US20100317005A1 - Modified Nucleotides and Methods for Making and Use Same - Google Patents

Modified Nucleotides and Methods for Making and Use Same Download PDF

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US20100317005A1
US20100317005A1 US12/724,392 US72439210A US2010317005A1 US 20100317005 A1 US20100317005 A1 US 20100317005A1 US 72439210 A US72439210 A US 72439210A US 2010317005 A1 US2010317005 A1 US 2010317005A1
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atp
phosphate
adp
nucleotide
labeled
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Susan H. Hardin
Hongyi Wang
Brent A. Mulder
Nathan K. Agnew
Tommie L. Lincecum, JR.
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Visigen Biotechnologies Inc
Life Technologies Corp
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Visigen Biotechnologies Inc
Life Technologies Corp
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    • C—CHEMISTRY; METALLURGY
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing

Definitions

  • the present invention relates to labeled nucleotides (NTPs), to method for making labeled NTPs and to method for using labeled NTPs such as a method of transferring the label to a target molecule.
  • NTPs labeled nucleotides
  • the present invention relates to gamma ( ⁇ ) phosphate labeled nucleotides (NTPs), where the label includes a detectable tag and an optional linker interposed between the tag and the ⁇ phosphate, to method for making ⁇ phosphate labeled NTPs and to method for transferring the ⁇ phosphate and the label to a target molecule including an oligonucleotide, a polynucleotide, a polypeptide, a protein, a monosaccharide, a polysaccharide, or mixtures or combinations thereof.
  • NTPs gamma ( ⁇ ) phosphate labeled nucleotides
  • nucleotide and polypeptide sequences with both radio and non-radio labels. These methods are routinely used to aid our understanding of molecular, cellular and intercellular dynamics and interaction.
  • nucleotide sequence DNA, RNA, DNA-RNA, etc.
  • peptide sequences polypeptides, proteins, enzymes, macro-assembly, etc.
  • ribozymes peptide-nucleotide mixed sequences, etc.
  • Fluorescence-based technologies are rapidly emerging as the methods of choice for nucleic acid labeling and detection.
  • a variety of molecular biology techniques have benefitted from fluorescent innovations.
  • DNA Dye-Terminator Sequencing has been revolutionized by the creation of individually identifiable color-coded bases thereby making system automation possible.
  • Assays such as Fluorescent In Situ Hybridization (FISH) and Quantitative/Real-time PCR (qPCR) also capitalized on the ability to monitor multiple fluorophores simultaneously. Furthermore, these new assays also make extensive use of the unique data provided by fluorophore-fluorophore interactions.
  • radio-isotopic labeling entails a variety of negative aspects which include: (1) health hazards related to exposure, (2) extra licensing requirements and permits, (3) contained storage requirements, (4) additional requirements for waste removal, and (5) the limited lifetime of a radioactive probe. Fluorescent technologies eliminate both health risks and regulatory paperwork and provide a greater degree of flexibility in experimental design.
  • nucleic acid labeling method In addition to fluorescence, another important nucleic acid labeling method is the attachment of biotin to DNA or RNA. A variety of nucleic acid and protein capture applications are developed that exploit the highly specific interaction between biotin and streptavidin.
  • magnetic column separations where biotinylated oligonucleotides are captured by streptavidin coated microbeads enable a straightforward way to separate biotinylated from non-biotinylated molecules as described herein.
  • This system permits the capture of DNA molecules, RNA molecules, DNA and RNA-binding proteins, and sequence specific transcripts.
  • tag or “label” means an atom or molecule that has a detectable property and is capable of being attached to a ⁇ phosphate of a nucleotide triphosphate.
  • detectable property means a physical or chemical property of a tag that is capable of independent detection and/or monitoring by an analytical technique after being attached to a target bio-molecule, i.e., the property is capable of being detected in the presence of the system under analysis.
  • the property can be light emission after excitation, quenching of a known emission sites, electron spin, radio activity (electron emission, positron emission, alpha particle emission, etc.), nuclear spin, color, absorbance, near JR absorbance, UV absorbance, far UV absorbance, etc.
  • analytical technique means an analytical chemical or physical instrument for detecting and/or monitoring the property. Such instruments are based on spectroscopic analytical methods such as electron spin resonance spectrometry, nuclear magnetic resonance (NMR) spectrometry, UV and visible light spectrometry, far IR, IR and near IR spectrometry, X-ray spectrometry, etc.
  • spectroscopic analytical methods such as electron spin resonance spectrometry, nuclear magnetic resonance (NMR) spectrometry, UV and visible light spectrometry, far IR, IR and near IR spectrometry, X-ray spectrometry, etc.
  • base means any natural or synthetic purine or pyrimidine or nucleotide analogs (e.g., 7-deaza-deoxyguanine) that is capable of forming nucleotides and sequences thereof, including, without limitation, adenine (A), cytosine (C), guanine (G), inosine (I), thymine (T), uracil (U), pseudouridine (Y), xanthine (X), Orotidine (O), 5-bromouridine (B), thiouridine (S), 5,6-dihydrouridine (D) or the like.
  • the natural or synthetic purines or pyrimidines may includes tags or may have been modified to have a particular detectable property such as enrichment with an NMR active nuclei.
  • bonded to means that chemical and/or physical interactions sufficient to maintain the label or tag at a given site of a target molecule.
  • the chemical and/or physical interactions include, without limitation, covalent bonding (preferred), ionic bonding, hydrogen bonding, apolar bonding, attractive electrostatic interactions, dipole interactions, or any other electrical or quantum mechanical interaction sufficient in toto to maintain the polymerizing agent in a desired region of the substrate.
  • nucleoside means a base bonded to a sugar such as a five carbon sugar e.g., ribose.
  • nucleotide means nucleoside bonded to at least one phosphate.
  • NMP means a nucleotide monophosphate
  • NDP means a nucleotide diphosphate
  • NTP means a nucleotide triphosphate
  • AMP means adenosine monophosphate
  • ADP means adenosine diphosphate
  • ATP means adenosine triphosphate
  • TMP means thymidine nucleotide monophosphate
  • TDP means thymidine nucleotide diphosphate
  • TTP means thymidine nucleotide triphosphate
  • CMP means cytidine nucleotide monophosphate
  • CDP means cytidine nucleotide diphosphate.
  • CTP means cytidine nucleotide triphosphate
  • GMP means guanine nucleotide monophosphate
  • GDP means guanine nucleotide diphosphate
  • GTP means guanine nucleotide triphosphate
  • PNA means a peptide nucleic acid
  • T-NTP means an NTP having an atomic and/or molecular tag bonded to the gamma phosphate of the NTP.
  • T-P means a phosphate (P) bonded to a tag (T).
  • L-NTP means an NTP having a linking reagent or linker bonded at its first end to the gamma phosphate of the NTP.
  • T-L-NTP means an L-NTP having a tag or label bonded to a second end of the linker.
  • T-L-P means a phosphate (P) bonded to the first end of the linker (L) and a tag (T) bonded to the second end of the linker (L).
  • ON means an oligonucleotide—a short sequence of nucleotides generally less than about 100 nucleotides, preferably less than about 50 nucleotides.
  • PN means a polynucleotide or nucleic acid—a long sequence of nucleotides generally over about 100 nucleotides.
  • PP means a polypeptide sequence, including at least two amino acids joined together via a peptide bond such as proteins, enzymes, protein assemblages, or the like.
  • PRN means a protein, which includes enzymes and protein assemblages.
