US 20100317005A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2010/0317005 A1 Hardin et al. (43) Pub. Date: Dec. 16, 2010

(54) MODIFIED NUCLEOTIDES AND METHODS (22) Filed: Mar. 15, 2010 FOR MAKING AND USE SAME Related U.S. Application Data (63) Continuation of application No. 11/007,794, filed on Dec. 8, 2004, now abandoned, which is a continuation (75) Inventors: Susan H. Hardin, College Station, in-part of application No. 09/901,782, filed on Jul. 9, TX (US); Hongyi Wang, Pearland, 2001. TX (US); Brent A. Mulder, (60) Provisional application No. 60/527,909, filed on Dec. Sugarland, TX (US); Nathan K. 8, 2003, provisional application No. 60/216,594, filed Agnew, Richmond, TX (US); on Jul. 7, 2000. Tommie L. Lincecum, JR., Publication Classification Houston, TX (US) (51) Int. Cl. CI2O I/68 (2006.01) Correspondence Address: (52) U.S. Cl...... 435/6 LIFE TECHNOLOGES CORPORATION (57) ABSTRACT CFO INTELLEVATE Labeled nucleotide triphosphates are disclosed having a label P.O. BOX S2OSO bonded to the gamma phosphate of the nucleotide triphos MINNEAPOLIS, MN 55402 (US) phate. Methods for using the gamma phosphate labeled nucleotide are also disclosed where the gamma phosphate labeled nucleotide are used to attach the labeled gamma phos (73) Assignees: LIFE TECHNOLOGIES phate in a catalyzed (enzyme or man-made catalyst) reaction to a target biomolecule or to exchange a phosphate on a target CORPORATION, Carlsbad, CA biomolecule with a labeled gamme phosphate. Preferred tar (US); VISIGEN get biomolecules are DNAs, RNAs, DNA/RNAs, PNA, BIOTECHNOLOGIES, INC. polypeptide (e.g., proteins enzymes, protein, assemblages, etc.), Sugars and polysaccharides or mixed biomolecules hav ing two or more of DNAs, RNAs, DNA/RNAs, polypeptide, (21) Appl. No.: 12/724,392 Sugars and polysaccharides moieties.

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OOOE+00 Patent Application Publication Dec. 16, 2010 Sheet 1 of 20 US 2010/0317005 A1 Patent Application Publication Dec. 16, 2010 Sheet 2 of 20 US 2010/0317005 A1

-e Patent Application Publication Dec. 16, 2010 Sheet 3 of 20 US 2010/0317005 A1 Patent Application Publication Dec. 16, 2010 Sheet 4 of 20 US 2010/0317005 A1

d.LN-T-L Patent Application Publication Dec. 16, 2010 Sheet 5 of 20 US 2010/0317005 A1

O-O-O-O-O-O-O-, +d.LN-T-L Patent Application Publication Dec. 16, 2010 Sheet 6 of 20 US 2010/0317005 A1

H

: Patent Application Publication Dec. 16, 2010 Sheet 7 of 20 US 2010/0317005 A1

ATP-1-ROX ROX 0.40

AU O20

OOO 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 MINUTES

O.30

AU 0.20 ATP-1-ROX ROX 0.10

0.00 OOO 5.00 10.00 15.00 20.00 25.00 30.00 35.00 MINUTES FIG. 5 Patent Application Publication Dec. 16, 2010 Sheet 8 of 20 US 2010/0317005 A1

0.40 0.35 O.30 0.25 AU O.20 O.15 O. 10 0.05 645.6 706.7 786.2 0.00

200.00 300.00 400.00 500.00 600.00 700.00

nm. FIG. 6

ATP/CAP ATP ATP EDAROX/CAP : ATP EDAROX Patent Application Publication Dec. 16, 2010 Sheet 9 of 20 US 2010/0317005 A1

d'OL doL-XOX)–> -OLI——>

UBOSOL|- Patent Application Publication Dec. 16, 2010 Sheet 10 of 20 US 2010/0317005 A1

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n O h H X O n1 Patent Application Publication Dec. 16, 2010 Sheet 11 of 20 US 2010/0317005 A1

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T4 PNK 5' End-labeling TimeCourse With ATP-L1-ROX

0.5 1 3 6 18 (Hrs) FIG. 12A

1.OO O.75 Relative Activity O5O O.25 O.OO (-) 0.5 1 Time (Hrs)

FIG. 12B Patent Application Publication Dec. 16, 2010 Sheet 14 of 20 US 2010/0317005 A1

T4 PNK Concentration Experiment

110 220 550 110 220 550 (uM) 10 Units 20 Units FIG. 13A

1.OO 0.75 % Activity % % 20 Units O.OO25 Y 3% 110 220 550 ATP-L1-ROX (uM)

FIG. 13B Patent Application Publication Dec. 16, 2010 Sheet 15 of 20 US 2010/0317005 A1

5' End-Labeling Improvements with PEG 8000 1.00 0.75 Re. Activity O.50 O.25 O.OO 4% 6%. 8%. 12%. 18% PEG 8000 FIG. 14

Linker Effects on 5' End-labeling with ATP-Li-ROX

Re Activity

LinkerS FIG. 15A Patent Application Publication Dec. 16, 2010 Sheet 16 of 20 US 2010/0317005 A1

Linker Effects on 5' End-labeling with ATP-Li-FLU 1.00

Sas O.75 Re. Activity 0.50 O.25 1. O.OO

FIG. 15B

5' ROX - 3' 3' - 5' A FIG. 1.6A Patent Application Publication Dec. 16, 2010 Sheet 17 of 20 US 2010/0317005 A1

5' ROX-oligonucleotide With duTP at 3' end

5' ROX-oligonucleotide -> . W.

FIG. 16B

ROX-Oligonucleotide-> Oligonucleotide

FIG. 17A Patent Application Publication Dec. 16, 2010 Sheet 18 of 20 US 2010/0317005 A1

Dr. O >< ? Q) O C --> 75 CD (5 JC CD Z oligonucleotide

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FIG. 17B Patent Application Publication Dec. 16, 2010 Sheet 19 of 20 US 2010/0317005 A1

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MODIFIED NUCLEOTDES AND METHODS 0009. In addition to fluorescence, another important FOR MAKING AND USE SAME nucleic acid labeling method is the attachment of biotin to DNA or RNA. A variety of nucleic acid and protein capture RELATED APPLICATIONS applications are developed that exploit the highly specific interaction between biotin and streptavidin. 0001. The present invention claims provisional priority to 0010. As an example, magnetic column separations where U.S. Provisional Patent Application Ser. No. 60/527,909, biotinylated oligonucleotides are captured by Streptavidin filed 8 Dec. 2003 and is a Continuation-in-part of U.S. patent coated microbeads enable a straightforward way to separate application Ser. No. 09/901,782 filed 9 Jul. 2001, which claim biotinylated from non-biotinylated molecules as described provisional priority to U.S. Provisional Patent Application herein. This system permits the capture of DNA molecules, Ser. No. 60/216,594, filed 7 Jul. 2000, all of which are incor RNA molecules, DNA and RNA-binding proteins, and porated herein by reference. sequence specific transcripts. 0011. The expanding need for higher throughput tech BACKGROUND OF THE INVENTION nologies suggests that the demand for both fluorescently labeled-ATPs and biotinylated-ATPs will increase. Thus, 0002 1. Field of the Invention there is a need in the art for reagents to label both oligonucle 0003. The present invention relates to labeled nucleotides otide and polynucleotides and polypeptide, proteins, (NTPs), to method for making labeled NTPs and to method enzymes, monosaccharides, polysaccharides, or mixtures or for using labeled NTPs such as a method of transferring the combinations thereof with labels having a readily detectable label to a target molecule. property. 0004 More particularly, the present invention relates to gamma (Y) phosphate labeled nucleotides (NTPs), where the DEFINITIONS label includes a detectable tag and an optional linker inter 0012. The term “tag” or “label means an atom or mol posed between the tag and the Y phosphate, to method for ecule that has a detectable property and is capable of being making Y phosphate labeled NTPs and to method for trans attached to a Y phosphate of a nucleotide triphosphate. ferring the Y phosphate and the label to a target molecule 0013 The term “detectable property” means a physical or including an oligonucleotide, a polynucleotide, a polypep chemical property of a tag that is capable of independent tide, a protein, a monosaccharide, a polysaccharide, or mix detection and/or monitoring by an analytical technique after tures or combinations thereof. being attached to a target bio-molecule, i.e., the property is 0005 2. Description of the Related Art capable of being detected in the presence of the system under 0006. Many labeling procedures have been developed analysis. The property can be light emission after excitation, over the years for labeling nucleotide and polypeptide quenching of a known emission sites, electron spin, radio sequences with both radio and non-radio labels. These meth activity (electron emission, positron emission, alpha particle ods are routinely used to aid our understanding of molecular, emission, etc.), nuclear spin, color, absorbance, near JR cellular and intercellular dynamics and interaction. However, absorbance, UV absorbance, far UV absorbance, etc. there are few relatively simple and versatile reagents and 0014. The term “analytical technique' means an analytical methods for attaching non-radio labels to both nucleotide chemical or physical instrument for detecting and/or moni sequence (DNA, RNA, DNA-RNA, etc.), peptide sequences toring the property. Such instruments are based on spectro (polypeptides, proteins, enzymes, macro-assembly, etc.) or scopic analytical methods such as electron spin resonance mixtures or combinations thereof (ribozymes, peptide-nucle spectrometry, nuclear magnetic resonance (NMR) spectrom otide mixed sequences, etc.). etry, UV and visible light spectrometry, far IR, IR and near IR 0007 Fluorescence-based technologies are rapidly spectrometry, X-ray spectrometry, etc. emerging as the methods of choice for nucleic acid labeling 0015 The term “base' means any natural or synthetic and detection. A variety of molecular biology techniques have purine or pyrimidine or nucleotide analogs (e.g., 7-deaza benefitted from fluorescent innovations. For example, DNA deoxyguanine) that is capable of forming nucleotides and Dye-Terminator Sequencing has been revolutionized by the sequences thereof, including, without limitation, adenine creation of individually identifiable color-coded bases (A), cytosine (C), guanine (G), inosine (I), thymine (T), uracil thereby making system automation possible. Assays such as (U), pseudouridine (Y), xanthine (X), Orotidine (O), 5-bro Fluorescent In Situ Hybridization (FISH) and Quantitative/ mouridine (B), thiouridine (S), 5,6-dihydrouridine (D) or the Real-time PCR (qPCR) also capitalized on the ability to like. The natural or synthetic purines or pyrimidines may monitor multiple fluorophores simultaneously. Furthermore, includes tags or may have been modified to have a particular these new assays also make extensive use of the unique data detectable property such as enrichment with an NMR active provided by fluorophore-fluorophore interactions. nuclei. 0008 Of vital importance is the added benefit provided to 0016. The term “bonded to means that chemical and/or personal health and safety by offering a robust alternative to physical interactions Sufficient to maintain the label or tag at the Scientific community's dependence on radioactive labels. a given site of a target molecule. The chemical and/or physical Specifically, radio-isotopic labeling entails a variety of nega interactions include, without limitation, covalent bonding tive aspects which include: (1) health hazards related to expo (preferred), ionic bonding, hydrogen bonding, apolar bond Sure, (2) extra licensing requirements and permits, (3) con ing, attractive electrostatic interactions, dipole interactions, tained storage requirements, (4) additional requirements for or any other electrical or quantum mechanical interaction waste removal, and (5) the limited lifetime of a radioactive Sufficient in toto to maintain the polymerizing agent in a probe. Fluorescent technologies eliminate both health risks desired region of the substrate. and regulatory paperwork and provide a greater degree of 0017. The term “nucleoside” means a base bonded to a flexibility in experimental design. Sugar Such as a five carbon Sugar e.g., ribose. US 2010/031 7005 A1 Dec. 16, 2010

