulting in unwanted and harmful contaminants. Contemporary Depurination Avoiding DNA and RNA synthesis utilizes solid-phase phosphoramidite chemistry to construct nucleotide sequences through a succession of phosphodiester linkages. By incorporating an acid labile 5’ protecting group, dimethoxytrityl (DMT), nucleotide assembly is accomplished through sequential automated cycles of deprotection, coupling, capping, and oxidation. The greatest potential for damage to the sequence occurs during the deprotection stage (detritylation) where the oligonucleotide is exposed to dilute acid that may induce purine hydrolysis. Adding further complexity, the depurination rate of single stranded DNA (as synthetically produced) is four-fold greater than native double stranded DNA. [3] Unlike nature, the synthetic process lacks the necessary mechanism to selectively clear apurinic sites from the full-length sequence. Consequently, to deliver a pure and error-free product, purification techniques are required to remove depurinated fragments and other remnant impurities. Crude synthetic oligonucleotides are purified by either trityl-on or trityl-off methodologies.
    [Show full text]
  • Depurination of Da and Dg Induced by 2-Bromopropane at the Physiological Condition
    The Journal of Applied Pharmacology, 15, 224-229(2007) Depurination of dA and dG Induced by 2-bromopropane at the Physiological Condition Pritam THAPA, Jyoti SHERCHAN, Radha KARKI, Tae Cheon JEONG, and Eung-Seok LEE* College of Pharmacy, Yeungnam University, Kyongsan 712-749, Korea (Received November 19, 2007; Accepted December 3, 2007) Abstract − Depurination, the release of purine bases from nucleosides by hydrolysis of the N-glycosidic bond, gives rise to alterations of the cell genome. Although, cells have evolved mechanisms to repair these lesions, unrepaired apurinic sites have been shown to have two biological consequences: lethality and base substitution errors. 2-Bromopropane (2-BP) is used as an intermediate in the synthesis of pharmaceuticals, dyes, and other organics. In addition, 2-BP has been used as a cleaning solvent in electronics industry. But, 2-BP was found to cause reproductive and hematopoietic disorders in local workers exposed to it. We observed massive depurination after incubation of 2’-deoxyadenosine (dA) and 2’-deoxyguanosine (dG) with the excess amount 2-BP at the physiological condition (pH 7.4, 37oC), which were analyzed by HPLC and LC-MS/MS. In addition, time and dose response relationship of depurination in dA and dG induced by 2-BP at the physiological condition were investigated. Keywords □ depurination, 1-bromopropane, 2-bromopropane, time and dose response relationship INTRODUCTION form tumor-intitiating H-ras mutations (Chakravarti et al., 2001) by inducing pre-replication repair that is error-prone and Depurination of nucleic acids, the release of purine bases forms mismatched heteroduplexes leading to transforming from nucleosides by hydrolysis of the N-glycosidic bond (Fig.
    [Show full text]
  • Depurination Causes Mutations in SOS-Induced Cells (Chemical Carcinogenesis/Mutagenesis/Fidelity of DNA Replication/SOS Repair) ROELAND M
    Proc. Nati. Acad. Sci. USA Vol. 78, No. 3, pp. 1773-1777, March 1981 Genetics Depurination causes mutations in SOS-induced cells (chemical carcinogenesis/mutagenesis/fidelity of DNA replication/SOS repair) ROELAND M. SCHAAPER AND LAWRENCE A. LOEB The Joseph Gottstein Memorial Cancer Research Laboratory, Department of Pathology, SM-30, University of Washington, Seattle, Washington 98195 Communicated by Earl P. Benditt, November 17, 1980 ABSTRACT Introduction of apurinic sites into dX174 am3 the apurinic sites. We show that, in accord with previous re- DNA leads to loss of biological activity when measured in a trans- ports, depurination is strongly inactivating but that, under cer- fection assay. For single-stranded DNA, approximately one apu- tain can indeed be rinic site constitutes a lethal hit; for double-stranded (RFI) DNA, conditions, depurination mutagenic. approximately 3.5 hits per strand are lethal. When the reversion frequency of am3 DNA is measured, no increase due to depuri- MATERIALS AND METHODS nation is observed above the background level. However, a large Bacteria and Bacteriophage. E. coli W6, obtained from M. increase in reversion frequency is observed when the same DNA Edgell (University of North Carolina) was used for the prepa- is assayed by using spheroplasts derived from bacteria previously ration of spheroplasts. E. HF4704 HF4714 exposed to UV light. The results suggest that apurinic sites are coli (su-) and (su1l) impediments to a replicating DNA polymerase; however, nucleo- were used to determine titers and reversion frequencies of tides can be incorporated opposite these sites under SOS-induced 4)X174 am3 DNA. The preparation of bacteriophage 4.X174 conditions.
    [Show full text]