  • AA means an amino acid either natural or synthetic and capable of forming peptide bonds.
  • T-ON means an oligonucleotide having an T-P bonded to the ON at either its 5′ or 3′ end.
  • T-L-ON means an oligonucleotide having an T-L-P bonded to the ON at either its 5′ or 3′ end.
  • T-PN means a polynucleotide having an T-P bonded to the PN at either its 5′ or 3′ end.
  • T-L-PN means a polynucleotide having an T-L-P bonded to the PN at either its 5′ or 3′ end.
  • T-P-PP means a polypeptide having an T-P bonded thereto.
  • T-L-P-PP means a polypeptide having an T-L-P bonded thereto.
  • T-P-PPN means a polypeptide having an T-P bonded thereto.
  • T-L-P-PPN means a polypeptide having an T-L-P bonded thereto.
  • PNPP means a biomolecule including both nucleosides, nucleotides, oligonucleotide or a polynucleotide and an amino acid, polypeptide or protein.
  • T-P-PNPP means an PNPP having an T-P bonded thereto.
  • T-L-P-PNPP means an PNPP having an T-L-P bonded thereto.
  • MES buffer means morpholinoethanesulfonic acid buffer.
  • TLC means thin layer chromatography
  • the present invention provides labeled nucleotides (T-NTPs or T-L-NTPs), where the label (T-P or T-L-P) is readily transferable to nucleotide sequences, amino acid sequences, saccharides or compositions comprising a nucleotide, an amino acid and/or a sugar.
  • the present invention also provides a method for making labeled nucleotides, where the label includes an atomic and/or molecular tag bonded to the gamma phosphate of the nucleotide (T-NTP5) comprising the steps of contacting an NTP with an atomic and/or molecular tag precursor to form the T-NTP.
  • T-NTP5 gamma phosphate of the nucleotide
  • the present invention also provides a method for making labeled nucleotides, where the label includes an atomic and/or molecular tag bonded to one end of a linker which is bonded to the gamma phosphate of the nucleotide (T-L-NTPs) comprising the steps of contacting an NTP with a linker precursor to form a linker modified NTP (L-NTP). The L-NTP is then contacted with an atomic and/or molecular tag to form the T-L-NTP.
  • the present invention provides a method for labeling oligonucleotides including the step of contacting a T-NTP or T-L-NTP with an oligonucleotide (ON) in the presence of a catalyst to form a tagged oligonucleotide (T-ON or T-L-ON).
  • the present invention also provides a method for labeling polypeptide (PP) including the step of contacting a T-NTP or T-L-NTP with the PP in the presence of a catalyst to form a tagged polypeptide (T-P-PP or T-L-P-PP).
  • PP polypeptide
  • the present invention provides a method for labeling proteins (PRN) including the step of contacting a T-NTP or T-L-NTP with the PRN in the presence of a catalyst to form a tagged protein (T-P-PRN or T-L-P-PRN).
  • PRN labeling proteins
  • the present invention provides a method for labeling a biomolecule including both nucleosides, nucleotides, oligonucleotide or a polynucleotide and an amino acid, polypeptide or protein (PNPP) including the step of contacting a T-NTP or T-L-NTP with the PNPP in the presence of a catalyst to form a tagged biomolecule (T-P-PNPP or T-L-P-PNPP).
  • PNPP amino acid, polypeptide or protein
  • the present invention provides a set of ATP-linker-fluorescent dye molecules and ATP-linker-biotin molecules.
  • the ATP molecules differ in the linker interposed between the ATP ⁇ -phosphate and the fluorescent dye or biotin.
  • the linkers differ in such properties as chain length, bulk or size, rigidity, and/or polarity.
  • the synthesis of the precursor ATP-linker molecules provides intermediates that are used for attachment of a variety of individual fluorescent dyes, biotin or other binding molecules. Specific commercially available dyes were investigated to determine the efficiency of the transfer reaction in applications that have commercial potential.
  • Such commercially available dyes includes; (1) Fluorescein [excitation ⁇ : 495 nm, emission ⁇ : 520 nm], (2) Cy3 [excitation ⁇ : 550 nm, emission ⁇ : 570 nm], (3) TAMRA [excitation ⁇ : 555 nm, emission ⁇ : 580 nm], (4) ROX [excitation ⁇ : 578 nm, emission ⁇ : 604 nm], and (5) Cy5 [excitation ⁇ : 649 nm, emission ⁇ : 670 nm].
  • the processes of this invention are capable of using a wide variety of ⁇ -phosphate-labeled nucleotides such as ATP.
  • the present invention also provides for the use of T4 PNK or other phosphatase or kinase enzymes to 5′ end-label oligonucleotides with a variety of fluorophore, biotin or other binding molecules using labeled ATP molecules.
  • the present invention also provides a screening assay to examine each of the ATP-Linker-Fluorophore and ATP-Linker-Biotin molecules in 5′ end-labeling reactions with T4 PNK.
  • the assay is based on the protocol for radio-isotopic 5′ end-labeling of oligonucleotides by T4 PNK. Fluorescent reactions are separated by PAGE and analysis is performed with a fluorescent gel scanning imager and software.
  • Biotin reactions are examined via covalent attachment of biotinylated oligonucleotide to DE81 filter paper, incubation in streptavidin-alkaline phosphatase conjugate, and color development in nitroblue tetrazolium chloride (NBT)/5-bromo-4-chloro-3-indolyl-phosphate (BCIP) solution.
  • Parameters that affect labeling efficiency include: (1) the incubation time, (2) labeling bias due to 5′ base sequence of the oligonucleotide, (3) concentration (ATP-Linker-Moiety) substrate, (4) T4 PNK amount and (5) oligonucleotide length.
  • a labeled-ATP Once a labeled-ATP is synthesized, it will then undergo quality control measures which entail: (1) TLC analysis to determine labeled-ATP synthesis integrity, (2) Spectrophotometric analysis to calculate labeling efficiency and, (3) Fluorometric analysis to verify the quantum yields of the ATP-labeled molecule and the fluorescently-labeled oligonucleotide.
  • the labeling efficiency are calculated by measuring the base:dye ratio on a NanoDrop spectrophotometer. This method uses Molecular Probes to calculate the labeling efficiency of their ULYSIS Nucleic Acid Labeling Kit (MP21650).
  • the NanoDrop spectrophotometer is used to measure the absorbance of the nucleic acid-dye conjugate at 260 nm ( ⁇ 260 ) and at a ⁇ max for the dye ( ⁇ dye ).
  • a measurement is also taken using the buffer alone at 260 nm and ⁇ max and these numbers are subtracted from the raw sample absorbances values.
  • CF 260 correction factor is introduced to correct for the dye contribution at the 260 nm reading.
  • the correction factor is given by:
  • ⁇ is an extinction coefficient of the dye and is unique for each dye.
  • the present invention provides a method for screening kinases including the step of providing one immobilized substrate or a plurality of immobilized substrates on a support.
  • the immobilized substrate(s) is then contacted with a solution including a kinase and a ⁇ -phosphate labeled NTP for a first time and at a first temperature sufficient to determine whether a transfer of the labeled phosphate from the ⁇ -phosphate labeled NTP to the substrate mediated by the kinase.
  • the support is washed for a second time and at a second temperature sufficient to remove the kinase and unreacted labeled NTP.
  • the support is analyzed to determine whether the substrate have been labeled with the labeled phosphate from the ⁇ -phosphate labeled NTP.