0.018. The term “nucleotide” means nucleoside bonded to 0050. The term T-L-P-PP means a polypeptide having an at least one phosphate. T-L-P bonded thereto. 0019. The term NMP means a nucleotide monophosphate. 0051. The term T-P-PPN means a polypeptide having an 0020. The term NDP means a nucleotide diphosphate. T-P bonded thereto. 0021. The term NTP means a nucleotide triphosphate. 0.052 The termT-L-P-PPN means a polypeptide having an 0022. The term AMP means adenosine monophosphate. T-L-P bonded thereto. 0023 The term ADP means adenosine diphosphate. 0053. The term PNPP means abiomolecule including both 0024. The term ATP means adenosine triphosphate. nucleosides, nucleotides, oligonucleotide or a polynucleotide 0025. The term TMP means thymidine nucleotide mono and an amino acid, polypeptide or protein. phosphate. 0054 The term T-P-PNPP means an PNPP having an T-P 0026. The term TDP means thymidine nucleotide diphos bonded thereto. phate. 0055. The term T-L-P-PNPP means an PNPP having an 0027. The term TTP means thymidine nucleotide triphos T-L-P bonded thereto. phate. 0056. The term “MES buffer” means morpholinoethane 0028. The term CMP means cytidine nucleotide mono sulfonic acid buffer. phosphate. 0057 The term TLC means thin layer chromatography. 0029. The term CDP means cytidine nucleotide diphos phate. SUMMARY OF THE INVENTION 0030 The term CTP means cytidine nucleotide triphos 0058. The present invention provides labeled nucleotides phate. (T-NTPs or T-L-NTPs), where the label (T-P or T-L-P) is 0031. The term GMP means guanine nucleotide mono readily transferable to nucleotide sequences, amino acid phosphate. sequences, Saccharides or compositions comprising a nucle 0032. The term GDP means guanine nucleotide diphos otide, an amino acid and/or a Sugar. phate. 0059. The present invention also provides a method for 0033. The term GTP means guanine nucleotide triphos making labeled nucleotides, where the label includes an phate. atomic and/or molecular tag bonded to the gamma phosphate 0034. The term PNA means a peptide nucleic acid. of the nucleotide (TNTP5) comprising the steps of contact 0035. The term T-NTP means an NTP having an atomic ing an NTP with an atomic and/or molecular tag precursor to and/or molecular tag bonded to the gamma phosphate of the form the TNTP. NTP. 0060. The present invention also provides a method for making labeled nucleotides, where the label includes an 0036. The term T-P means a phosphate (P) bonded to a tag atomic and/or molecular tag bonded to one end of a linker (T). which is bonded to the gamma phosphate of the nucleotide 0037. The term L-NTP means an NTP having a linking (T-L-NTPs) comprising the steps of contacting an NTP with reagent or linker bonded at its first end to the gamma phos a linker precursor to form a linker modified NTP (L-NTP). phate of the NTP. The L-NTP is then contacted with anatomic and/or molecular 0038. The term T-L-NTP means an L-NTPhaving a tag or tag to form the T-L-NTP. label bonded to a second end of the linker. 0061 The present invention provides a method for label 0039. The termT-L-P means a phosphate(P) bonded to the ing oligonucleotides including the step of contacting a T-NTP first end of the linker (L) and a tag (T) bonded to the second or T-L-NTP with an oligonucleotide (ON) in the presence of end of the linker (L). a catalyst to form a tagged oligonucleotide (T-ON or T-L- 0040. The term ON means an oligonucleotide—a short ON). sequence of nucleotides generally less than about 100 nucle 0062. The present invention also provides a method for otides, preferably less than about 50 nucleotides. labeling polypeptide (PP) including the step of contacting a 0041. The term PN means a polynucleotide or nucleic T-NTP or T-L-NTP with the PP in the presence of a catalyst to acid—a long sequence of nucleotides generally over about form a tagged polypeptide (T-P-PP or T-L-P-PP). 100 nucleotides. 0063. The present invention provides a method for label 0042. The term PP means a polypeptide sequence, includ ing proteins (PRN) including the step of contacting a TNTP ing at least two amino acids joined together via a peptide bond or T-L-NTP with the PRN in the presence of a catalyst to form Such as proteins, enzymes, protein assemblages, or the like. a tagged protein (T-P-PRN or T-L-P-PRN). 0043. The term PRN means a protein, which includes 0064. The present invention provides a method for label enzymes and protein assemblages. ing a biomolecule including both nucleosides, nucleotides, 0044. The term AA means an amino acid either natural or oligonucleotide or a polynucleotide and an amino acid, synthetic and capable of forming peptide bonds. polypeptide or protein (PNPP) including the step of contact 0045. The termTON means an oligonucleotide having an ing a T-NTP or T-L-NTP with the PNPP in the presence of a T-P bonded to the ON at either its 5' or 3' end. catalyst to form a tagged biomolecule (T-P-PNPP or T-L-P- 0046. The term T-L-ON means an oligonucleotide having PNPP). an T-L-P bonded to the ON at either its 5' or 3' end. 0065. The present invention provides a set of ATP-linker 0047. The term T-PN means a polynucleotide having an fluorescent dye molecules and ATP-linker-biotin molecules. T-P bonded to the PN at either its 5' or 3' end. The ATP molecules differ in the linker interposed between the 0048. The termT-L-PN means a polynucleotide having an ATP Y-phosphate and the fluorescent dye or biotin. The link T-L-P bonded to the PN at either its 5' or 3' end. ers differ in Such properties as chain length, bulk or size, 0049. The term T-P-PP means a polypeptidehaving an T-P rigidity, and/or polarity. The synthesis of the precursor ATP bonded thereto. linker molecules provides intermediates that are used for US 2010/031 7005 A1 Dec. 16, 2010

attachment of a variety of individual fluorescent dyes, biotin port. The immobilized substrate(s) is then contacted with a or other binding molecules. Specific commercially available solution including a kinase and a Y-phosphate labeled NTP for dyes were investigated to determine the efficiency of the a first time and at a first temperature sufficient to determine transfer reaction in applications that have commercial poten whether a transfer of the labeled phosphate from the Y-phos tial. Such commercially available dyes includes: (1) Fluores phate labeled NTP to the substrate mediated by the kinase. cein excitation w: 495 nm, emission w: 520 nm), (2) Cy3 Next, the Support is washed for a second time and at a second excitation w: 550 nm, emission w: 570 nm), (3) TAMRA temperature Sufficient to remove the kinase and unreacted excitation w: 555 nm, emission w: 580 nm), (4) ROX exci labeled NTP. After washing, the support is analyzed to deter tation w: 578 nm, emission w: 604 nm, and (5) Cy5 excita mine whether the substrate have been labeled with the labeled tion w: 649 nm, emission w: 670 nm. Of course, the processes phosphate from the Y-phosphate labeled NTP. of this invention are capable of using a wide variety of Y-phos 0071. The present invention provides a method for screen phate-labeled nucleotides such as ATP. ing kinase Substrate candidates including the step of provid 0066. The present invention also provides for the use of T4 ing an immobilized kinase on a Support. The Support is then PNK or other phosphatase or kinase enzymes to 5' end-label contacted with a solution including one or more kinase Sub oligonucleotides with a variety of fluorophore, biotin or other strate candidates and a labeled NTP for the kinase. The sub binding molecules using labeled ATP molecules. strate candidates are then analyzed for the presence or 0067. The present invention also provides a screening absence of the label. assay to examine each of the ATP-Linker-Fluorophore and 0072 The present invention provides a method including ATP-Linker-Biotin molecules in 5' end-labeling reactions the step of contacting a solution comprising non-modified with T4 PNK. The assay is based on the protocol for radio nucleotides or deoxynucleotides and modified nucleotides or isotopic 5' end-labeling of oligonucleotides by T4PNK. Fluo deoxynucleotides, where the enzyme selectively degrades the rescent reactions are separated by PAGE and analysis is per non-modified nucleotides or deoxynucleotides. formed with a fluorescent gel Scanning imager and software. 0073. The present invention provides a method for moni Biotin reactions are examined via covalent attachment of toring an NTP dependent reaction including the step of Sup biotinylated oligonucleotide to DE81 filter paper, incubation plying to a system in which an NTP dependent reaction in Streptavidin-alkaline phosphatase conjugate, and color occurs, a Y-phosphate labeled nucleotide (NTP) and monitor development in nitroblue tetrazolium chloride (NBT)/5- ing the label during the reaction. bromo-4-chloro-3-indolyl-phosphate (BCIP) solution. Parameters that affect labeling efficiency include: (1) the DESCRIPTION OF THE DRAWINGS incubation time, (2) labeling bias due to 5' base sequence of 0074 The invention can be better understood with refer the oligonucleotide, (3) concentration (ATP-Linker-Moiety) ence to the following detailed description together with the substrate, (4) T4 PNKamount and (5) oligonucleotide length. appended illustrative drawings in which like elements are 0068. Once a labeled-ATP is synthesized, it will then numbered the same: undergo quality control measures which entail: (1) TLC 0075 FIG. 1A depicts a general scheme for preparing analysis to determine labeled-ATP synthesis integrity, (2) gamma phosphate tagged NTPs; Spectrophotometric analysis to calculate labeling efficiency 0076 FIG. 1B depicts a general scheme for preparing and, (3) Fluorometric analysis to verify the quantum yields of gamma phosphate tagged NTPs with a linker interposed the ATP-labeled molecule and the fluorescently-labeled oli between the gamma phosphate and the tag: gonucleotide. 0077 FIG. 1C a general scheme for preparing a gamma 0069. The labeling efficiency are calculated by measuring phosphate tagged ATP with a linker interposed between the the base:dye ratio on a NanoDrop spectrophotometer. This gamma phosphate and the tag: method uses Molecular Probes to calculate the labeling effi 0078 FIG. 2A a general scheme for preparing 3' and/or 5' ciency of their ULYSIS Nucleic Acid Labeling Kit tagged oligonucleotides using the tagged NTPs of this inven (MP21650). The NanoDrop spectrophotometer is used to tion; measure the absorbance of the nucleic acid-dye conjugate at 007.9 FIG. 2B another general scheme for preparing 3' 260 nm (720) and at a for the dye (...). A measurement and/or 5' tagged oligonucleotides using the tagged NTPs of is also taken using the buffer alone at 260 nm and W and this invention or for exchanging an untagged phosphate group these numbers are subtracted from the raw sample absor for a tagged phosphate group; bances values. To correct for the dye contribution at the 260 0080 FIG. 3 a general scheme for preparing phosphory nm reading a correction factor is introduced (CF). The lated polypeptide or proteins using the tagged NTPs of this correction factor is given by: invention; CFA2 for the free dye. A for the free dye I0081 FIG. 4 depicts an HPLC chromatogram of the reac tion product ATP-EDA-ROX at 576 nm: By applying the correction factor to the following equation, I0082 FIG. 5 depicts an HPLC chromatogram of the reac an accurate absorbance measurement can be obtained: tion product ATP-EDA-ROX at 259 mm: A base-A260-(AixCF260) I0083 FIG. 6 depicts a UV spectrum of ATP-EDA-ROX: Finally, the base:dye ratio is given by: I0084 FIG. 7A depict TLC monitoring of reactions of ATP and ATP-EDA-Rox with CLAP: base:dye ratio=(A baseXAde)(4d. exebase) I0085 FIG. 8 depicts PAGE monitoring of T4 PNK 5' end where e is an extinction coefficient of the dye and is unique for labeling of a TOP oligonucleotide using ATP-EDA-ROX: each dye. I0086 FIG.9 depicts gel plates of T4 PNK 5' end labeling 0070 The present invention provides a method for screen of a TOP oligonucleotide using ATP-EDA-ROX: ing kinases including the step of providing one immobilized I0087 FIG. 10 depicts a plot of CNT vs. pmol for ROX-T- Substrate or a plurality of immobilized Substrates on a Sup Top: US 2010/031 7005 A1 Dec. 16, 2010

0088 FIG. 11 depicts a plot of CNT vs. ng Top oligonucle 0099. The present invention broadly relates to a composi otide and different T4 PNK concentrations; tion for efficiently labeling oligonucleotide, polynucleotides, I0089 FIGS. 12A&B depict plots of T4 PNK 5' end-label polypeptide, proteins and/or biomolecules including both a ing timecourse using ATP-L1-ROX; nucleoside, nucleotide, oligonucleotide, and/or polynucle 0090 FIGS. 13A&B depict plots of T4 PNK concentra otide and a polypeptide and/or protein, where the composi tion effects on 5' end-labeling using ATP-L1-ROX: tion includes an NTP, a linker and a tag, where the linker is 0091 FIG. 14 depicts plots of T4 PNK 5' end-labeling t bonded at one end to a gamma phosphate of the NTP and at using ATP-L1-ROX in the presence of PEG 8000; the other end to the tag. 0092 FIGS. 15A&B depict plots of linker effects on T4 0100. The present invention broadly relates to a method PNK 5' end-labeling using ROX labeled ATPs: for efficiently labeling oligonucleotide, polynucleotides, 0093 FIGS. 16A&B depict extension reactions of a ROX polypeptide, proteins and/or biomolecules including both a labeled oligonucleotide; nucleoside, nucleotide, oligonucleotide, and/or polynucle 0094 FIGS. 17A&B depict the relatively activity of an otide and a polypeptide and/or protein, where the method exonuclease against a 5' end-labeled oligonucleotide and an includes the step of contacting oligonucleotide, polynucle un-labeled oligonucleotide; otides, polypeptide, proteins and/or biomolecules including 0095 FIG. 18 depicts three developed filter paper disks both a nucleoside, nucleotide, oligonucleotide, and/or poly showing T4 PNK 5' end-labeling using ATP-L1-biotin com nucleotide and a polypeptide and/or protein with a labeled pared to a negative control and a synthetic biotin labeled NTP (T-L-NTP) to form a labeled oligonucleotide, poly oligonucleotide; and nucleotides, polypeptide, proteins and/or biomolecules 0096 FIG. 19 depicts 5' end-labeling an RNA oligonucle including both a nucleoside, nucleotide, oligonucleotide, otide with ATP-L2-Cy3 and hybridization to a DNA microar and/or polynucleotide and a polypeptide and/or protein in the ray. presence of a catalyst, where the contacting transfers the gamma phosphate, linker and tag to the oligonucleotide, DETAILED DESCRIPTION OF THE INVENTION polynucleotides, polypeptide, proteins and/or biomolecules 0097. The inventors have found a versatile, inexpensive including both a nucleoside, nucleotide, oligonucleotide, and efficient technique for labeling nucleic acids, polypep and/or polynucleotide and a polypeptide and/or protein. tides and/or biomolecules including both nucleic acids and 0101 For decades T4 polynucleotide kinase (T4PNK) has amino acids with atomic and/or molecular tags having a been essential for phosphorylating, either radioactively or detectable property and reagents for accomplishing the tag non-radioactively, the 5'-end of oligonucleotides for subse ging reaction. The techniques involves the preparation of quent use in a variety of molecular biology applications. T4 labeled NTPs capable of transferring their label to a target PNK has two distinct functions: (1) transfer of the Y-phos nucleotide, polypeptide, Saccharide, and/or a biomolecule phate of adenosine triphosphate (ATP) or other nucleoside including one or combinations of a nucleoside, nucleotide, triphosphates to the 5’ hydroxyl end of a polynucleotide and oligonucleotide or a polynucleotide and an amino acid, (2) 3'-phosphatase activity that is independent of ATP and polypeptide or protein, combinations of a nucleoside, nucle able to hydrolyze 2',3'-cyclic phosphodiesters. otide, oligonucleotide or a polynucleotide and a monosaccha 0102 The dual functionality of the enzyme can be ride or polysaccharide and combinations of an amino acid, explained by its physiological role within the T4 bacterioph polypeptide or protein and a monosaccharide or polysaccha age life cycle. Upon infection by T4, some strains of Escheri ride. The inventors have found that novel fluorescently chia coli have the capability to initiate a suicide defense tagged ATP molecules and biotin-tagged ATP molecules can mechanism that causes the specific cleavage of bacterial be prepare and used by T4 Polynucleotide Kinase (T4 PNK) lysine tRNA (tRNA) and results in the abrogation of protein in gamma-phosphate transfer reactions to the 5' end of oligo synthesis. In response, the phage initiates tRNA repair via nucleotides, giving the end-user the ability to quickly label an the bacteriophage encoded T4 PNK and T4 RNA ligase. T4 oligonucleotide with a desired fluorophore or a binding mol PNK specifically phosphorylates the 5'-hydroxyl group and ecule Such as biotin. simultaneously reprocesses the 3' end by opening the 2',3'- 0098. The present invention relates to the modification of cyclic phosphate and then removing the 3'-phosphate. This the gamma-phosphate of a nucleotide, preferably ATP and results in a suitable substrate for T4 RNA ligase which can GTP, to form gamma-phosphate labeled nucleotides, which then repair the tRNA lesion and allow continued phage can Subsequently be used to transfer the labeled gamma phos propagation. phate moiety to a target substrate such as DNA, RNA, RNA/ 0103 Both of T4 PNK's actions, phosphorylation and DNA, protein, polypeptide, Sugars, polysaccharides or bio dephosphorylation, have been exploited by molecular biolo molecules including DNA, RNA, polypeptides, Sugars or gists for the purpose of radio-labeling nucleic acids for use as polysaccharides. The label can include an atomic and/or hybridization probes, sequencing primers, transcript map molecular tag having a separately detectable property Such as ping, and for the cold phosphorylation of DNA ends for a fluorescent tag, a biotinylated tag, electrochemical tag, lan cloning. However, only limited attempts have been made to thanide or actinide series containing tag, radical tag or para develop non-radioactive, gamma-labeled ATP Substrates that magnetic tag, nmr tag ('C, N, or other isotopically could be used for 5' end-labeling nucleic acids. enriched atom or molecular tags) or any other tag capable of 0104 Currently, an oligonucleotide is typically labeled detection. The label can also include a linker interposed during its chemical synthesis and the label may be directed to molecularly between the gamma-phosphate and the tag, the 3' end, 5' end, or at an internal position. Additionally, a which may influence the efficiency of the transfer reaction, label may be added post-synthesis by polymerase incorpora and this efficiency may be specific for the transfer reaction tion of a base-labeled dNTP onto the 3' end of a duplexed under study. Additionally, the transfer reaction may be influ molecule or via terminal deoxy transfer activity (TdT). At enced by the identity of the tag. present, chemical attachment of a dye during the process of US 2010/031 7005 A1 Dec. 16, 2010

oligonucleotide synthesis or via amino-chemistry after Syn electrochemical probes or tags, or other similar molecular thesis are the only methods used to add a fluorescent moiety tags or probes. Fluorescent dyes include, without limitation, to the oligonuleotides 5'-end. The necessity of chemically Such as d-Rhodamine acceptor dyes including Cy5, dichloro labeling the 5'-end can be explained by the inherent direction R110, dichloro R6G, dichloroTAMRA), dichloro ROX ality of DNA synthesis by a polymerase. To initiate DNA or the like, fluorescein donor dye including fluorescein, synthesis, a polymerase must be able to access the 3'-end of 6-FAM, or the like: Acridine including Acridine orange, Acri the DNA at the primer-template junction. Fluorescent dyes dine yellow, Proflavin, pH7, or the like: Aromatic Hydrocar that have been attached at the 3'-nucleotide's base, sugar, or bon including 2-Methylbenzoxazole, Ethyl p-dimethylami alpha phosphate can severely alter the conformation of the DNA, subsequently inhibiting or dramatically reducing DNA nobenzoate, Phenol, Pyrrole, benzene, toluene, or the like: synthesis efficiency. Explanations for the affect that fluoro Arylmethine Dyes including Auramine O. Crystal violet, phore presence has on DNA structural perturbations involve H2O, Crystal violet, glycerol, Malachite Green or the like: the notable size of the fluorescent dye and its hydrophobic Coumarin dyes including 7-Methoxycoumarin-4-acetic acid, nature. Coumarin 1, Coumarin 30, Coumarin 314, Coumarin 343, 0105. Although fluorescence is rapidly emerging as the Coumarin 6 or the like: Cyanine Dye including 1,1'-diethyl technology of choice, it can be cost prohibitive. For example, 2,2'-cyanine iodide, Cryptocyanine, Indocarbocyanine (C3) a standard 25 base oligonucleotide that is fluorescently dye, Indodicarbocyanine (C5)dye, Indotricarbocyanine (C7) 5'-end-labeled using current methods can range in price from dye, Oxacarbocyanine (C3)dye, Oxadicarbocyanine (C5) S98.75 to upwards of S600 at the 250 nmole scale, depending dye, Oxatricarbocyanine (C7)dye, Pinacyanol iodide, Stains on the choice of fluorophore, its place of attachment, and the all. Thiacarbocyanine (C3)dye, ethanol. Thiacarbocyanine level of purity desired. The time needed for the labeled oli (C3)dye, n-propanol. Thiadicarbocyanine (C5)dye. Thiatri gonucleotide order to arrive can range between 3 to 10 days, carbocyanine (C7)dye, or the like; Dipyrrin dyes including depending on the complexity of the synthesis. Additionally, N,N'-Difluoroboryl-1,9-dimethyl-5-(4-iodophenyl)-dipyr inefficiencies in the coupling of the fluorophore to the oligo rin, N,N'-Difluoroboryl-1,9-dimethyl-5-(4-(2-trimethylsi nucleotide may result in a large fraction of the product being lylethynyl), N,N'-Difluoroboryl-1,9-dimethyl-5-pheny unlabeled, and require its re-synthesis resulting in further dipyrrin, or the like; Merocyanines including delay. 4-(dicyanomethylene)-2-methyl-6-(p-dimethylami 0106. The Labeled ATPs of this invention are ideally nostyryl)-4H-pyran (DCM), acetonitrile, 4-(dicyanomethyl Suited for used in the following assays and detection proce ene)-2-methyl-6-(p-dimethylaminostyryl)-4H-pyran dures: absorbance assays, fluorescence intensity assays, fluo (DCM), methanol, 4-Dimethylamino-4'-nitrostilbene, Mero rescence polarization assays, time resolved fluorescence cyanine 540, or the like; Miscellaneous Dye including 4.6- assays, fluorescence resonance energy transfer (FRET) Diamidino-2-phenylindole (DAPI), 4',6-Diamidino-2-phe assays, or other assays. nylindole (DAPI), dimethylsulfoxide, 7-Benzylamino-4- 0107 The present invention also relates to a kit adapted to nitrobenz-2-oxa-1,3-diazole, Dansylglycine, H2O, Dansyl 5' end-label an target oligonucleotide with a fluorophore or glycine, dioxane, Hoechst 33258, DMF, Hoechst 33258, biotin or other binding molecules. The reagents of this inven HO, Luciferyellow CH, Piroxicam, Quinine sulfate, 0.05 M tion can be used with either previously synthesized or newly H2SO4, Quinine sulfate, 0.5 MH2SO4, Squarylium dye III, synthesized oligonucleotides in labeling reactions and tailor or the like; Oligophenylenes including 2,5-Diphenyloxazole experiments on the fly by selecting an appropriate fluores (PPO), Biphenyl, POPOP. p-Quaterphenyl, p-Terphenyl, or the like: Oxazines including Cresyl violet perchlorate, Nile cently labeled ATP or biotin labeled ATP. Blue, methanol, Nile Red, Nile blue, ethanol, Oxazine 1, Suitable Reagents Listings Oxazine 170, or the like: Polycyclic Aromatic Hydrocarbons including 9,10-Bis(phenylethynyl)anthracene, 9,10-Diphe 0108 Suitable atomic tags for use in this invention nylanthracene, Anthracene, Naphthalene, Perylene, Pyrene, include, without limitation, any atomic element amenable to or the like; polyene/polyynes including 1,2-diphenylacety attachment to a specific site in a target or dNTP, especially lene, 1,4-diphenylbutadiene, 1,4-diphenylbutadiyne, 1.6- Europium shift agents, NMR active atoms or the like. Diphenylhexatriene, Beta-carotene, Stilbene, or the like: 0109 Suitable molecular tags for use in this invention Redox-active Chromophores including Anthraquinone, include, without limitation, any molecule amenable to attach Azobenzene, Benzoquinone, Ferrocene, Riboflavin, Tris(2. ment to a specific site of a target PN or PP, such as fluorescent 2'-bipyridypruthenium(II), Tetrapyrrole, Bilirubin, Chloro molecules, quenching molecules, Europium shift agents, phylla, diethyl ether, Chlorophylla, methanol, Chlorophyllb. NMR active molecules, Raman active molecules, near IR Diprotonated-tetraphenylporphyrin, Hematin, Magnesium active molecules, or the like. octaethylporphyrin, Magnesium octaethylporphyrin 0110 Suitable NMR tags include any active NMR nuclei. (MgOEP), Magnesium phthalocyanine (MgPc), PrOH, Mag Exemplary examples of NMR active nuclei include, without nesium phthalocyanine (MgPe), pyridine, Magnesium tet limitation, H, C, N, F, Si, 7 Fe, 'Rh, etc. ramesitylporphyrin (MgTMP), Magnesium tetraphenylpor 0111 Suitable molecular tags for use in this invention phyrin (MgTPP), Octaethylporphyrin, Phthalocyanine (Pc), include, without limitation, any molecule amenable to attach Porphin, Rox, TAMRA, Tetra-t-butylazaporphine, Tetra-t- ment to a specific site in a target or dNTP, especially fluores butylnaphthalocyanine, Tetrakis(2,6-dichlorophenyl)por cent dyes or molecules that quench the fluorescence of the phyrin, Tetrakis(o-aminophenyl)porphyrin, Tetramesitylpor fluorescent dyes, paramagnetic molecules such as those dis phyrin (TMP), Tetraphenylporphyrin (TPP), Vitamin B12, closed in U.S. Pat. Nos: 6,458,758; 6,436,640; 6,410,255; Zinc octaethylporphyrin (ZnOEP), Zinc phthalocyanine 6,316, 198; 6,303,315; 5,840,701; 5,833,601; 5,824,781; (ZnPc), pyridine, Zinc tetramesitylporphyrin (ZnTMP), Zinc 5,817,632; 5,807,831; 5,804,561; 5,741,893; 5,725,8395; tetramesitylporphyrin radical cation, Zinc tetraphenylpor 706,805; and 5,494,030, incorporated herein by reference, phyrin (ZnTPP), or the like: Xanthenes including Eosin Y.