  • the present invention provides a method for screening kinase substrate candidates including the step of providing an immobilized kinase on a support.
  • the support is then contacted with a solution including one or more kinase substrate candidates and a labeled NTP for the kinase.
  • the substrate candidates are then analyzed for the presence or absence of the label.
  • the present invention provides a method including the step of contacting a solution comprising non-modified nucleotides or deoxynucleotides and modified nucleotides or deoxynucleotides, where the enzyme selectively degrades the non-modified nucleotides or deoxynucleotides.
  • the present invention provides a method for monitoring an NTP dependent reaction including the step of supplying to a system in which an NTP dependent reaction occurs, a ⁇ -phosphate labeled nucleotide (NTP) and monitoring the label during the reaction.
  • NTP ⁇ -phosphate labeled nucleotide
  • FIG. 1A depicts a general scheme for preparing gamma phosphate tagged NTPs
  • FIG. 1B depicts a general scheme for preparing gamma phosphate tagged NTPs with a linker interposed between the gamma phosphate and the tag;
  • FIG. 1C a general scheme for preparing a gamma phosphate tagged ATP with a linker interposed between the gamma phosphate and the tag;
  • FIG. 2A a general scheme for preparing 3′ and/or 5′ tagged oligonucleotides using the tagged NTPs of this invention
  • FIG. 2B another general scheme for preparing 3′ and/or 5′ tagged oligonucleotides using the tagged NTPs of this invention or for exchanging an untagged phosphate group for a tagged phosphate group;
  • FIG. 3 a general scheme for preparing phosphorylated polypeptide or proteins using the tagged NTPs of this invention
  • FIG. 4 depicts an HPLC chromatogram of the reaction product ATP-EDA-ROX at 576 nm
  • FIG. 5 depicts an HPLC chromatogram of the reaction product ATP-EDA-ROX at 259 nm
  • FIG. 6 depicts a UV spectrum of ATP-EDA-ROX
  • FIG. 7A depict TLC monitoring of reactions of ATP and ATP-EDA-Rox with CLAP
  • FIG. 8 depicts PAGE monitoring of T4 PNK 5′ end labeling of a TOP oligonucleotide using ATP-EDA-ROX;
  • FIG. 9 depicts gel plates of T4 PNK 5′ end labeling of a TOP oligonucleotide using ATP-EDA-ROX;
  • FIG. 10 depicts a plot of CNT vs. pmol for ROX-T-Top
  • FIG. 11 depicts a plot of CNT vs. ng Top oligonucleotide and different T4 PNK concentrations
  • FIGS. 12A&B depict plots of T4 PNK 5′ end-labeling timecourse using ATP-L1-ROX
  • FIGS. 13A&B depict plots of T4 PNK concentration effects on 5′ end-labeling using ATP-L1-ROX
  • FIG. 14 depicts plots of T4 PNK 5′ end-labeling t using ATP-L1-ROX in the presence of PEG 8000;
  • FIGS. 15A&B depict plots of linker effects on T4 PNK 5′ end-labeling using ROX labeled ATPs
  • FIGS. 16A&B depict extension reactions of a ROX labeled oligonucleotide
  • FIGS. 17A&B depict the relatively activity of an exonuclease against a 5′ end-labeled oligonucleotide and an un-labeled oligonucleotide
  • FIG. 18 depicts three developed filter paper disks showing T4 PNK 5′ end-labeling using ATP-L1-biotin compared to a negative control and a synthetic biotin labeled oligonucleotide;
  • FIG. 19 depicts 5′ end-labeling an RNA oligonucleotide with ATP-L2-Cy3 and hybridization to a DNA microarray.
  • the inventors have found a versatile, inexpensive and efficient technique for labeling nucleic acids, polypeptides and/or biomolecules including both nucleic acids and amino acids with atomic and/or molecular tags having a detectable property and reagents for accomplishing the tagging reaction.
  • the techniques involves the preparation of labeled NTPs capable of transferring their label to a target nucleotide, polypeptide, saccharide, and/or a biomolecule including one or combinations of a nucleoside, nucleotide, oligonucleotide or a polynucleotide and an amino acid, polypeptide or protein, combinations of a nucleoside, nucleotide, oligonucleotide or a polynucleotide and a monosaccharide or polysaccharide and combinations of an amino acid, polypeptide or protein and a monosaccharide or polysaccharide.
  • T4 Polynucleotide Kinase T4 PNK
  • T4 PNK T4 Polynucleotide Kinase
  • the present invention relates to the modification of the gamma-phosphate of a nucleotide, preferably ATP and GTP, to form gamma-phosphate labeled nucleotides, which can subsequently be used to transfer the labeled gamma phosphate moiety to a target substrate such as DNA, RNA, RNA/DNA, protein, polypeptide, sugars, polysaccharides or biomolecules including DNA, RNA, polypeptides, sugars or polysaccharides.
  • a target substrate such as DNA, RNA, RNA/DNA, protein, polypeptide, sugars, polysaccharides or biomolecules including DNA, RNA, polypeptides, sugars or polysaccharides.
  • the label can include an atomic and/or molecular tag having a separately detectable property such as a fluorescent tag, a biotinylated tag, electrochemical tag, lanthanide or actinide series containing tag, radical tag or paramagnetic tag, nmr tag ( 13 C, 15 N, or other isotopically enriched atom or molecular tags) or any other tag capable of detection.
  • the label can also include a linker interposed molecularly between the gamma-phosphate and the tag, which may influence the efficiency of the transfer reaction, and this efficiency may be specific for the transfer reaction under study. Additionally, the transfer reaction may be influenced by the identity of the tag.
  • the present invention broadly relates to a composition for efficiently labeling oligonucleotide, polynucleotides, polypeptide, proteins and/or biomolecules including both a nucleoside, nucleotide, oligonucleotide, and/or polynucleotide and a polypeptide and/or protein, where the composition includes an NTP, a linker and a tag, where the linker is bonded at one end to a gamma phosphate of the NTP and at the other end to the tag.
  • the present invention broadly relates to a method for efficiently labeling oligonucleotide, polynucleotides, polypeptide, proteins and/or biomolecules including both a nucleoside, nucleotide, oligonucleotide, and/or polynucleotide and a polypeptide and/or protein, where the method includes the step of contacting oligonucleotide, polynucleotides, polypeptide, proteins and/or biomolecules including both a nucleoside, nucleotide, oligonucleotide, and/or polynucleotide and a polypeptide and/or protein with a labeled NTP (T-L-NTP) to form a labeled oligonucleotide, polynucleotides, polypeptide, proteins and/or biomolecules including both a nucleoside, nucleotide, oligonucleotide, and/or polynucleotide and
  • T4 polynucleotide kinase has been essential for phosphorylating, either radioactively or non-radioactively, the 5′-end of oligonucleotides for subsequent use in a variety of molecular biology applications.
  • T4 PNK has two distinct functions: (1) transfer of the ⁇ -phosphate of adenosine triphosphate (ATP) or other nucleoside triphosphates to the 5′ hydroxyl end of a polynucleotide and (2) 3′-phosphatase activity that is independent of ATP and able to hydrolyze 2′,3′-cyclic phosphodiesters.
  • ATP adenosine triphosphate
  • 3′-phosphatase activity that is independent of ATP and able to hydrolyze 2′,3′-cyclic phosphodiesters.
  • T4 bacteriophage life cycle Upon infection by T4, some strains of Escherichia coli have the capability to initiate a suicide defense mechanism that causes the specific cleavage of bacterial lysine tRNA (tRNA lys ) and results in the abrogation of protein synthesis.
  • the phage initiates tRNA lys repair via the bacteriophage encoded T4 PNK and T4 RNA ligase.
  • T4 PNK specifically phosphorylates the 5′-hydroxyl group and simultaneously reprocesses the 3′ end by opening the 2′,3′-cyclic phosphate and then removing the 3′-phosphate. This results in a suitable substrate for T4 RNA ligase which can then repair the tRNA lys lesion and allow continued phage propagation.
  • an oligonucleotide is typically labeled during its chemical synthesis and the label may be directed to the 3′ end, 5′ end, or at an internal position. Additionally, a label may be added post-synthesis by polymerase incorporation of a base-labeled dNTP onto the 3′ end of a duplexed molecule or via terminal deoxy transfer activity (TdT).
  • TdT terminal deoxy transfer activity
  • chemical attachment of a dye during the process of oligonucleotide synthesis or via amino-chemistry after synthesis are the only methods used to add a fluorescent moiety to the oligonuleotides 5′-end. The necessity of chemically labeling the 5′-end can be explained by the inherent directionality of DNA synthesis by a polymerase.
  • a polymerase To initiate DNA synthesis, a polymerase must be able to access the 3′-end of the DNA at the primer-template junction. Fluorescent dyes that have been attached at the 3′-nucleotide's base, sugar, or alpha phosphate can severely alter the conformation of the DNA, subsequently inhibiting or dramatically reducing DNA synthesis efficiency. Explanations for the affect that fluorophore presence has on DNA structural perturbations involve the notable size of the fluorescent dye and its hydrophobic nature.
  • a standard 25 base oligonucleotide that is fluorescently 5′-end-labeled using current methods can range in price from $98.75 to upwards of $600 at the 250 nmole scale, depending on the choice of fluorophore, its place of attachment, and the level of purity desired.
  • the time needed for the labeled oligonucleotide order to arrive can range between 3 to 10 days, depending on the complexity of the synthesis. Additionally, inefficiencies in the coupling of the fluorophore to the oligonucleotide may result in a large fraction of the product being unlabeled, and require its re-synthesis resulting in further delay.
  • the Labeled ATPs of this invention are ideally suited for used in the following assays and detection procedures: absorbance assays, fluorescence intensity assays, fluorescence polarization assays, time resolved fluorescence assays, fluorescence resonance energy transfer (FRET) assays, or other assays.
  • absorbance assays fluorescence intensity assays, fluorescence polarization assays, time resolved fluorescence assays, fluorescence resonance energy transfer (FRET) assays, or other assays.
  • FRET fluorescence resonance energy transfer
  • the present invention also relates to a kit adapted to 5′ end-label an target oligonucleotide with a fluorophore or biotin or other binding molecules.
  • the reagents of this invention can be used with either previously synthesized or newly synthesized oligonucleotides in labeling reactions and tailor experiments on the fly by selecting an appropriate fluorescently labeled ATP or biotin labeled ATP.
  • Suitable atomic tags for use in this invention include, without limitation, any atomic element amenable to attachment to a specific site in a target or dNTP, especially Europium shift agents, NMR active atoms or the like.
  • Suitable molecular tags for use in this invention include, without limitation, any molecule amenable to attachment to a specific site of a target PN or PP, such as fluorescent molecules, quenching molecules, Europium shift agents, NMR active molecules, Raman active molecules, near IR active molecules, or the like.
  • Suitable NMR tags include any active NMR nuclei.
  • Exemplary examples of NMR active nuclei include, without limitation, 1 H, 13 C, 15 N, 19 F, 29 Si, 57 Fe, 103 Rh, etc.
  • Suitable molecular tags for use in this invention include, without limitation, any molecule amenable to attachment to a specific site in a target or dNTP, especially fluorescent dyes or molecules that quench the fluorescence of the fluorescent dyes, paramagnetic molecules such as those disclosed in U.S. Pat. Nos: 6,458,758; 6,436,640; 6,410,255; 6,316,198; 6,303,315; 5,840,701; 5,833,601; 5,824,781; 5,817,632; 5,807,831; 5,804,561; 5,741,893; 5,725,8395; 706,805; and 5,494,030, incorporated herein by reference, electrochemical probes or tags, or other similar molecular tags or probes.
  • Fluorescent dyes include, without limitation, such as d-Rhodamine acceptor dyes including Cy5, dichloro[R110], dichloro[R6G], dichloro[TAMRA], dichloro[ROX] or the like, fluorescein donor dye including fluorescein, 6-FAM, or the like; Acridine including Acridine orange, Acridine yellow, Proflavin, pH 7, or the like; Aromatic Hydrocarbon including 2-Methylbenzoxazole, Ethyl p-dimethylaminobenzoate, Phenol, Pyrrole, benzene, toluene, or the like; Arylmethine Dyes including Auramine O, Crystal violet, H 2 O, Crystal violet, glycerol, Malachite Green or the like; Coumarin dyes including 7-Methoxycoumarin-4-acetic acid, Coumarin 1, Coumarin 30, Coumarin 314, Coumarin 343, Coumarin 6 or the like; Cyanine Dye including 1,1′-diethyl
  • Suitable phosphorylation catalysts or enzymes are any naturally occurring, human modified or synthetic molecule or molecular assembly that can phosphorylate 5′ and/or 3′ hydroxy terminated oligonucleotides or polynucleotides, phosphorylate peptide or proteins or that can exchange an existing phosphate with a labeled phosphate of this invention.
  • Exemplary examples include kinases.
  • Preferred kinases include, without limitation, T4 Polynucleotide Kinase, Abl (mouse), Abl, Abl (T315I), ALK, AMPK (rat), Arg (mouse), Aurora-A, Axl, Blk (mouse), Bmx, BTK, CaMKII (rat), CaMKIV, CDK1/cyclinB, CDK2/cyclinA, CDK2/cyclinE, CDK3/cyclinE, CDK5/p35, CDK6/cyclinD3, CDK7/cyclinH/MAT1, CHK1, CHK2, CK1 (yeast), CK1 ⁇ , CK2, c-RAF, CSK, cSRC, EGFR, EphB2, EphB4, Fes, FGFR3, Flt3, Fms, Fyn, GSK3 ⁇ , GSK3 ⁇ , IGF-1R, IKK ⁇ , IKK ⁇ , IR, JNK1 ⁇ 1, JNK2 ⁇ 2, JNK3, Lck, Ly
  • CA: ATP+L-methionine+H(2)O phosphate+diphosphate+S-adenosyl-L-methionine); ID 2.5.1.17 Cob(I)alamin adenosyltransferase.
  • CA: ATP+cob(I)alamin+H(2)O phosphate+diphosphate+adenosylcobalamin); ID 2.6.99.1 dATP(dGTP)—DNA purine transferase.
  • CA: dATP+depurinated DNA ribose triphosphate+DNA
  • CA: ATP+D-hexose ADP+D-hexose 6-phosphate
  • ID 2.7.1.3 Ketohexokinase.
  • CA: ATP+D-mannose ADP+D-mannose 6-phosphate); ID 2.7.1.8 Glucosamine kinase.
  • CA: ATP+glucosamine ADP+glucosamine phosphate); ID 2.7.1.10 Phosphoglucokinase.
  • CA: ATP+D-fructose 1-phosphate ADP+D-fructose 1,6-bisphosphate); ID 2.7.1.11 6-phosphofructokinase.