US 2010/031 7005 A1 Dec. 16, 2010 kinase. (CA: ATP+acetyl-CoA carboxylase=ADP+ acetyl 2.7.2.6 Formate kinase. (CA: ATP+formate=ADP+formyl CoA carboxylasephosphate); ID 2.7.1.129 Myosin heavy phosphate); ID 2.7.2.7 Butyrate kinase. (CA: ATP+2- chainkinase. (CA: ATP+ myosin heavy-chain=ADP+ butanoate=ADP+butanoyl phosphate); ID 2.7.2.8 Acetyl myosin heavy-chain phosphate); ID 2.7.1.130 glutamate kinase. (CA: ATP+N-acetyl-L-glutamate=ADP+ Tetraacyldisaccharide 4'-kinase. (CA: ATP+2,3-bis(3-hy N-acetyl-L-glutamate 5-phosphate); ID 2.7.2.11 Glutamate droxytetradecanoyl)-D-glucosarninyl-(beta-D-16)-2,3-bis 5-kinase. (CA: ATP+L-glutamate=ADP+L-glutamate (3-hydroxytetradecanoyl)-D-glucosaminyl beta 5-phosphate); ID 2.7.2.13 Glutamate 1-kinase. (CA: ATP+L- phosphate=ADP+2.3.2',3'-tetrakis(3- glutamate=ADP+alpha-L-glutamyl phosphate); ID 2.7.2.14 hydroxytetradecanoyl)-D-glucosaminyl-1,6-beta-D- Branched-chain-fatty-acid kinase. (CA: ATP-2- glucosamine 1,4'-bisphosphate); ID 2.7.1.131 Low-density methylpropanoate=ADP+2-methylpropanoyl phosphate); ID lipoprotein receptorkinase. (CA: ATP+low-density lipopro 2.73.1 Guanidoacetate kinase. (CA: ATP tein receptor L-serine=ADP+low-density lipoprotein recep guanidoacetate=ADP+phosphoguanidoacetate); ID 2.7.3.2 torO-phospho-L-serine); ID 2.7.1.132 Tropomyosin kinase. Creatine kinase. (CA: ATP+creatine=ADP+phosphocreat (CA: ATP+tropomyosin ADP+tropomyosinC)-phospho ine):ID 2.7.3.3 Arginine kinase. (CA: ATP+L- L-serine); ID 2.7.1.134 Inositol-tetrakisphosphate 1-kinase. arginine=ADP+N-phospho-L-arginine); ID 2.7.3.4 Tauro (CA: ATP+ 1D-myo-inositol 3,4,5,6- cyamine kinase. (CA: ATP+taurocyamine=ADP+N- tetrakisphosphate=ADP+ 1D-myo-inositol 1,3,4,5,6-pentak phosphotaurocyamine); ID 2.7.3.5 Lombricine kinase. (CA: isphosphate); ID 2.7.1.135 Tau protein kinase. (CA: ATP+ ATP+lombricine=ADP+N-phospholombricine); ID 2.7.3.6 tau protein-ADP+tau proteinO-phospho-L-serine); ID Hypotaurocyamine kinase. (CA: ATP 2.7.1.136 Macrollide 2'-kinase. (CA: ATP hypotaurocyamine ADP+N(omega)-phosphohypotauro oleandomycin=ADP+oleandomycin 2'-O-phosphate); ID cyamine); ID 2.7.3.7 Opheline kinase. (CA: ATP+guanidino 2.7.1.137 Phosphatidylinositol 3-kinase. (CA: ATP+1-phos ethyl methyl phosphate=ADP+N'-phosphoguanidinoethyl phatidyl-1D-myo-inositol=ADP+1-phosphatidyl-1D-myo methyl phosphate); ID 2.7.3.8 Ammonia kinase. (CA: ATP+ inositol 3-phosphate); ID 2.7.1.138 Ceramide kinase. (CA: NH(3)=ADP+-phosphoramide); ID 2.7.3.10 Agmatine ATP+ceramide ADP+ceramide 1-phosphate); ID 2.7.1.140 kinase. (CA: ATP+agmatine=ADP+N(4)-phosphoagma 1D-myo-inositol-tetrakisphosphate 5-kinase. (CA: ATP+ 1D tine); ID 2.7.3.11 Protein-histidine pros-kinase. (CA: ATP+ myo-inositol 1,3,4,6-tetrakisphosphate=ADP+ 1D-myo protein L-histidine=ADP+protein N(pi)-phospho-L-histi inositol 1,3,4,5,6-pentakisphosphate); ID 2.7.1.141 RNA dine); ID 2.7.3.12 Protein-histidine tele-kinase. (CA: ATP+ polymerase-subunit kinase. (CA: ATP+DNA-directed RNA protein L-histidine=ADP+protein N(tau)-phospho-L- polymerase=ADP+phospho-DNA-directed RNA poly histidine); ID 2.7.4.1 Polyphosphate kinase. (CA: ATP+ merase); ID 2.7.1.144 Tagatose-6-phosphate kinase. (CA: {phosphate}(N)=ADP+-phosphate}(N+1)); ID 2.7.4.2 ATP+D-tagatose 6-phosphate=ADP+D-tagatose 1,6-bispho Phosphomevalonate kinase. (CA: ATP+(R)-5- sphate); ID 2.7.1.145 Deoxynucleoside kinase. (CA: ATP+ phosphomevalonate=ADP+(R)-5-diphosphomevalonate); 2'-deoxynucleoside=ADP+2'-deoxynucleoside 5'-phos ID 2.7.4.3 Adenylate kinase. (CA: ATP+AMP=ADP+ADP): phate); ID 2.7.1.148 4-(cytidine 5'-diphospho)-2-C-methyl ID 2.7.4.4 Nucleoside-phosphate kinase. (CA: ATP+nucleo D-erythritol kinase. (CA: ATP+4-(cytidine 5'-diphospho)-2- side phosphate=ADP+nucleoside diphosphate); ID 2.7.4.6 C-methyl-D-erythritol=ADP+2-phospho-4-(cytidine Nucleoside-diphosphate kinase. (CA: ATP+nucleoside 5'-diphospho)-2-C-methyl-D-erythritol); ID 2.7.1.149 diphosphate=ADP+nucleoside triphosphate); ID 2.7.4.7 1-phosphatidylinositol-5-phosphate 4-kinase. (CA: ATP+1- Phosphomethylpyrimidine kinase. (CA: ATP+4-amino-2- phosphatidyl-1D-myo-inositol 5-phosphate=ADP+1-phos methyl-5-phosphomethylpyrimidine=ADP+4-amino-2-me phatidyl-1D-myo-inositol 4,5-bisphosphate); ID 2.7.1.150 thyl-5-diphosphomethylpyrimidine); ID 2.7.4.8 Guanylate 1-phosphatidylinositol-3-phosphate 5-kinase. (CA: ATP+1- kinase. (CA: ATP+GMP=ADP+GDP); ID 2.74.9 Thymidy phosphatidyl-1D-myo-inositol 3-phosphate=ADP+1-phos late kinase. (CA: ATP+thymidine 5'-phosphate=ADP+thy phatidyl-1D-myo-inositol 3.5-bisphosphate); ID 2.7.1.151 midine 5'-diphosphate); ID 2.7.4.11 (Deoxy)adenylate Inositol-polyphosphate multikinase. (CA: ATP+1D-myo kinase. (CA: ATP+dAMP=ADP+dADP); ID 2.7.4.12 T2-in inositol 1,4,5-trisphosphate=ADP+ 1D-myo-inositol 1,4,5,6- duced deoxynucleotide kinase. (CA: ATP+dGMP (or dTMP) tetrakisphosphate=ATP+ 1D-myo-inositol 1,4,5,6- =ADP+dGDP (or dTDP)); ID 2.7.4.13 (Deoxy)nucleoside tetrakisphosphate=ADP+ 1D-myo-inositol 1,3,4,5,6- phosphate kinase. (CA: ATP+deoxynucleoside pentakisphosphate); ID 2.7.1.152 Inositol-hexakisphosphate phosphate=ADP+deoxynucleoside diphosphate); ID kinase. (CA: ATP+myo-inositol hexakisphosphate=ADP+ 2.74.14 Cytidylate kinase. (CA: ATP+(d)CMP=ADP+(d) diphospho-myo-inositol pentakisphosphate (isomeric con CDP); ID 2.7.4.15 Thiamine-diphosphate kinase. (CA: ATP+ figuration unknown)); ID 2.7.1.153 Phosphatidylinositol-4, thiamine diphosphate=ADP+thiamine triphosphate); ID 2.7. 5-bisphosphate 3-kinase. (CA: ATP+1-phosphatidyl-1D 4.16 Thiamine-phosphate kinase. (CA: ATP+thiamine myo-inositol 4,5-bisphosphate=ADP+1-phosphatidyl-1D phosphate=ADP+thiamine diphosphate); ID 2.7.4.18 Farne myo-inositol 3,4,5-trisphosphate); ID 2.7.1.154 syl-diphosphate kinase. (CA: ATP+farnesyl Phosphatidylinositol-4-phosphate 3-kinase. (CA: ATP+1- diphosphate=ADP+farnesyltriphosphate); ID 2.7.4.19 5-me phosphatidyl-1D-myo-inositol 4-phosphate=ADP+1-phos thyldeoxycytidine-5'-phosphate kinase. (CA: ATP+5-meth phatidyl-1D-myo-inositol 3,4-bisphosphate); ID 2.7.2.1 yldeoxycytidine 5'-phosphate=ADP+5-methyldeoxycyti Acetate kinase. (CA: ATP+acetate=ADP+acetyl phosphate); dine diphosphate); ID 2.7.6.1 Ribose-phosphate ID 2.7.2.2 Carbamate kinase. (CA: ATP+NH(3)+CO(2) diphosphokinase. (CA: ATP-D-ribose 5-phosphate=AMP+ =ADP+carbamoyl phosphate); ID 2.7.2.3 Phosphoglycerate 5-phospho-alpha-D-ribose 1-diphosphate); ID 2.7.6.2 Thia kinase. (CA: ATP+3-phospho-D-glycerate=ADP+3-phos mine diphosphokinase. (CA: ATP+thiamine=AMP+thiamine pho-D-glyceroyl phosphate); ID 2.7.2.4 Aspartate kinase. diphosphate); ID 2.7.6.3 2-amino-4-hydroxy-6-hydroxym (CA: ATP+L-aspartate=ADP+4-phospho-L-aspartate); ID ethyldihydropteridine diphosphokinase. (CA: ATP+2-

US 2010/031 7005 A1 Dec. 16, 2010

thereof; (b) one or more carbonatoms are replaced by a hetero by measuring separation of tagged gamma phosphate from atom-containing groups selected from the group consisting of intact nucleotide, where intact nucleotide produces minimal —C(O)NH (amido group), —NHC(O)NH (uryl group), signal, but cleaved products produce detectable signals that or the like; (c) one or more hydrogen atoms are are replaced increase or change over time and these changes indicate that by a hetero atom selected from the group consisting of O, S, the activity being monitored is occurring or has occurred. P. and mixtures thereof; (d) one or more hydrogen atoms are replaced by a hetero atom-containing groups selected from High Throughput Screening of Kinase Substrate Specificity the group consisting of C(O)NH- (amido group), —NHC 0117 The gamma-labeled NTPs of this invention can be (O)NH (uryl group), or the like; (e) and mixture or combi used to perform kinase activity assays on multiple candidate nations thereof. Preferred hetero atom analogs include com Substrates along with positive and negative controls, all in pounds of the general formula —CH2(CH), ECH (CH), parallel. One preferred method involves immobilizing the ECH (CH)— where E is O, S, or P. n is an integer having a controls and candidate Substrate polypeptides in an array on a value between 0 and 10 and m is an integer having a value Substrate, such as a polypeptide array. The kinase and labeled between 0 and 10. Preferred alkenyl group include —(CH) NTP of this invention are allowed to contact the immobilized — where k is an integer having a value between 1 and 16. species under conditions to promote the kinase activity result Preferred arenyl groups includes phenylene, divalent naphth ing in the transfer of the labeled phosphate of the labeled-NTP ylene, divalent antracene, or similer polycondensed aromat to the candidate substrate polypeptide. Knowledge of the ics or mixtures or combinations thereof, where divalent Substrates that are phosphorylated and the corresponding means that the amine, phosphine or thiol groups are attached sites of phosphorylation of the candidate substrates provide at two different sites of the aromatic molecules. Preferred information about the recognition target of the assayed alka-arenyl groups include dialkyenylbenzenes, dialkylenyl kinase. naphthalenes, dialkenyl antracenes or similar condensed 0118. It is possible that different kinases will preferen fused aromatics with two dialkenyl groups. Preferred ara tially interact with specific fluorophores and/or linkers pro alkenyl group include phenyl Substituted alkenyl group or the viding a measure of specificity to the reaction. Thus, T-NTPs like. can be designed or tailored for use by a specific kinase. Articulation of Reagent Usages Labeling the 5' End of Oligonucleotides Mapping Phosphorylation Site Within a Protein 0115 The labeled-5' phosphate of the gamma phosphate 0119 The T-NTPs or T-L-NTPs of this invention are con labeled nucleotide (T-NTP or T-L-NTP) is transferred by T4 tacted with a polypeptide in the presence of a catalyst, pref polynucleotide kinase (PNK) to the unlabeled, 5' end of an erably a protein kinase, to generate a phosphorylated, labeled oligonucleotide. Alternatively, phosphate exchange reaction polypeptide. The phosphorylated, labeled polypeptide can be methodology can be used to transfer the labeled phosphate of cleaved, enabling determination of the site of phosphoryla the T-NTP or T-L-NTP to the 5' end of an oligonucleotide. tion. Thus, the TNTPs or T-L-NTPs of this invention are used to catalytically or enzymatically label oligonucleotides. The Incorporation of Kinase Modifiable Site in Recombinant Pro enzymatically or catalytically labeled oligonucleotides of this teins invention are well-suited as probes taking the place of a 0.120. A fusion protein encoding polynucleotide sequence chemically-synthesized fluorescent probes, or in any appli including a protein encoding region encoding a protein of cation where one needs to track an oligonucleotide (for interest and at least one phophorylation site encoding example, a 'P-labeled probe). Multiple fluorophores can be sequence ligated to either 5' end or 3' end or both the 5' and 3' added into a single reaction to an oligonucleotide solution, ends of the protein encoding region, where the phophoryla where each fluorophore has a different color and each result tion site encoding sequence encoded amino acid sequences ing color coded oligonucleotide can be monitored in parallel. that correspond to a kinase phosphorylation amino acid The enzymatically labeled oligonucleotides of this invention sequence. The expressed fusion protein can then be contacted can also be used to prepare probes Such as TaqMan probes, with a T-NTP or T-L-NTP of this invention and the kinase for molecular beacons, etc. Because this reaction allows for phosphorylating the fused phosphorylation site(s) producing existing oligonucleotide or polynucleotide to be labeled after a specifically labeled fusion protein, where the label is preparation, whereas in the past, labeled oligonucleotide and designed to not significantly impact protein folding or native polynucleotides had to be specially synthesized, the present activity, but gives rise to site-specifically labeled target pro invention allows labeling of pre-existing unlabeled oligo teins. If multiple fluorophores are attached to the fusion pro nucleotides or polynucleotides. Thus, researchers can rapidly tein, then the labels can be either the same or different colors. attach a fluorescent label or other type of label to an on-hand Such labeled fusion proteins can be used to monitor the oligonucleotide. Using the technology of this invention pro expression, activation, activity and deactivation of these vides researchers with a minimal time (generally overnight) fusion proteins in the expressed cells, cell cluster, tissue or method for labeling existing oligonucleotides as compared to Organ. the delays encountered when a fluorescently labeled oligo nucleotide is ordered, a typically 3 to 10 day delay. Enzymati Color Coded Nucleotide and Polypeptide Markers cally labeled oligonucleotides can be used in any application in which chemically-labeled fluorescent or radioactively-la I0121 Because the T-P moiety of the T-NTP or the T-L-P beled oligonucleotides would be used. moiety of the T-L-NTPs of this invention are readily trans ferred to the 5' and/or 3' ends of polynucleotides, the T-NTPs Reaction Monitoring or T-L-NTPs of this invention can be used to create color 0116. The gamma-labeled NPTs of this s invention can be coded polynucleotide (DNA, RNA, or DNA/RNA) molecular used to monitor reaction by monitoring modified nucleotide weight markers for use in DNA, RNA or DNA/RNA analyses US 2010/031 7005 A1 Dec. 16, 2010