  • CA: ATP+D-fructose 6-phosphate ADP+D-fructose 1,6-bisphosphate); ID 2.7.1.12 Gluconokinase.
  • CA: ATP+L-ribulose ADP+L-ribulose 5-phosphate); ID 2.7.1.17 Xylulokinase.
  • CA: ATP+D-xylulose ADP+D-xylulose 5-phosphate); ID 2.7.1.18 Phosphoribokinase.
  • CA: ATP+D-ribose 5-phosphate ADP+D-ribose 1,5-bisphosphate; ID 2.7.1.19 Phosphoribulokinase.
  • CA: ATP+D-ribulose 5-phosphate ADP+D-ribulose 1,5-bisphosphate
  • ID 2.7.1.20 Adenosine kinase.
  • CA: ATP+4-methyl-5-(2-hydroxyethyl)-thiazole ADP+4-methyl-5-(2-phosphoethyl)-thiazole); ID 2.7.1.51 L-fuculokinase.
  • CA: ATP+L-fuculose ADP+L-fuculose 1-phosphate); ID 2.7.1.52 Fucokinase.
  • CA: ATP+6-deoxy-L-galactose ADP+6-deoxy-L-galactose 1-phosphate); ID 2.7.1.53 L-xylulokinase.
  • CA: ATP+L-xylulose ADP+L-xylulose 5-phosphate); ID 2.7.1.54 D-arabinokinase.
  • CA: ATP+D-arabinose ADP+D-arabinose 5-phosphate); ID 2.7.1.55 Allose kinase.
  • CA: ATP+D-allose ADP+D-allose 6-phosphate); ID 2.7.1.56 1-phosphofructokinase.
  • CA: ATP+D-fructose 1-phosphate ADP+D-fructose 1,6-bisphosphate); ID 2.7.1.58 2-dehydro-3-deoxygalactonokinase.
  • CA: ATP+2-dehydro-3-deoxy-D-galactonate ADP+2-dehydro-3-deoxy-D-galactonate 6-phosphate); ID 2.7.1.59 N-acetylglucosamine kinase.
  • CA: ATP+N-acetyl-D-glucosamine ADP+N-acetyl-D-glucosamine 6-phosphate); ID 2.7.1.60 N-acylmannosamine kinase.
  • CA: ATP+N-acyl-D-mannosamine ADP+N-acyl-D-mannosamine 6-phosphate); ID 2.7.1.64 Inositol 3-kinase.
  • CA: ATP+myo-inositol ADP+1D-myo-inositol 3-phosphate); ID 2.7.1.65 Scyllo-inosamine kinase.
  • CA: ATP+1-amino-1-deoxy-scyllo-inositol ADP+1-amino-1-deoxy-scyllo-inositol 4-phosphate); ID 2.7.1.66 Undecaprenol kinase.
  • CA: ATP+undecaprenol ADP+undecaprenyl phosphate); ID 2.7.1.67 1-phosphatidylinositol 4-kinase.
  • CA: ATP+1-phosphatidyl-1D-myo-inositol ADP+1-phosphatidyl-1D-myo-inositol 4-phosphate); ID 2.7.1.68 1-phosphatidylinositol-4-phosphate 5-kinase.
  • CA: ATP+1-phosphatidyl-1D-myo-inositol 4-phosphate ADP+1-phosphatidyl-1D-myo-inositol 4,5-bisphosphate
  • ID 2.7.1.70 Protamine kinase.
  • CA: ATP+[protamine] ADP+[protamine]O-phospho-L-serine); ID 2.7.1.71 Shikimate kinase.
  • CA: ATP+ethanolamine ADP+O-phosphoethanolamine); ID 2.7.1.83 Pseudouridine kinase.
  • CA: ATP+pseudouridine ADP+pseudouridine 5′-phosphate); ID 2.7.1.84 Alkylglycerone kinase.
  • CA: ATP+O-alkylglycerone ADP+O-alkylglycerone phosphate); ID 2.7.1.85 Beta-glucoside kinase.
  • CA: ATP+cellobiose ADP+6-phospho-beta-D-glucosyl-(1,4)-D-glucose); ID 2.7.1.86 NADH kinase.
  • CA: ATP+sphinganine ADP+sphinganine 1-phosphate); ID 2.7.1.92 5-dehydro-2-deoxygluconokinase.
  • CA: ATP+5-dehydro-2-deoxy-D-gluconate ADP+6-phospho-5-dehydro-2-deoxy-D-gluconate); ID 2.7.1.93 Alkylglycerol kinase.
  • CA: ATP+1-O-alkyl-sn-glycerol ADP+1-O-alkyl-sn-glycerol 3-phosphate); ID 2.7.1.94 Acylglycerol kinase.
  • CA: ATP+acylglycerol ADP+acyl-sn-glycerol 3-phosphate); ID 2.7.1.95 Kanamycin kinase.
  • CA: ATP+kanamycin ADP+kanamycin 3′-phosphate); ID 2.7.1.99 [Pyruvate dehydrogenase(lipoamide)]kinase.
  • CA: ATP+[pyruvate dehydrogenase (lipoamide)] ADP+[pyruvate dehydrogenase (lipoamide)]phosphate); ID 2.7.1.100 5-methylthioribose kinase.
  • CA: ATP+[3-hydroxy-3-methylglutaryl-CoA reductase (NADPH)] ADP+[3-hydroxy-3-methylglutaryl-CoA reductase (NADPH)]phosphate); ID 2.7.1.110 Dephospho-[reductase kinase]kinase.
  • CA: ATP+dephospho[[3-hydroxy-3-methylglutaryl-CoA reductase (NADPH)]kinase] ADP+[[3-hydroxy-3-methylglutaryl-CoA reductase (NADPH)]kinase]); ID 2.7.1.112 Protein-tyrosine kinase.
  • CA: ATP+[caldesmon] ADP+[caldesmon]phosphate); ID 2.7.1.122 Xylitol kinase.
  • CA: ATP+xylitol ADP+xylitol 5-phosphate); ID 2.7.1.123 Calcium/calmodulin-dependent protein kinase.
  • CA: ATP+protein ADP+O-phosphoprotein); ID 2.7.1.124 Tyrosine 3-monooxygenase kinase.
  • CA: ATP+[tyrosine-3-monooxygenase] ADP+[tyrosine-3-monooxygenase]phosphate); ID 2.7.1.125 Rhodopsin kinase.
  • CA: ATP+[rhodopsin] ADP+[rhodopsin]phosphate); ID 2.7.1.126 [Beta-adrenergic-receptor]kinase.
  • CA: ATP+[beta-adrenergic receptor] ADP+[beta-adrenergic receptor]phosphate); ID 2.7.1.127 Inositol-trisphosphate 3-kinase.
  • CA: ATP+1D-myo-inositol 1,4,5-trisphosphate ADP+1D-myo-inositol 1,3,4,5-tetrakisphosphate); ID 2.7.1.128 [Acetyl-CoA carboxylase]kinase.
  • CA: ATP+1D-myo-inositol 3,4,5,6-tetrakisphosphate ADP+1D-myo-inositol 1,3,4,5,6-pentakisphosphate); ID 2.7.1.135 [Tau protein]kinase.
  • CA: ATP+[tau protein] ADP+[tau protein]O-phospho-L-serine); ID 2.7.1.136 Macrolide 2′-kinase.
  • CA: ATP+oleandomycin ADP+oleandomycin 2′-O-phosphate); ID 2.7.1.137 Phosphatidylinositol 3-kinase.
  • CA: ATP+1-phosphatidyl-1D-myo-inositol ADP+1-phosphatidyl-1D-myo-inositol 3-phosphate); ID 2.7.1.138 Ceramide kinase.
  • CA: ATP+ceramide ADP+ceramide 1-phosphate); ID 2.7.1.140 1D-myo-inositol-tetrakisphosphate 5-kinase.
  • CA: ATP+1D-myo-inositol 1,3,4,6-tetrakisphosphate ADP+1D-myo-inositol 1,3,4,5,6-pentakisphosphate); ID 2.7.1.141 [RNA-polymerase]-subunit kinase.
  • CA: ATP+[DNA-directed RNA polymerase] ADP+phospho-[DNA-directed RNA polymerase]); ID 2.7.1.144 Tagatose-6-phosphate kinase.
  • CA: ATP+D-tagatose 6-phosphate ADP+D-tagatose 1,6-bisphosphate); ID 2.7.1.145 Deoxynucleoside kinase.
  • CA: ATP+2′-deoxynucleoside ADP+2′-deoxynucleoside 5′-phosphate); ID 2.7.1.148 4-(cytidine 5′-diphospho)-2-C-methyl-D-erythritol kinase.
  • CA: ATP+1-phosphatidyl-1D-myo-inositol 4,5-bisphosphate ADP+1-phosphatidyl-1D-myo-inositol 3,4,5-trisphosphate); ID 2.7.1.154 Phosphatidylinositol-4-phosphate 3-kinase.
  • CA: ATP+1-phosphatidyl-1D-myo-inositol 4-phosphate ADP+1-phosphatidyl-1D-myo-inositol 3,4-bisphosphate); ID 2.7.2.1 Acetate kinase.
  • CA: ATP+acetate ADP+acetyl phosphate); ID 2.7.2.2 Carbamate kinase.
  • CA: ATP+NH(3)+CO(2) ADP+carbamoyl phosphate); ID 2.7.2.3 Phosphoglycerate kinase.
  • CA: ATP+3-phospho-D-glycerate ADP+3-phospho-D-glyceroyl phosphate); ID 2.7.2.4 Aspartate kinase.
  • CA: ATP+L-aspartate ADP+4-phospho-L-aspartate); ID 2.7.2.6 Formate kinase.
  • CA: ATP+formate ADP+formyl phosphate); ID 2.7.2.7 Butyrate kinase.
  • CA: ATP+2-butanoate ADP+butanoyl phosphate); ID 2.7.2.8 Acetylglutamate kinase.
  • CA: ATP+N-acetyl-L-glutamate ADP+N-acetyl-L-glutamate 5-phosphate); ID 2.7.2.11 Glutamate 5-kinase.
  • CA: ATP+L-glutamate ADP+L-glutamate 5-phosphate); ID 2.7.2.13 Glutamate 1-kinase.
  • CA: ATP+L-glutamate ADP+alpha-L-glutamyl phosphate); ID 2.7.2.14 Branched-chain-fatty-acid kinase.
  • CA: ATP+2-methylpropanoate ADP+2-methylpropanoyl phosphate); ID 2.7.3.1 Guanidoacetate kinase.
  • CA: ATP+hypotaurocyamine ADP+N(omega)-phosphohypotaurocyamine); ID 2.7.3.7 Opheline kinase.
  • CA: ATP+guanidinoethyl methyl phosphate ADP+N′-phosphoguanidinoethyl methyl phosphate); ID 2.7.3.8 Ammonia kinase.
  • CA: ATP+NH(3) ADP+phosphoramide); ID 2.7.3.10 Agmatine kinase.
  • CA: ATP+agmatine ADP+N(4)-phosphoagmatine); ID 2.7.3.11 Protein-histidine pros-kinase.
  • CA: ATP+AMP ADP+ADP); ID 2.7.4.4 Nucleoside-phosphate kinase.
  • CA: ATP+nucleoside phosphate ADP+nucleoside diphosphate); ID 2.7.4.6 Nucleoside-diphosphate kinase.
  • CA: ATP+nucleoside diphosphate ADP+nucleoside triphosphate); ID 2.7.4.7 Phosphomethylpyrimidine kinase.
  • CA: ATP+4-amino-2-methyl-5-phosphomethylpyrimidine ADP+4-amino-2-methyl-5-diphosphomethylpyrimidine); ID 2.7.4.8 Guanylate kinase.
  • CA: ATP+GMP ADP+GDP); ID 2.7.4.9 Thymidylate kinase.
  • CA: ATP+thymidine 5′-phosphate ADP+thymidine 5′-diphosphate); ID 2.7.4.11 (Deoxy)adenylate kinase.
  • CA: ATP+dAMP ADP+dADP); ID 2.7.4.12 T2-induced deoxynucleotide kinase.
  • CA: ATP+dGMP (or dTMP) ADP+dGDP (or dTDP)
  • ID 2.7.4.13 Deoxy)nucleoside-phosphate kinase.
  • CA: ATP+deoxynucleoside phosphate ADP+deoxynucleoside diphosphate); ID 2.7.4.14 Cytidylate kinase.
  • CA: ATP+(d)CMP ADP+(d)CDP); ID 2.7.4.15 Thiamine-diphosphate kinase.
  • CA: ATP+thiamine diphosphate ADP+thiamine triphosphate); ID 2.7.4.16 Thiamine-phosphate kinase.
  • CA: ATP+thiamine phosphate ADP+thiamine diphosphate); ID 2.7.4.18 Farnesyl-diphosphate kinase.
  • CA: ATP+farnesyl diphosphate ADP+farnesyl triphosphate); ID 2.7.4.19 5-methyldeoxycytidine-5′-phosphate kinase.
  • CA: ATP+5-methyldeoxycytidine 5′-phosphate ADP+5-methyldeoxycytidine diphosphate); ID 2.7.6.1 Ribose-phosphate diphosphokinase.
  • CA: ATP+D-ribose 5-phosphate AMP+5-phospho-alpha-D-ribose 1-diphosphate); ID 2.7.6.2 Thiamine diphosphokinase.
  • CA: ATP+GTP AMP+guanosine 3′-diphosphate 5′-triphosphate); ID 2.7.7.1 Nicotinamide-nucleotide adenylyltransferase.
  • CA: ATP+nicotinamide ribonucleotide diphosphate+NAD(+)); ID 2.7.7.2 FMN adenylyltransferase.
  • CA: ATP+FMN diphosphate+FAD); ID 2.7.7.3 Pantetheine-phosphate adenylyltransferase.
  • CA: ATP+pantetheine 4′-phosphate diphosphate+3′-dephospho-CoA); ID 2.7.7.4 Sulfate adenylyltransferase.
  • CA: ATP+sulfate diphosphate+adenylylsulfate); ID 2.7.7.18 Nicotinate-nucleotide adenylyltransferase.
  • CA: ATP+nicotinate ribonucleotide diphosphate+deamido-NAD(+)); ID 2.7.7.19 Polynucleotide adenylyltransferase.
  • CA: N ATP+ ⁇ nucleotide ⁇ (M) N diphosphate+ ⁇ nucleotide ⁇ (M+N)); ID 2.7.7.25 tRNA adenylyltransferase.
  • CA: ATP+ ⁇ tRNA ⁇ (N) diphosphate+ ⁇ tRNA ⁇ (N+1)); ID 2.7.7.27 Glucose-1-phosphate adenylyltransferase.