similar to the kaleidoscope markers used in protein molecular physical interactions to maintain the kinase and the Substrate size standards. The use of color-coded polynucleotide mark in their associated State. The chemical and/or physical inter ers allows for unambiguous identification of the molecular actions can be hydrogen bonding, covalent bonding, ionic weight and/or size of each separated polynucleotide frag bonding, electrostatic attractions, or mixtures or combina ment. The same process can be used to form polypeptide tions thereof. This is useful for identifying interacting part markers provided that the polypeptide sequence includes at ners (i.e., the specific protein(s) phoshorylated by a specific least one site capable of being phosphorylated using a T-NTP kinase). or T-L-NTP of this invention. Kinase Substrate Screening. Using Modified NTPs Labeling Restriction Fragments 0.126 The modified NTPs of this invention can be 0122). Using the T-NTPs or T-L-NTPs of this invention, designed for use with proteins that require the unmodified restriction nucleotide fragments can be labeled through phos NTP in order to bind and/or transform a substrate protein or phate exchange reaction using PNK (e.g., T4-PNK) for polypeptide. The modified NTP can be designed to interact example to form labeled restriction nucleotide fragments. with the protein and to induce a conformation change in the These labeled restriction nucleotide fragments can be used to protein that would facilitate its binding to its substrate protein visualize and/or identify the fragments and do not involve or polypeptide. Preferably, the interaction is irreversible lock staining the fragments such as with ethidium bromide and the ing the protein in its active conformation. For example, if the DNA is viewed with longer wavelength light, thereby mini protein which depends on the unmodified NTP for activity is mizing DNA damage. So that the fragments are still active a kinase, then the modified NTP would lock the kinase in its fragments for Subsequent use. Complete labeling is not essen active conformation, the conformation that permits binding tial for visualization (i.e., only sufficient labeling for visual and/or transformation of its Substrate protein and/or polypep ization and/or identification), ensuring that pristine DNA tide. Thus, the modified NTP can be designed to lock the fragments can be isolated, as needed, for downstream protein in a conformation with an altered affinity for a second manipulation. substrate. This activated protein can then either be attached to a Support through a reactive group Such as a His-affinity Purifying Labeled Oligonucleotides tag Nickel resin, biotin—streptavidin, or antibody or the 0123. Because the labeling reaction produces labeled protein can be preattached and activated by the modified NTP material as well as unlabeled material, T-NTPs or T-L-NTPs by passing a solution containing the modified NTP over the and unreacted NTPs, purification may be needed. The unre Substrate having preattached protein. The substrate having acted NTPs and T-NTPs or T-L-NTPs can be separated from the activated protein, can then be used to screen a library of the labeled targets and unreacted targets using a sizing col potential Substrates by passing a solution of the Substrates umn (or similar strategy). Purifying the labeled target can be over the support. The substrates will then be separated into done enzymatically using an enzyme that distinguishes bound and unbound Substrates, unbound pass through over between 5'-labeled target nucleotides and non-labeled target the support with little or no delay, while bound substrates stay nucleotides through recognition of the 5' end to specifically attached to the activated protein. The bound substrates can degrade the non-labeled nucleotide. Lambda exonuclease and then be eluted off the support and identified. This procedure is phosphodiesterase 2 (PDE2) are candidate 5' to 3' exonu similar to the identification of unknown proteins isolated by cleases. Alternatively, HPLC purification may be used to their affinity to a known protein via the yeast two hybrid isolate the labeled nucleotide target. Spin sizing columns or system. similar techniques can be used in the purification process. 0127. The modified NTPs can also be used to examine at the affinity of known interactions where the binding of one Quick Assay of a Wide Range of ATP-utilizing Enzymes substrate is dependent on the binding of an NTP by using a 0.124. The labeled nucleotides of this invention, especially modified NTP to trap the enzyme in a state that would have an labeled ATP and GTP can be used as probes in ATP-utilizing increased affinity for a given Substrate and monitoring the or GTP-utilizing enzymatic processes to determine specific binding affinity between the two substrates (e.g., Biacore or activity, kinetics, mechanisms useful for biochemist and gel filtration). enzymologist. Enzymes could be NMP kinases, NDP kinases, Sugar kinases, and/or etc (see attached list of APT Dye-ATP Yeast Strain Cell Free Lysate Kinase Utilization utilizing Enzymes or variants thereof). These reactions are Assay usually coupled to a second NAD(H)-dependent enzyme for I0128. The present modified ATP are ideally suited for use accurate specific activity determination. The labeled nucle in Yeast strain cell free lysate kinase utilization assays under otides of this invention can be used to investigate the activity, Native Condition similar to the process described in Kolpdz kinetics and mechanism of action of Sugar kinase mediated ie, P. A. Young, R. A. 35 Epitope Tagging and Protein phosphorylations, a large class of biomolecules. Surveillance. Methods of Enzymology, vol. 194, pp 508-519. The procedure includes the steps of: (1) inoculate the yeast Suicide Tags strain in 10 ml YPD media and grow at 30° C. overnight until 0125 Attach a suicide tag to the gamma-phosphate of an cells reach log phase growth (~1x10" cells (a O.D. oo); (2) NTP. If the tagged gamma phosphate is transferred to a Sub harvest 2 mL of the log phase culture in an Eppendorf micro strate Such as a target protein, the protein kinase and the centrifuge tube; (3) pellet the cells at RT at 3000xg. Decant substrate are held in their associated state with sufficient supernatant; (4) resuspend pellet in 200 mL of Buffer A at 4° strength for post reaction purification and/or identification, C. which comprises 10% glycerol. 20 mM Hepes pH 7.9, 10 where term held means that the suicide tag interacts with both mM EDTA (maybe omitted, phosphatase inhibitor), 1 mM the kinase and the substrate with sufficient chemical and/or DTT, 0.5 mg/mL BSA, 100 mM Ammonium Sulfate, and 1 US 2010/031 7005 A1 Dec. 16, 2010

mM PMSF (other protease inhibitors may be added); (5) add EDA to form an ethylene diamine modified ATP (EDA-ATP). about 50 ml of 425-600 micron glass beads; (6) vortex vig Then in a second step 144, the EDA-ATP is reacted with a tag orously, 7x30s, alternate with 30s on ice; (7) centrifuge 5 min. T via a second amino group of the EDA to forman tagged ATP at 3000xg; (8) aliquot supernatant to a new Eppendorf micro (T-EDA-ATP), where the tag is preferably a fluorescent tag. centrifuge tube; (9) centrifuge 5 min. at 3000xg: (10) aliquot General Method for Labeling ONs or PNs with T-L-NTPs Supernatant to a new Eppendorf microcentrifuge tube; (11) 0.134 Referring now to FIG. 2A, a block diagram of a add dye-dATP to a final working concentration of 100 mM; method for tagging a synthetic 5' and 3' hydroxy terminated (12) incubate at 30°C. for a predetermined time-course not to oligonucleotide (SON) at either the 5' end using a tagged exceed 24 h; (13) take 10 mL aliquots at the specified time nucleotide triphosphate (T-L-NTP) of this invention, gener points; (14) add 10 mL of 2x denaturing gel loading buffer. ally 200, is shown to involve reacting a T-L-NTP comprising (place at -20°C.); (15) load the 20 mL time points (step a Base, a Sugar and three phosphate moieties (P., P and P.) 13+14) onto a 10% SDS-PAGE gel with appropriate markers with a SON in a step 202 in the presence of a catalyst or and electrophorese at 200V until dye front is ~1 cm from the enzyme. The catalyst or enzyme adds the tagged phosphate bottom: (16) visualize/document phosphorylation by laser (T-L-P) of the T-L-NTP to the 5' end, This same general light imaging at the specified dye excitation wavelength. (DO procedure works equally well with T-NTPs of this invention. THIS STEP FIRST, DO NOT STAIN); (17) stain gel using a 0.135 Referring now to FIG. 2B, a block diagram of a local coomassie protocol to visualize and document protein method for tagging a natural or synthetic 5" phosphate and 3' band pattern; and (18) overlay and compare laser-light image hydroxy terminated oligonucleotide (NON) at the 5' end vs. Coomassie stain. using a tagged nucleotide triphosphate (T-L-NTP) of this Dye-ATP (modified) Rapid Transformation Protocol for invention, generally 250, is shown to involve reacting a T-L- Yeast Strains NTP comprising a Base, a Sugar and three phosphate moi 0129. The present modified ATP can be used in rapid eties (P, P and P.) with a NON in a step 252 in the presence transformation protocol yeast strain assays similar to the pro of a catalyst or enzyme. The catalyst or enzyme exchanges the cedure described in Geitz, R. D., Woods, R. A. (2002) Trans 5' phosphate of the NON with the tagged phosphate (T-L-P) formation ofYeast by the LiAc/ssCarrier DNA/PEG Method. of the T-L-NTP. Methods of Enzymology 350: 87-96. The procedure includes General Method for Labeling PPs or PRNs with T-L-NTPs the steps of: (1) inoculate the yeast strain in 10 ml YPD media 0.136 Referring now to FIG. 3, a block diagram of a and grow at 30° C. overnight until cells reach log phase method for tagged polypeptides using a tagged nucleotide growth (~1x10" cells (a O.D. oo); (2) harvest 2 mL of the log triphosphate (T-L-NTP) of this invention, generally 300, is phase culture in an Eppendorfmicrocentrifuge tube; (3) pellet shown to involve reacting a T-L-NTP comprising a Base, a the cells at RT at 3000xg. Decant supernatant; (4) resuspend Sugar and three phosphate moieties (P., P and P.) with a the cells by adding the following components in the order polypeptide PN in a step 302 in the presence of a catalyst or listed: 240 mL of PEG 4000 50% w/v. 36 mL of lithium enzyme. The catalyst or enzyme adds the tagged phosphate acetate (LiAc) 1.0M.36 mL of 1.1 mM of Dye-dATP, and 48 (T-L-P) of the TL-NTP to a site AA m+1 of the PP, where the mL dEIO; (5) incubate the mixture in a water bath at 42°C. exact site of phosphorylation depends on the catalyst or for 45 minutes to 1 hour; (6) prepare a 1:40 dilution in deion ized HO; (7) spot 5 mL of the 1:40 diluent oil to a clean glass enzyme used in the reaction. This same general procedure slide; (8) spread to disseminate the cells on the slide (do not works equally well with T-NTPs of this invention. cover); and (9) visualize transformation efficiency via laser light microscopy at the dye specific excitation wavelength. Experimental Section 0130. The modified nucleotide reagents can be introduced into any cell or organism for monitoring NTP dependent General Synthetic Procedure for Preparing y-Phosphate cellular or organ functions. Modified ATP 0.137 The following synthetic scheme was used to prepare General and Specific Methods for T-L-NTP Preparation a variety of Y-Phosphate-Modified ATPs: 0131 Referring now to FIG. 1A, a block diagram of a method for making the tagged NTPs of this invention, gener ally 100, is shown to involve reacting a nucleotide triphos NH2 phate (NTP) comprising a Base, a Sugar and three phosphate a N moieties (P, P and P) in a first step 102 with a tag T to form N y O O O a tagged NTP (T-NTP). sus B. B. B. HeHE-R-EH 0132 Referring now to FIG. 1B, a block diagram of a N O O1 No1 No1 YOH method for making the tagged NTPs of this invention, gener OH OH OH ally 120, is shown to include a nucleotide triphosphate (NTP) comprising a Base, a Sugar and three phosphate moieties (P. HO OH P and P.) which is reacted in a first step 122 with a linker L NH2 to form a linker modified NTP (L-NTP), where the linker L is bonded to the gamma phosphate P. After the linker L is a N Sa Dye/Biotin bonded to the NTP to form the L-NTP, the L-NTP is reacted JXN P P P He in a second step 124 with a tag T to form a tagged NTP N ONo1 No1 No1 NE-R-EH DMF, (T-L-NTP). OH OH OH 1M NaHCO 0.133 Referring now to FIG. 1C, a method for preparing a pH 9 tagged ATP generally 140, is shown to include an ATP which OHOH is reacted in a first step 142 with an ethylene diamine linker US 2010/031 7005 A1 Dec. 16, 2010 16

set forth in the Reagent Listing Section. As stated previously, -continued the linker can be diamines, diphosphines, dithiols, or mixed NH2 amine, phosphine and thiols. The linkers can also be carbon a N chains having leaving groups to promote direct carbon phos phate bonding and direct carbon to dye bonding. Because the Sa X O linkers can have different lengths, charges, sizes, shapes, and N N OYO1 P.YO1 P.YO1 PYE-R-E1Dye/Biotin l polarities, and other physical properties, the exact linker for OH OH OH use in a particular application may depend on different vari ables. However, it is expected that any of the modified ATPs OH OH of this invention will work in all circumstances, each will be better suited for some application while other will be better Suited to other applications. where E is a main group element selected from the group consisting of N, O, P, S and combinations thereof and where Example 1 R is a carbon-containing group having between 1 and about 0.138. This example illustrates the preparation of a ATP 20 main group atoms selected from the group consisting of B. L1-ROX or ATP-EDA-ROX, where the EDA is attached to C, N, O, P, S and combinations thereof, with the valency being the Y-phosphate of ATP as shown in the following reaction completed by H. A more detailed explanation of the linker is scheme:

NH2

21 N N y O O O ls N | | NH2CH2CH2NH2 N O o1 No1 No1 Non DEC, MBS pH5.7 OH OH OH

HO OH

NH2

2 N N y O O O sus ROX SE N O o1 No1 No1 Nencient, HerDMF, IM NaHCO, pH 9 OH OH OH

OH OH

NH2

21 N

N JXN P P P. H N O o1 No1 No1 NCHCH OH OH OH N

OH OH US 2010/031 7005 A1 Dec. 16, 2010

0139 Preparation of ATP-L1 or ATP-EDA 0140. A mixture of 7 mg, 12.7 umol of ATP and 10 mg, 52 -continued umol of DEC in 0.1M, pH 5.7, 1.5 mL of MES buffer was (L.2) stirred at room temperature for 10 min. 7 mg, 53 umol of HN ethylene diamine hydrochloride (EDA.HCl) in 0.1M, pH 5.7, 2 mL of MES buffer was added and the mixture was stirred for NH 2-3 hr. The pH was maintained between 5.65-5.75 during this (L3) time. The reaction was monitored on Thin Layer Chromatog 1n-N-11-N" raphy (TLC). The reaction mixture was then lyophilized and (L4) the pellet was dissolved in 1 mL water. The resulting solution was subjected to HPLC purification using a Waters HPLC system on a Supelco C18 column using a TEAA-acetonitrile buffer system; or on a Waters Protein-Pak Q using a 0145. In these structures, one of the amine protons is miss NHHCO, MeOH/HO buffer system. The product peak ing from each terminal nitrogen atom because the linkers was collected and lyophilized to yield a white powder which bond to the Y-phosphate of ATP and the fluorescent dye was dissolved in 200 uL water and quantitated by spectro through the terminal nitrogen atoms of the diamine linkers. photometry at 259 mm; 24 mM. The yield of the ATP-EDA The twelve y-phosphate-modified-ATPs are tabulated Table intermediate was 38%. 1. 0141 Preparation of ATP-L1-ROX or ATP-EDA-ROX 0142. 42 uL. 1 umol of the ATP-EDA intermediate of TABLE 1 Example 1 was dissolved in 1M, pH 9.0, 70 uL of NaHCO buffer and 2.5 mg, 4 umol of ROX-NHS was dissolved in 100 Prepared ATPs and Their Excitation and Emission Properties uL of dry DMF. These two solutions were mixed and set on a Modified ATPs Excitation (2) Emission (2) shaker overnight. The reaction was monitored via HPLC on a ATP-L1-Fluorescein 495 526 C18 column using a TEAA-acetonitrile buffer system. After ATP-L2-Fluorescein 495 S18 lyophilization, the pellet was dissolved in 0.3 mL of water and ATP-L3-Fluorescein 495 52O the solution was passed through a Sephadex G-25 column (1* ATP-L4-Fluorescein 495 521 15 cm). The first eluted fraction was lyophilized. A 1 mL ATP-L1-Tamra S4O 582 ATP-L1-Rox 581 612 water solution of the pellet was subjected to HPLC purifica ATP-L2-Rox 581 609 tion on a Supelco C18 column using a TEAA-acetonitrile ATP-L3-Rox 581 607 buffer system. The product peak, having a retention time 11 ATP-LA-Rox 581 609 minutes, was collected and lyophilized to give a pellet. The ATP-L1-Cy3 550 S62 pellet was dissolved in 5 mM, 780 uL of HEPES buffer and ATP-L1-Cy5 646 663 quantified by 576 nm reading on a spectrometer. The mea ATP-L1-biotin na na Sured concentration of the Solution was 1.1 mM representing a yield of 86% of ATP-EDA-ROX. MALDI-Mass: 1124 Removal of Unlabeled Nucleotides from Labeled Nucle (M-3+Na+K); 1102 (M+K), 1058 (M-HO--K). 1.1 mM, 1 uL otides of the ATP-EDA-ROX compound was treated with 65 U/mL, 0146. Once a Y-phosphate-modified nucleotide or deoxy 1 LL of phosphodiesterase (PDE) in 10 LIL of a 1x buffer nucleotide was prepared, then unlabeled nucleotide ordeoxy comprising 110 mM Tris.HCl pH 8.9, 110 mM NaCl and 15 nucleotide can be removed by treating the solution with an mM MgCl2 at room temperature for 20 minutes and then appropriate phosphatase. For example, if the nucleotide is analyzed with TLC. The products were characterized as AMP ATP, then calf intestinal phosphatase (CIAP) or shrimp alka and pyrophosphate (Ppi)-EDA-ROX by comparison with line phosphatase (SAP) can be used to specifically remove the authentic samples. unmodified ATP leaving the Y-phosphate-modified-ATP 0143 An HPLC chromatogram of ATP-1-ROX synthesis intact. In many applications, Y-phosphate-modified-ATPs reaction (576 nm) to monitor the mobility of molecules linked free from their corresponding natural analog are prepared. to the fluorophore or free dye is shown in FIG. 4. HPLC The removal of the unmodified ATPs greatly enhances suc chromatogram of ATP-1-ROX synthesis reaction (259 nm) to cessful implementation of the methodology of this invention. monitor oligonucleotide mobility is shown in FIG. 5. UV This invention provides an effective process using phos spectrum of ATP-1-ROX is shown in FIG. 6. phatases (e.g., CIAP and SAP) to remove non-modified NTPs or dNTPs from Y-phosphate-modified-NTPs and dNTPs. Examples 2-10 0147 The general procedure for purifying a Y-phosphate modified nucleotide involves treating 1 mM, 1 mL, 1 nmol of 0144. Using the general scheme, a set of twelve y-phos the crude Y-phosphate-modified nucleotide with 1 U/mL, 1 phate-modified-ATPs using four different linker were synthe uL. 1U of CIAP in 10 mL or 20 mL of a 1x buffer comprising sized. The four linkers designated L1 or ethylene diamine 50 mM Tris.HCl pH 9.3, 1 mM MgCl, 0.1 mM ZnCl, 1 mM (EDA), L2, L3 and L4 and having the structures shown below, spermidine, at room temperature for 20 minutes. The treated were prepared and tested for activity in 5" labeling of nucle sample was then used in a Subsequent reaction as described otides: herein. It is also possible to remove CIAP by heat-shock and centrifugal filtration.

(L1) Example 11 NH N1,N1 0.148. This example illustrates the purification of ATP ROX with CIAP US 2010/031 7005 A1 Dec. 16, 2010

0149 ATP (10 mM, 2 uL. 20 nmol) and ATP-ROX (1.1 amount of T4 PNK to two microliters (20U) and the amount mM, 2 LL, 2.2 nmol) were treated with CIAP (1 U/uL. 1 uL. of oligonucleotide to one microgram as shown in FIGS. 10 1U) in 1x buffer (10 uD) at room temperature for 20 minutes. and 11. Controls were the same reaction mixtures without enzyme. After 25 min, aliquots (1 L) from the samples were analyzed Example 13 with appropriate TLC chromatography and fluorescence 0155 This example illustrates the purification of end imaging or UV-shadowing. The result is shown below in FIG. labeling of the 5' end of an oligonucleotide using T4 PNKand 7. ATP-L1-ROX or ATP-EDA-ROX 0156 CENTRI-SEP Columns (Princeton Separations, Example 12 Inc. Cat. #CS-900) were used to remove unwanted dye-ATP and dye breakdown from the 5' dye-labeled oligonucleotide 0150. This example illustrates the end labeling of the 5' product. A ten microliter reaction was performed with the end of an oligonucleotide using T4 PNK and ATP-ROX. ATP-L1-ROX and the TOP oligonucleotide and purified with a CENTRI-SEP column according to a protocol based on the manufacture's protocol as set forth at their website at hup:// Objective www.prinsep.com/html/products/centri sep/single column/ 0151. To determine ifT4 PolyNucleotide Kinase is able to protocoli, where the modification replaced the 750xg for 2 use our ATP-1-Dye molecules as substrates for the 5' end minute centrifugation step with a 500xg for 3 minutes cen labeling of an oligonucleotide. trifugation step. Additionally, ten microliters of sterile milli-Q water were added to the ten microliter end-labeling Utility reaction to bring the final volume to twenty microliters. 0152 The ability to enzymatically add a fluorophore to the Removal of Dye Terminators Prior to Sequencing 5' end of an oligonucleotide has tremendous applicability for 0157. Several methods have been identified for removing a wide variety of molecular biological techniques. The initial labeled-ATP and free dye from the labeled oligonucleotide labeled phosphate transfer experiment was set-up as a stan (DNA, RNA, RNA/DNA) products of this invention. These dard end-labeling reaction whereby one microliter of a 1.1 methods include phenol-chloroform extraction, phenol-chlo mM solution of ATP-L1-ROX was used in a ten microliter roform extraction and ethanol precipitation, and widely used reaction using 10U of T4 PNK to end-label 100 nanograms of spin-column removal processes (e.g., Qiagen, CentriSep. an in-house “TOP oligonucleotide having the nucleotide etc.). sequence SEQ. ID NO. 2 of 5'gg TACTAAgCggCC gCATg 3'. The reaction was incubated at 37° C. for 30 minutes, Examining ATP-L1-ROX in 5' End-labeling Oligonucleotide loading dye was added, and the sample was loaded onto a Reactions 20% denaturing polyacrylamide gel along with a chemically 0158. The following figures display experimental results synthesized (MWG) 5' fluorescein labeled TOP oligonucle investigating T4 PNK’s ability to utilize the set of fluores otide (FITC-TOP) as a size marker. Scanning the gel with cently labeled ATP molecules in 5'-end-labeling reactions of Texas Red channel revealed no labeled oligonucleotide in the oligonucleotides. All reactions have undergone column puri reaction lane which corresponded to 19mer TOP. FITC scan fication prior to electrophoresis on a 20% denaturing poly ning of the gel clearly reveals the FITC-TOPoligonucleotide, acrylamide gel to remove excess fluorescent-ATP and free indicating that the end-labeling reaction did not work. dye which obscures the 5' fluorescently end-labeled oligo 0153. The negative result prompted us to consider that our nucleotide product. For each experiment, the most intense ATP-11-ROX molecule contained unlabeled ATP that carried (major) band was assigned a relative activity value of 1.0 and over from synthesis. Unlabeled ATP is thought to out-com all other bands were normalized to this value. pete the ATP-L1-ROX molecule for T4 PNK binding and thereby reduce the yield of 5' ROX-labeled oligonucleotide. Reaction Time To insure purity, the ATP-L1-ROX product was treated with CLAP to remove unlabeled ATP. Additionally, to circumvent 0159. The initial experiment was designed to investigate a potentially slower reaction rate of T4 PNK with ATP-1- the time required to maximize T4 PNK's ability to 5' end ROX substrate, an overnight incubation at 37°C. was con label a 19-base oligonucleotide with ATP-L1-ROX as shown ducted. A CTAP reaction was performed containing one FIGS. 12A&B. A significant improvement in 5' end-labeling microliter of 1.1 mM solution of crude ATP-L1-ROX, 1 occurred when reactions were allowed to continue overnight microliter of CIAP (available from Promega), one microliter or for about 18 hours. Since reactions that were incubated for of 10x CLAP buffer (available from Promega), and seven longer than 18 hours (not shown) did not improve labeling, 18 microliters of sterile milli-Q water. This reaction was incu hours is designated as the standard labeling reaction time. bated at 37° C. for 30 minutes. Five microlliters of the CLAP treated ATP-L1-ROX was added to 1 microliter T4 PNK Timecourse Experiment Examining 5' End-labeling Effi (available from Promega), 1 microliter of 10x PNK buffer ciency (available from Promega), 100 nanograms of the TOP oligo 0160 Referring now to FIG.12A, a timecourse investigat nucleotide in a total volume often microliters. The reaction ing 5' end-labeling of a 19-base oligonucleotide with APT was incubated at 37°C. overnight. Results from this reaction L1-ROX. Reactions were incubated at 37 degrees Celsius for are shown in FIGS. 8 and 9 clearly evidencing 5' end labeling. 0.5, 1, 3, 6, and 18 hours. All reactions were column purified 0154) In follow up reactions, the inventors determined that and electrophoresed on a 20% denaturing gel. The negative more efficient 5' end-labeling was achieved by increasing the control (-) does not contain T4 PNK. Referring now to FIG. US 2010/031 7005 A1 Dec. 16, 2010