  • CA: ATP+alpha-D-glucose 1-phosphate diphosphate+ADP-glucose); ID 2.7.7.42 Glutamate-ammonia-ligase adenylyltransferase.
  • CA:ADP+ATP phosphate+P(1),P(4)-bis(5′-adenosyl)tetraphosphate); ID 2.7.7.54 Phenylalanine adenylyltransferase.
  • CA: ATP+L-phenylalanine diphosphate+N-adenylyl-L-phenylalanine); ID 2.7.7.55 Anthranilate adenylyltransferase.
  • CA: ATP+anthranilate diphosphate+N-adenylylanthranilate); ID 2.7.7.58 (2,3-dihydroxybenzoyl)adenylate synthase.
  • CA Exonucleolytic cleavage (in the presence of ATP) in either 5′- to 3′- or 3′- to 5′-direction to yield 5′-phosphooligonucleotides); ID 3.1.21.3 Type I site-specific deoxyribonuclease.
  • CA Endonucleolytic cleavage of DNA to give random double-stranded fragments with terminal 5′-phosphate; ATP is simultaneously hydrolyzed); ID 3.4.21.53 Endopeptidase La.
  • CA Hydrolysis of large proteins such as globin, casein and denaturated serum albumin, in presence of ATP); ID 3.4.21.92 Endopeptidase Clp.
  • CA Hydrolysis of proteins to small peptides in the presence of ATP and magnesium.
  • Alpha-casein is the usual test substrate.
  • oligopeptides shorter than five residues are cleaved (such as succinyl-Leu-Tyr-
  • CA: ATP+H(2)O+oligosaccharide(Out) ADP+phosphate+oligosaccharide(In)); ID 3.6.3.19 Maltose-transporting ATPase.
  • CA: ATP+H(2)O+maltose(Out) ADP+phosphate+maltose(In)); ID 3.6.3.20 Glycerol-3-phosphate-transporting ATPase.
  • CA: ATP+H(2)O+glycerol-3-phosphate(Out) ADP+phosphate+glycerol-3-phosphate(In)); ID 3.6.3.21 Polar-amino-acid-transporting ATPase.
  • CA: ATP+H(2)O+lipopolysaccharide(In) ADP+phosphate+lipopolysaccharide(Out)); ID 3.6.3.40 Teichoic-acid-transporting ATPase.
  • CA: ATP+H(2)O+teichoic acid(In) ADP+phosphate+teichoic acid(Out)); ID 3.6.3.41 Heme-transporting ATPase.
  • CA: ATP+H(2)O+heme(In) ADP+phosphate+heme(Out)); ID 3.6.3.42 Beta-glucan-transporting ATPase.
  • CA: ATP+(R)-5-diphosphomevalonate ADP+phosphate+isopentenyl diphosphate+CO(2)); ID 4.1.1.49 Phosphoenolpyruvate carboxykinase (ATP).
  • CA: ATP+(6S)-6-beta-hydroxy-1,4,5,6-tetrahydronicotinamide-adenine dinucleotide ADP+phosphate+NADH); ID 4.6.1.1 Adenylate cyclase.
  • CA: ATP+biotin+CoA AMP+diphosphate+biotinyl-CoA); ID 6.2.1.12 4-coumarate—CoA ligase.
  • CA: ATP+4-coumarate+CoA AMP+diphosphate+4-coumaroyl-CoA); ID 6.2.1.13 Acetate—CoA ligase (ADP-forming).
  • CA: ATP+acetate+CoA ADP+phosphate+acetyl-CoA); ID 6.2.1.14 6-carboxyhexanoate—CoA ligase.
  • CA: ATP+6-carboxyhexanoate+CoA AMP+diphosphate+6-carboxyhexanoyl-CoA); ID 6.2.1.15 Arachidonate—CoA ligase.
  • CA: ATP+arachidonate CoA AMP+diphosphate+arachidonoyl-CoA); ID 6.2.1.16 Acetoacetate—CoA ligase.
  • CA: ATP+acetoacetate+CoA AMP+diphosphate+acetoacetyl-CoA); ID 6.2.1.17 Propionate—CoA ligase.
  • CA: ATP+an omega-dicarboxylic acid AMP+diphosphate+an omega-carboxyacyl-CoA); ID 6.2.1.24 Phytanate—CoA ligase.
  • CA: ATP+phytanate+CoA AMP+diphosphate+phytanoyl-CoA); ID 6.2.1.25 Benzoate—CoA ligase.
  • CA: ATP+benzoate+CoA AMP+diphosphate+benzoyl-CoA); ID 6.2.1.26 O-succinylbenzoate—CoA ligase.
  • CA: ATP+O-succinylbenzoate+CoA AMP+diphosphate+O-succinylbenzoyl-CoA); ID 6.2.1.27 4-hydroxybenzoate—CoA ligase.
  • CA: ATP+4-hydroxybenzoate+CoA AMP+diphosphate+4-hydroxybenzoyl-CoA); ID 6.2.1.28 3-alpha,7-alpha-dihydroxy-5-beta-cholestanate—CoA ligase.
  • ID 6.2.1.33 4-chlorobenzoate-CoA ligase.
  • CA: 4-chlorobenzoate+CoA+ATP 4-chlorobenzoyl-CoA+AMP+diphosphate
  • ID 6.2.1.34 Trans-feruloyl-CoA synthase.
  • CA: Ferulic acid+CoA+ATP trans-feruloyl-CoA+products of ATP breakdown); ID 6.3.1.1 Aspartate—ammonia ligase.
  • CA: ATP+L-aspartate+NH(3) AMP+diphosphate+L-asparagine); ID 6.3.1.2 Glutamate—ammonia ligase.
  • CA: ATP+L-glutamate+NH(3) ADP+phosphate+L-glutamine); ID 6.3.1.4 Aspartate—ammonia ligase (ADP-forming).
  • CA: ATP+L-aspartate+NH(3) ADP+phosphate+L-asparagine); ID 6.3.1.5 NAD(+) synthase.
  • CA: ATP+L-glutamate+L-cysteine ADP+phosphate+gamma-L-glutamyl-L-cysteine); ID 6.3.2.3 Glutathione synthase.
  • CA: ATP+gamma-L-glutamyl-L-cysteine+glycine ADP+phosphate+glutathione); ID 6.3.2.4 D-alanine-D-alanine ligase.
  • CA: ATP+2 D-alanine ADP+phosphate+D-alanyl-D-alanine); ID 6.3.2.6 Phosphoribosylaminoimidazole-succinocarboxamide synthase.
  • CA: ATP+gamma-L-glutaniyl-L-cysteine+beta-alanine ADP+phosphate+gamma-L-glutamyl-L-cysteinyl-beta-alanine); ID 6.3.2.24 Tyrosine—arginine ligase.
  • CA: ATP+L-tyrosine+L-arginine AMP+diphosphate+L-tyrosyl-L-arginine); ID 6.3.2.25 Tubulin—tyrosine ligase.
  • ID 6.3.2.26 N-(5-amino-5-carboxypentanoyl)-L-cysteinyl-D-valine synthase.
  • CA: L-2-aminohexanedioate+L-cysteine+L-valine+3 ATP N-[L-5-amino-5-carboxypentanoyl]-L-cysteinyl-D-valine+3 AMP+3 diphosphate
  • CA: ATP+5-formyltetrahydrofolate ADP+phosphate+5,10-methenyltetrahydrofolate); ID 6.3.3.3 Dethiobiotin synthase.