12B, a graphical representation of quantitated bands from gel labeled ATP) by stabilizing the enzyme via macromolecular imaging. Gels are scanned with a BIORAD Molecular Imager crowding. Based on this earlier report, polyethylene glycol FX Pro at the fluorophores specific emission wavelength filter (PEG 8000) was added to end-labeling reactions to determine setting. BIORAD Quantity One Quantitation software is used ifa similar affect is observed when fluorescently-labeled ATP to analyze bands. Band values are obtained by highlighting a is used as the label source as shown in FIG. 14. Increases in band's area and then Subtracting background value (equiva the amount of 5' ROX-labeled oligonucleotide are observed lent gel image area without a band) to obtain an optical as a result of increased PEG in the reaction. Further increases density value. in PEG past 12% did not improve the sefficiency of the fluorescent labeling reaction. Oligonucleotide 5' End-labeling Reaction: Method Details 0161 Reactions were assembled by adding 1 g of a Macromolecular Crowding Improves 5' End-labeling 19-base oligonucleotide (5' GGTACTAAGCGGCCGCATG 0166 Referring now to FIG. 14, an effect following addi 3'), 1 ul of Promega 10x Kinase buffer (700 mM Tris-HCl, pH tion of polyethylene glycol 8000 (PEG 8000) on the amount 7.6., 100 mM MgCl2, and 50 mM DTT), 2 nmol ATP-Linker of labeled oligonucleotide. 5' end-labeling reactions contain Fluorophore (or ATP-Linker-Biotin), 12% Polyethylene gly ing different concentrations of PEG 8000, (4, 6, 8, 12, and col8000, Promega T4 PNK(20U), and sterile milliO water to 18%) were examined with a 19-base oligonucleotide and a final volume of 10 ul. Reactions were incubated overnight APT-L1-ROX. The graph represents quantitated bands deter (18 hours) at 37 degrees Celsius in a BioRad 96 microtube mined via gel imaging and analysis. iCyclerthermocycler with heated-lid and purified with a Cen Chemical Properties that Affect the Labeling Reaction tri-SEP column (Princeton Separations, Inc.) following the 0167. In addition to examining reaction parameters to manufacture's protocol with the exceptions: (1) columns begin optimizing the 5' end-labeling reaction, Some of the were centrifuged at 500xg for 3 minutes (2) reaction volumes chemical properties of the preliminary set of fluorescently were adjusted to 20 ul with sterile MilliO water prior to labeled-ATPs were also investigated. The crystal structure of loading. T4 PNK reveals that the active site resembles a shallow tunnel in which agamma-phosphate of ATP can be positioned on one Product Analysis of 5' Fluorescently-labeled Oligonucle side of the tunnel for an in-line phosphoryl transfer to the otides: Method Details 5'-OH of a nucleic acid substrate on the opposite side. Two 0162 Reaction products can be directly loaded onto a 20% features of the labeled-ATP which may influence the phos denaturing polyacrylamide gel for electrophoresis or purified phoryl transfer by T4 PNK are the chemical characteristics of through a CentriSEP spin-column to remove excess ATP the fluorophore and the linker attachment. Their structural Linker-Fluorophore or ATP-Linker-Biotin that has not been size and/or rigidity may be critical to enzymatic activity, utilized in the reaction. Samples are electrophoresed at 50W. depending on potential steric constraints within the active site 50 degrees Celsius, for a period of approximately 1-1.5 hours tunnel. and then the gel is scanned with a BIORAD Molecular Imager 0168 Evidence which supports the differential effects that FX Pro at the fluorophores specific emission wavelength filter a DNA substrate has upon T4 PNK's end-labeling ability is setting. Subsequent gel analysis is performed with BIORAD the enzyme’s decreased activity when confronted with Quantity One Quantitation software. recessed 5' ends and nicks in double stranded DNA, and an apparent bias it exhibits towards preferentially phosphorylat Enzyme Amount ing a 5' terminal guanosine base over other bases. Other considerations include the effect that hydrophobicity and 0163 To determine the optimal amount of T4 PNKneeded polarity may have upon catalysis. This is particularly impor for 5' end-labeling with a fluorescent-ATP, two different con tant due to the hydrophobic nature of many of the fluorescent centrations (10U and 20U) of T4 PNK were tested with dyes. T4 PNK's active site surface is almost entirely com increasing concentrations of APT-L1-ROX as shown in FIGS. posed of charged or polar residues, however several hydro 13 A-B. The results demonstrate that 20 Units of T4 PNK phobic residues are involved in formation of the walls. In increases the efficiency of 5' end-labeling at all concentra order to ascertain whether the four linkers have differential tions of APT-L1-ROX tested. No improvements were effects on 5' end-labeling, two sets of the fluorescently observed when greater than 20U of T4 PNK were tested (data labeled-ATPs (ATP-Linker-ROX and ATP-Linker-Fluores not shown). cein) were examined shown in FIGS. 15A and B. Oligonucle otide labeling with the ROX-labeled ATPs show that T4 Optimizing Enzyme Concentration PNK's efficiency is higher when linker L1 joins the ATP and 0164 Referring now to FIG. 13A, a 5' end-labeling of a fluorophore, whereas linker L2 increases oligonucleotide 19-base oligonucleotide examining optimal T4 PNK activity labeling when fluorescein is used. Examining each of the (10U and 20U). Three concentrations of APT-L1-ROX (110, fluorescent dyes (Fluorescein, ROX, TAMRA, Cy3, and Cy5) 220, and 550 micromolar) were tested. Reactions were incu with linkers of different composition will enable the optimi bated at 37 degrees Celsius for 18 hours, column purified, and zation of 5' end-labeling oligonucleotides for these dyes and electrophoresed on a 20% denaturing gel. Referring now to will allow a better understanding of the enzyme’s phosphoryl FIG.13B. Graphical representation of quantitated bands from transfer mechanism. gel imaging. Examining Linker Effects on 5' End-labeling Volume Exclusion Agents (0169. Referring now to FIGS. 15A&B, the differential 0.165 High molecular weight polymers are reported to effect of L1, L2, L3, and L4 on T4 PNK's utilization of promote T4 PNK activity (i.e., transfer of they'P of a radio fluorescently labeled ATP molecules in the 5' end-labeling US 2010/031 7005 A1 Dec. 16, 2010 20 reaction. The reactions examining each of the four linkers hypothesized that a fluorescent dye attached at the 5' end of an were tested with a 19-base oligonucleotide and the set of oligonucleotide might provide protection against degradation (FIG. 15A) ATP-L-ROX (220 and 440 micromolar) mol by lambda exonuclease relative to one containing a 5' phos ecules and (FIG. 15B) ATP-L-Fluorescein molecules (220 phate added. The experiment where a ROX-labeled oligo and 440 micromolar). Reactions were incubated at 37°C. for nucleotide and unlabeled oligonucleotide were treated with 18 hours, column purified, and electrophoresed on a 20% lambda exonuclease for different periods of time is shown in denaturing gel, not shown. The graph represents quantitated FIG. 17A. Intact ROX-oligonucleotide (Red) and unlabeled bands from gel imaging and analysis. oligonucleotide (Green) are visible at timepoint 0. The amount of each remaining after treatment with lambda exo Specificity of Labeling at the 5' End of the Oligonucleotide nuclease is compared as shown in FIG. 17B. This experiment (0170 To determine whether the 3' end of the labeled oli demonstrates that the presence of the fluorophore and linker gonucleotide was available for enzymatic synthesis, DNA masks the 5' end of the labeled oligonucleotide, making it less polymerase extension reactions were performed. Since poly Susceptible to degradation by lambda exonuclease, and pro merases are restricted to incorporation at the 3' end, a 19-base vides a method to enrich for the desired labeled product. oligonucleotide was 5' end-labeled with APT-L1-ROX and then hybridized to a 20-base complementary oligonucleotide 5' Fluorescently Labeled Oligonucleotide is Protected containing a single base overhang A as shown in FIG.16A. Against Lambda Exonuclease This overhanging A was used by the polymerase as a tem 0.174 Referring now to FIG. 17A&B, depict examining plate for the incorporation of an incoming base-labeled duTP lambda exonuclease activity on ROX-labeled versus unla at the ROX-oligonucleotide's 3' end as shown in FIG. 16B. beled oligonucleotide. Looking at FIG. 17A, a 5' end-labeled Incorporation was determined by 19-base ROX-oligonucle ROX-oligonucleotide and unlabeled oligonucleotide that otide conversion into a 20-base ROX-oligonucleotide and have been treated with lambda exonuclease for 0 to 10 min confirmed by its altered migration in the denaturing gel as utes at 37° C. Reactions were electrophoresed and oligo shown in FIG. 16B. This experiment not only demonstrates nucleotide integrity monitored by ROX emission for the the applicability of enzymatic fluorescent labeling of an oli ROX-oligonucleotide (RED) and SyBR Gold staining for gonucleotide with the fluorescent-ATP reagents, but also unlabeled oligonucleotide (GREEN); gel images overlayed. reveals the potential utility for the creation of tailor-made The two green bands observed at timepoint 0 for the unla probes with fluorescent dyes or fluorescent dyes and quench beled oligonucleotide represent intact 19-base and 18-base ers at either end, such as a TaqMan or Molecular Beacon oligonucleotide (a byproduct of incomplete oligonucleotide probe. synthesis, -1). Looking at FIG. 17B, a graph represents quan titated bands, the intact band at timepoint 0 min. for ROX-1- Confirmation of ROX-Labeling at the Oligonucleotides 5' oligonucleotide and unlabeled oligonucleotide (19-base) End have each been assigned the relative activity of 1.0, with all (0171 Referring now to FIG. 16A, a Schematic of the 5' other bands normalized to their resp. control values. end-labeled ROX-oligonucleotide duplexed to a complemen Lambda Exonuclease Treatment of a 19-base Oligonucle tary 20-base oligonucleotide and used as primer-template for otide 5' End-labeled with ATP-L1-ROX: Method Details incorporation of a base-labeled duTP. Referring now to FIG. 0.175 Standard 5' end-labeling reactions were performed 16B, a gel electrophoresis of samples (Lane 1): . ROX-oligo with ATP-L1-ROX with the exception that after overnight nucleotide with an incorporated base-labeled duTP at its 3' incubation natural ATP was added to a final concentration of end (Lane 2): ROX-oligonucleotide without the base-labeled 1 mM and reactions were allowed to incubate for an addi dUTP incorporated. Reaction products were detected using a tional 2 hours at 37 degrees Celsius. The reactions were then ROX emission filter. purified on CentriSep columns and the eluent was vacuum dried. Treatment consisted of adding 5 ul of 10x lambda Primer Extension Reactions: Method Details exonuclease buffer (1x buffer: 67 mM Glycine-KOH, 2.5 0172 Oligonucleotides were annealed to form duplex by mM MgCl, 50 g/ml BSA, pH 9.4 at 25°C.), 5 ul (25U) of incubating primer and complementary template at equimolar lambda exonuclease (NeW England Biolabs, MA), and then amounts in a BioRad 96 microtube iCycler thermocycler with incubated for specified timepoints at 37 degrees Celsius. heated-lid starting at 96 degrees Celsius and slow-cooling to Timepoint aliquots were immediately removed to 5 ul stop 20 degrees Celsius over a one hour period. Primer extension solution (88% formamide, 1% bromophenol blue, 0.6M reactions contain the duplexed molecules, 1 ul HIV reverse EDTA). Samples were electrophoresed on a 20% denaturing transcriptase (1 mg/ml), 1 ul of 10x Promega Taq buffer (100 gel and the gel was scanned for ROX emission (red channel) mM Tris-HCl (pH 9.0 at 25° C), 500 mM KC1, 1.5 mM and stained with SYBR Gold (Invitrogen/Molecular Probes) MgCl, and 1% Triton X-100), and 200 uMdUTP-Alexa 488 (green channel). (Invitrogen/Molecular Probes). The reaction was incubated in a 37° C. water bath for 2 hours. PDE2 Treatment for Removing Unlabeled Single-Stranded DNA or RNA Preferential Elimination of Unlabeled Oligonucleotide (0176 The inventors also identified an alternative to the 0173 Experiments were conducted to examine if unla lambda exonuclease treatment for clearing unlabeled, single beled oligonucleotide could be preferentially eliminated stranded DNA and RNA from our T4 PNK labeling reactions: post-5' end-labeling. The enzyme used to degrade the unla effectively raising the specific activity of the labeling reac beled oligonucleotide was lambda exonuclease, since the tion. The inventors have found that the phosphodiesterases 2 enzyme preferentially degrades phosphorylated DNA in the (PDE2) enzyme has several advantages over the Lambda 5' to 3’ direction if a 5' phosphate is present. The inventors exonuclease including: (1) no cold-phosphorylation of the US 2010/031 7005 A1 Dec. 16, 2010 unlabeled oligonucleotide is required, thus making pre-treat quantitated with a BioRad Gel Documentation System and ment of the reaction with natural ATP unnecessary, and (2) analysis is performed with BIORAD Quantity One Quantita PDE2 will work on both DNA and RNA substrates. tion software. Calculating Labeling Efficiency. 5' End-labeling an Oligo 5' End-Labeling an Oligonucleotide Using ATP-L1-Biotin nucleotide with ATP-L1-ROX: Method Details 0177. Due to the favorable results observed with the fluo 0180. A standard method utilized by Molecular Probes/ rescently labeled-ATPs, a biotin-labeled ATP was designed Invitrogen (ULYSIS: Calculating the labeling efficiency of and synthesized for 5' end-labeling an oligonucleotide. The Nucleic Acid Labeling Kit, MP21650) to determine the label molecule, APT-L1-biotin, is comprised ofa biotin attached by ing efficiency of nucleic acids was performed to examine the linker L1 to the gamma-phosphate of ATP. As described for 5' end-labeling of an oligonucleotide with VisiCen's ROX the fluorescently tagged-Y-ATP products, the attached biotin labeled ATP. A reaction was performed as described previ linker moiety is transferred with the gamma-phosphate to the ously (see Oligonucleotide 5' end-labeling reaction: Method 5' end of the oligonucleotide by T4 PNK. Detection of 5' details) and a 2 microliter sample volume of the purified biotin-labeling of the oligonucleotide was observed by modi ROX-labeled oligonucleotide, 50 microMolar ATP-L1-ROX, fying an immunoblot analysis that is based on the strong and resuspension buffer were analyzed on a NanoDrop interaction between biotin and streptavidin. Column purified ND-1000 spectrophotometer (NanoDrop Technologies, Inc. (CentriSEP) 5’ biotinylated oligonucleotide was covalently USA) according to the manufacturer's recommendations. attached to Whatman DE81 filter paper disc by spotting and Absorbances were measured at 260 nm (for nucleic acid) allowed to air dry. Filter discs were processed with a strepta and 584 nm (, for ROX dye) to calculate the Base: Dye vidin-alkaline-phosphatase conjugate and Subsequently ratio. Also, the absorbance of the ATP-L1-ROX was mea developed with NBT/BCIP reagent. Displayed are three sured at these wavelengths to correct for the dye contribution DE81 filter discs that have undergone color development as at the 260 nm reading, i.e., a correction factor (CF). Equa shown in FIG. 18. Any residual APT-L1-biotin not utilized in tions are given in Research and Design Methods section, see the reaction was removed by CentriSEP column purification, Determining the relative use of the labeled-ATP product in as indicated by the lack of a spot on the negative control disc. oligonucleotide labeling reactions. The positive control (center filter) has been spotted with a dilution series of a chemically synthesized biotin-labeled oli TABLE 2 gonucleotide (Integrated DNA Technologies, Inc.) and is Calculating the Base:Dye Ratio for a 19 base DNA Oligonucleotide used as a comparison for labeling. 5' end-labeled with ATP-L1-ROX 5' End-Labeling a 19-Base Oligonucleotide with APT-L1 Biotin Reagent A260 A584 0.178 Referring now to FIG. 18, a 5' end-labeling experi ROX-Oligonucleotide O.6SOS O.O220 ment of a 19-base oligonucleotide with APT-LI-biotin is ATP-L1-ROX O.0983 O.S388 shown. Three DE81 filters that have been color-processed CF260 O.18 (Left disc) Negative control: reaction without T4 PNK, (Cen Reagent Extinction Coef. (e) ter disc) Positive control: Chemically synthesized biotin-la beled oligonucleotide (Integrated DNA Technologies, Inc.) ROX Dye 82OOO spotted are 1, 0.1, and 0.01 pmol indicated below each spot, SSDNA 8919 (Right) APT-L1-biotin experiment: reaction containing T4 Reagents in Ratio Ratio Value PNK. Base:Dye 82:1 Product Analysis of 5' Biotin-Labeled Oligonucleotides: Oligonucleotide:Dye 4.3:1 Method Details (0179 Reaction products are CentriSEP column purified 5' End-Labeling RNA for Microarray Hybridization: Method and the eluted sample is vacuum dried and resuspended in 5ul Details of sterile milliO water. The reaction is spotted onto Whatman DE81 filter paper in 1 Jul aliquots with air drying between 0181. A 20 base RNA oligonucleotide was 5' end-labeled spottings. Filters are incubated in 10 mL of 1x phosphate with ATP-2-Cy3 for the purpose of examining labeling via buffered saline (PBS) solution for 10 minutes at room tem signal intensity of the hybridized oligonucleotide to a DNA perature with agitation. The filter is then incubated in 1x microarray. One microgram of the RNA oligonucleotide was PBS+0.1% bovine serum albumin (BSA) as a block for 30 used in a standard 5' end-labeling reaction.(see Oligonucle minutes at room temperature with agitation. Filters then otide 5' end-labeling reaction: Method details) and then underwent 3 ten minute washes with 1x PBS for 10 minutes hybridized for 18 hours to the array. The array was composed at room temperature with agitation and were placed in a of oligos consisting of the perfect match (PM), single base Promega Streptavidin-alkaline phosphatase conjugate that mismatch (MM1), two mismatched bases (MM2), a single was diluted /sooo in 1x PBS for an overnight incubation at 4 base deletion (Del 1), two deletions (Del2), or background degrees Celsius with agitation. Three washes are conducted spot which contains no oligonucleotide (BGD). The signal and the filters then underwent incubation in PromegaWestern intensity profile and corresponding array cross-section illus Blue substrate (NBT/BCIP) at room temperature, covered trate that the Cy3-oligonucleotide is efficiently labeled and with foil. The appearance of dark spots was monitored, and specifically hybridizes to the array. This demonstrates proof occurred within 1-15 minutes. Color reactions were termi of principle for direct labeling of RNA and use of same in a nated by washing filters in sterile milliO water. Spots can be microarray experiment, as shown in FIG. 19, below. US 2010/031 7005 A1 Dec. 16, 2010 22

0182 FIG. 19 depicts 5' end-labeling an RNA oligonucle comprise a plurality of Y-phosphate labeled ATPs where the otide with ATP-L2-Cy3 and hybridization to a DNA microar fluorophore and linkers are different. ray. Intensity diagram of a DNA microarray cross-section using a Cy3-5' end-labeled-RNA oligonucleotide for hybrid Labeled-ATP Synthesis and Characterization ization. Intensity signal corresponding to individual microar 0187 Details of the labeled-ATP synthesis were described ray spot is shown for each. Microarray layout consists of above. Once a labeled-ATP is synthesized, it undergoes qual BGD (Background), Perfect Match (PM), Mismatch of 1 base ity control Screening. First, Thin Layer Chromatography (MM1), Mismatch of 2 bases (MM2), Deletion of 1 base (TLC) is run on the product to ensure product integrity by (Dell), and Deletion of 2 bases (Del2). comparing the intact labeled-ATP to phosphodiesterase Develop Standard Method to Directly Label mRNA for Use (PDE) treated labeled-ATP. PDE treatment produces a cleav in Microarray Studies age event between the bond of the alpha and beta phosphates of the ATP, and results in the decomposition of the intact 0183 The labeling techniques used in current microarray molecule. Product breakdown can be observed on TLC as technology to determine information about the levels of spots which migrate differently in comparison to the intact numerous transcripts in parallel are not reproducible, prima molecule. Second, fluorometric analysis is run to verify the rily due to labeling and hybridization biases. The present excitation and emission wavelengths of the intact molecule. labeling technique will allow microarry researchers to The inventors have observed a discernible quenching effect directly label RNA and to characterize the labeling efficiency between the ATP intermediate and the fluorescent dye that to determine if the labeled targets are adequate to detect their allows us to confirm the integrity of the labeled-ATP via a transcript of interest, prior to committing a costly microarray slight shift in its emission max wavelength and intensity to a suboptimal experiment (due to insufficient labeling of the provides us with an additional quality control. target). 0184 One preferred application for the labeling technique Example 14 of this invention is in direct labeling of RNA for use in 0188 This example illustrates the preparation of ATP-L2 microarray studies. The present technique transfers a labeled Cy5(TEA"). Although this example illustrates the prepara phosphate of any-phosphate labeled ATP to the 5'OH (the 5' tion of ATP-L2-Cy5(TEA), the preparation works equally end) of a nucleic acid such as DNA, RNA, RNA/DNA mixed well with L3 and L4. biomolecules, ribozymes, or other biomolecules having a 5'OH DNA or RNA terminus. To expose the 5'OH of a Preparation of ATP(TEA") mRNA, the mRNA is fragmented prior to labeling. For other biomolecules, fragmentation may or may not be necessary (0189 TEAB buffer (1M, 1L) was made from triethy depending on the type of DNA or RNA containing biomol lamine (139 mL) and dry ice (-200 g). Dowex resin (H) (100 ecule. Further, for labeling consistency and associated repro g) is treated with water (1L), ethanol (300 mL), water (2 L), ducibility, the fragmentation process preferably produces TEAB (1 M, 1L), water (5L), to prepare the DoweX resin fragments having an average length of about 200 bases, the (TEA") (-95 g). ATPNa (57 umol) is transformed to ATP. targeted size for cDNA used in microarray hybridization TEA by passing through Dowex resin (TEA) (2 g). The experiments. Fragmentation conditions that produce the collected fractions are tested with a UV lamp and combined desired consistency in RNA length can be determined by first solution is lyophilized. The pellet is dissolved in water and its exposing total RNA and subsequently mRNA in an alkaline concentration is measured on UV spectrometer (174 buffer having a pH 9 for increasing amounts of time, and mM*300 uL, 52 umol). examining the resulting size distribution of the fragmented (0190. Preparation of ATP-L2 (TEA") RNA via denaturing polyacryamide gel electrophoresis. The 0191 ATPTEA (20 umol) was rotavaped to dryness with fragmentation conditions is designed to produce similarly TEA (20 uL) and then rotavaped with methanol (100 uL) sized mRNA for use in the end-labeling reaction and mini three times. The pellet was dried on high vacuum overnight. mize error. Dry ice (3 kg)/acetone (500 mL) bath was used as the trap for 0185. The fragmented RNA are then end-labeled as the pump. DCC (75umol) is weighed out and dried on high described above. The end labeling efficiency is then deter vacuum overnight. L2 (200umol) is treated with TEA (20 uL) mined for high, medium and low abundance transcripts. To and methanol (100LL) and rotavaped to dryness. It was dried determine an efficiency index for unknown RNA fragments, on vacuum overnight. Pyridine (100 mL) was refluxed with end labeling actin as an abundant RNA marker, glyceralde CaH (8 g) and distilled onto 4A molecular sieves (5 g) under hydes-3-phosphate dehydrogenase(GAPDH) as a moder argon (50 mL distillate). Methanol (100 mL) was refluxed ately abundant RNA marker and hypoxanthine-guanine phos with Magnesium (5g) and distilled onto 4A molecular sieves phoribosyltransferase(HPRT) as a low abundance RNA under argon (50 mL distillate). DMF (100 mL) was refluxed marker. with CaH (8 g) and distilled (50 mL) onto 4A molecular sieves under argon (50 mL distillate). Labeled-ATP Syntheses 0.192 The following procedure was carried out under argon. Dried DCC was dissolved in dry DMF/MeOH (200 0186 The present invention also relates to a library of uL/20LL) and transferred to dry ATP TEA and stirred at room Y-phosphate labeled ATPs or other NTP. One preferred class temperature for 3-4 hours. Pyridine (17 uL) was added and of libraries comprise a plurality of Y-phosphate labeled ATPs the solution was stirred for 5 minutes before evaporated on where the linker is the same and the fluorophore is different. high vacuum (protected by dry ice-acetone trap). This takes Another preferred class of libraries comprise a plurality of 1-2 hours. L2 in DMF (200-300 uL) was then added to the Y-phosphate labeled ATPs where the fluorophore is the same pellet and the resulting Solution was stirred at room tempera and the linker is different. Another preferred class of libraries ture overnight. After water (1 mL) was added, the reaction US 2010/031 7005 A1 Dec. 16, 2010

mixture was lyophilized. Water (1.5 mL) was added to the ture for 3 hours and pH was maintained at ~5.7 over the time. pellet and the solution was centrifuged 14000 rpm3 minutes. The reaction was monitored by silica TLC. When completed, The Supernatant was then passed through a red 22 um Syringe the solution was adjusted to pH-7.5 and rotavaped. The pellet filter. The sample was purified on HPLC (SAX column) with was dissolved in water (1.5 mL) and the sample was purified TEAB/H2O as elution buffer system (-200 mL of 1M on HPLC (C18) with TEAA (100mM,200 mL)/CHCN (100 TEAB). In some cases two such purifications were needed for mL) as elution system. If needed, the sample was split into this amount of material. The collected product fraction was two halves for two purifications. The product fraction was then lyophilized. The pellet was dissolved in HEPES buffer (5 lyophilized and the pellet was dissolved in HEPES buffer (5 mM, pH 8.5) and its concentration was measured on UV spectrometer (200 uL*48 mM, 9.6 umol). Its purity was mM, pH 8.5). Concentration was determined on UV spec evaluated on silica TLC plate visualized by UV lamp and trometer (200 uL*24 mM, 4.8 umol). Yield was 37%. Its ninhydrin stain. Yield was 48%. purity was evaluated on silica TLC. (0193 ATP-L2-Cy5 (TEA") (0198 ATP-L1-Cy5(TEA") (0194 ATP-2 (0.8 umol. 17 uL) in NaHCO, buffer (1M, pH (0199 APT-L1 (1 umol) in NaHCO, buffer (1 M, pH9, 50 9, 40 uL) and Cy5-NHS (1 umol) in DMF (42 uL) were mixed uL) and Cy5-NHS (1.3 umol) in DMF (100 uL) were mixed and the resulting mixture is set on the shaker to react for 7 and the resulting mixture was set on the shaker and reacted for hours. Water (1.5 mL) was added and the mixture is lyo 6 hr. Water (1.5 mL) was added and the mixture is lyophilized. philized. A water solution (200 uL) of the pellet was passed A water solution (200 uL) of the pellet was passed through a through a 128 cm Sephadex G-25 column. The right frac 128 cm Sephadex G-25 column. The right fractions were tions are collected and the combined solution was lyophi collected and the combined solution was lyophilyzed. The lyzed. The pellet is dissolved in TEAA (100 mM, 1.5 mL) and pellet was dissolved in TEAA (100 mM, 1.2 mL) and purified purified on HPLC (C18) with TEAA (200 mL, 100 mM)/ on HPLC (C18) with TEAA (100 mM,200 mL)/CHCN (100 CHCN. The product fraction is collected and lyophilized. mL) as elution system. The product fraction was collected The pellet was dissolved in HEPES buffer (5 mM, pH8.5). Its and lyophilized. The pellet was dissolved in HEPES buffer (5 concentration was determined on UV spectrometer at 646 mm mM, pH8.5). Its concentration was determined on UV spec at (80 uL*2.8 mM, 0.22 umol). Yield was 28%. If necessary, a 646 nm (1.1 mM*470 uL). Yield is 52%. Enzymatic assay phosphate assay was carried out as an independent concen was done and analyzed on TLC. MS was determined if nec tration determination. Enzymatic assay was done and ana essary. lyzed on TLC (silica or PEI cellulose). Mass Spectrometry (0200. The above examples also work for GTP and other was determined if necessary. NTPs. Example 15 0201 All references cited herein are incorporated by ref erence. While this invention has been described fully and 0.195. This example illustrates the preparation of ATP-L1 completely, it should be understood that, within the scope of Cy5 (TEA"). This reaction does not work efficiently for the the appended claims, the invention may be practiced other linker L2, L3, and L4. Although ethylene diamine was used as wise than as specifically described. Although the invention the linker, the preparation is well suited for other alkenyl has been disclosed with reference to its preferred embodi diamines. ments, from reading this description those of skill in the art (0196 ATP-L1(TEA") (100 mL) as Elution System may appreciate changes and modification that may be made 0.197 ATPNa (12.7 umol) was reacted with L1 (110 which do not depart from the scope and spirit of the invention umol) in the presence of EDC (110 umol) at room tempera as described above and claimed hereafter.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 3

<21 Os SEQ ID NO 1 &211s LENGTH: 5 212s. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide

<4 OOs SEQUENCE: 1 Leu Tyr Lieu. Tyr Trp 1. 5

<21 Os SEQ ID NO 2 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: US 2010/031 7005 A1 Dec. 16, 2010 24

- Continued <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide

<4 OOs, SEQUENCE: 2 ggtact aagc ggcc.gcatg 19

<210s, SEQ ID NO 3 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic oligonucleotide

<4 OOs, SEQUENCE: 3 ggtact aagc ggcc.gcatg 19

1-43. (canceled) resonance (NMR) spectrometry instrument, a UV and visible 44. The method of claim 80, wherein the nucleotides are light spectrometry instrument, a far IR, IR or near IR spec the same or different. trometry instrument, and an X-ray spectrometromety instru 45. The method of claim 44, wherein the gamma labeled ment. nucleotide or nucleotide analog comprises a nucleotides-Se 56-74. (canceled) lected from the group consisting of adenine triphosphate 75. The method of claim 80, wherein the phosphatase (ATP), cytosine triphosphate (CTP), guanine triphosphate comprises an alkaline phosphatase. (GTP), inosine triphosphate (ITP), thymine triphosphate 76. The method of claim 75, wherein the intestinal phos (TTP), uridine triphosphate (UTP), pseudouridine triphos phatase is selected from the group consisting calf intestinal phate (YTP), xanthine triphosphate (XTP), Orotidine triph alkaline phosphatase, shrimp alkaline phosphatase and mix osphate (OTP), 5-bromouridine triphosphate (BTP), thiouri tures or combinations thereof. dine triphosphate (STP), 5,6-dihydrouridine triphosphate 77. The method of claim 80, wherein the solution is con (DTP), dATP, dGTP, dTTP, dUTP, dCTP and analogs thereof. tained in a container. 46-48. (canceled) 78. The method of claim 55, wherein the solution is view 49. The method of claim 80, wherein the gamma label of able within a viewing field of a camera, the detectable prop the gamma labeled nucleotide or nucleotide analog comprises erty is fluorescence and the fluorescence is detectable in a a linker and a fluorescent dye. single pixel or pixel-bin of the viewing field of the camera. 50. The method of claim 49, wherein each linker has the 79. The method of claim 80, wherein the-label of the same or different properties, where the properties are selected gamma labeled nucleotide or nucleotide analog comprises a from the group consisting of chain length, bulk or size, rigid fluorescent dye. ity, polarity and combinations thereof. 80. A method comprising: 51. The method of claim 50, wherein the linker has the purifying one or more gamma labeled nucleotides or nucle general formula -E-R-E-, where E is a main group element otide analogs from a solution comprising both unlabeled selected from the group consisting of C, N, O, P S and and gamma labeled nucleotides or nucleotide analogs by combinations thereof and R is a carbon-containing group contacting the Solution with a phosphatase to produce having between 1 and about 20 main group atoms selected one or more purified gamma labeled nucleotides or from the group consisting of B, C, N, O, P S and combina nucleotide analogs; tions thereof, with the valency being completed by H. using the one or more purified gamma labeled nucleotides 52. The method of claim 80, wherein the detecting com or nucleotide analogs in a polymerase reaction; prises detecting a detectable property of the gamma label of detecting each of one or more incorporations of a gamma the gamma labeled nucleotide or nucleotide analog. labeled nucleotide or nucleotide analog into a nascent 53. The method of claim 80, wherein the detectable prop nucleic acid strand by the polymerase; and erty is selected from the group consisting of light emission, determining the base identity of the at least one incorpo emission frequency, emission duration, emission intensity, rated nucleotide or nucleotide analog. quenching, electron spin, radio-activity, nuclear spin, color, 81. The method of claim 80, wherein one or more gamma absorbance, near IR absorbance, UV absorbance, and far UV labeled nucleotides or nucleotide analogs comprise a deoxy absorbance. nucleotide or analog thereof. 54. The method of claim 52, wherein the analytical tech 82. The method of claim 80, wherein one or more gamma nique comprises an analytical chemical or physical instru labeled nucleotides or nucleotide analogs comprise a fluores ment for detecting and/or monitoring the property. cent or fluorogenic label attached to or associated with the 55. The method of claim 54, wherein the instrument is Y-(gamma) phosphate of the nucleotide or nucleotide analog. selected from the group consisting of a camera, an electron spin resonance spectrometry instrument, a nuclear magnetic c c c c c