  • CA: ATP+7,8-diaminononanoate+CO(2) ADP+phosphate+dethiobiotin); ID 6.3.4.1 GMP synthase.
  • CA: ATP+xanthosine 5′-phosphate+NH(3) AMP+diphosphate+GMP); ID 6.3.4.2 CTP synthase.
  • CA: ATP+UTP+NH(3) ADP+phosphate+CTP); ID 6.3.4.3 Formate—tetrahydrofolate ligase.
  • CA: ATP+formate+tetrahydrofolate ADP+phosphate+10-formyltetrahydrofolate); ID 6.3.4.5 Argininosuccinate synthase.
  • CA: ATP+L-citrulline+L-aspartate AMP+diphosphate+L-argininosuccinate); ID 6.3.4.6 Urea carboxylase.
  • CA: ATP+urea+CO(2) ADP+phosphate+urea-l-carboxylate); ID 6.3.4.7 Ribose-5-phosphate—ammonia ligase.
  • CA: 2 ATP+NH(3)+CO(2)+H(2)O 2 ADP+phosphate+carbamoyl phosphate); ID 6.3.4.17 Formate-dihydrofolate ligase.
  • CA: ATP+formate+dihydrofolate ADP+phosphate+10-formyldihydrofolate); ID 6.3.5.1 NAD(+) synthase (glutamine-hydrolyzing).
  • CA: ATP+deamido-NAD (+)+L-glutamine+H(2)O AMP+diphosphate+NAD(+)+L-glutamate); ID 6.3.5.2 GMP synthase (glutamine-hydrolyzing).
  • CA: ATP+xanthosine 5′-phosphate+L-glutamine+H(2)O AMP+diphosphate+GMP+L-glutamate); ID 6.3.5.3 Phosphoribosylformylglycinamidine synthase.
  • CA: ATP+N(2)-formyl-N(1)-(5-phospho-D-ribosyl)glycinamide+L-glutamine+H(2)O ADP+phosphate+2-(formamido)-N(1)-(5-phospho-D-ribosyl)acetamidine+L-glutamate); ID 6.3.5.4 Asparagine synthase (glutamine-hydrolyzing).
  • CA: ATP+L-aspartate+L-glutamine AMP+diphosphate+L-asparagine+L-glutamate); ID 6.3.5.5 Carbamoyl-phosphate synthase (glutamine-hydrolyzing).
  • CA: 2 ATP+L-glutamine+CO(2)+H(2)O 2 ADP+phosphate+L-glutamate+carbamoyl phosphate); ID 6.3.5.6 Asparaginyl-tRNA synthase (glutamine-hydrolyzing).
  • CA: ATP+Aspartyl-tRNA(Asn)+L-glutamine ADP+phosphate+Asparaginyl-tRNA(Asn)+L-glutamate); ID 6.3.5.7 Glutaminyl-tRNA synthase (glutamine-hydrolyzing).
  • CA: ATP+propanoyl-CoA+HCO(3)( ⁇ ) ADP+phosphate+(S)-methylmalonyl-CoA); ID 6.4.1.4 Methylcrotonyl-CoA carboxylase.
  • CA: ATP+3-methylcrotonyl-CoA+HCO(3)( ⁇ ) ADP+phosphate+3-methylglutaconyl-CoA); ID 6.4.1.5 Geranoyl-CoA carboxylase.
  • CA: ATP+geranoyl-CoA+HCO(3)( ⁇ ) ADP+phosphate+3-(4-methylpent-3-en-1-yl)pent-2-enedioyl-CoA); ID 6.4.1.6 Acetone carboxylase.
  • Suitable linkers include, without limitation, ⁇ , ⁇ -diamines, ⁇ , ⁇ -disphosphines, ⁇ -amine- ⁇ -phosphine, ⁇ -phosphine- ⁇ -thiols or mixture or combinations thereof.
  • Preferred ⁇ , ⁇ -diamines are compounds of the general formula H 2 N—R—NH 2 , where R is selected from the group consisting of a linear or branched alkenyl group, an arenyl group, a linear or branched ara-alkenyl group, a linear or branched alka-arenyl group and hetero atom analogs thereof.
  • the hetero atom analogs comprises the recited groups where: (a) one or more carbon atoms are replaced by a hetero atom selected from the group consisting of O, S, P, and mixtures thereof; (b) one or more carbon atoms are replaced by a hetero atom-containing groups selected from the group consisting of —C(O)NH— (amido group), —NHC(O)NH— (uryl group), or the like; (c) one or more hydrogen atoms are are replaced by a hetero atom selected from the group consisting of O, S, P, and mixtures thereof; (d) one or more hydrogen atoms are replaced by a hetero atom-containing groups selected from the group consisting of —C(O)NH— (amido group), —NHC(O)NH— (uryl group), or the like; (e) and mixture or combinations thereof.
  • Preferred hetero atom analogs include compounds of the general formula —CH 2 (CH 2 ) n [ECH 2 (CH 2 ) n ] m ECH 2 (CH 2 )— where E is O, S, or P, n is an integer having a value between 0 and 10 and m is an integer having a value between 0 and 10.
  • Preferred alkenyl group include —(CH 2 ) k — where k is an integer having a value between 1 and 16.
  • Preferred arenyl groups includes phenylene, divalent naphthylene, divalent antracene, or similer polycondensed aromatics or mixtures or combinations thereof, where divalent means that the amine, phosphine or thiol groups are attached at two different sites of the aromatic molecules.
  • Preferred alka-arenyl groups include dialkyenyl benzenes, dialkylenyl naphthalenes, dialkenyl antracenes or similar condensed fused aromatics with two dialkenyl groups.
  • Preferred ara-alkenyl group include phenyl substituted alkenyl group or the like.
  • T-NTP or T-L-NTP The labeled-5′ phosphate of the gamma phosphate labeled nucleotide (T-NTP or T-L-NTP) is transferred by T4 polynucleotide kinase (PNK) to the unlabeled, 5′ end of an oligonucleotide.
  • PNK polynucleotide kinase
  • phosphate exchange reaction methodology can be used to transfer the labeled phosphate of the T-NTP or T-L-NTP to the 5′ end of an oligonucleotide.
  • the T-NTPs or T-L-NTPs of this invention are used to catalytically or enzymatically label oligonucleotides.
  • the enzymatically or catalytically labeled oligonucleotides of this invention are well-suited as probes taking the place of a chemically-synthesized fluorescent probes, or in any application where one needs to track an oligonucleotide (for example, a 32 P-labeled probe).
  • Multiple fluorophores can be added into a single reaction to an oligonucleotide solution, where each fluorophore has a different color and each resulting color coded oligonucleotide can be monitored in parallel.
  • the enzymatically labeled oligonucleotides of this invention can also be used to prepare probes such as TaqMan probes, molecular beacons, etc.
  • the present invention allows labeling of pre-existing unlabeled oligonucleotides or polynucleotides.
  • researchers can rapidly attach a fluorescent label or other type of label to an on-hand oligonucleotide.
  • Using the technology of this invention provides researchers with a minimal time (generally overnight) method for labeling existing oligonucleotides as compared to the delays encountered when a fluorescently labeled oligonucleotide is ordered, a typically 3 to 10 day delay.
  • Enzymatically labeled oligonucleotides can be used in any application in which chemically-labeled fluorescent or radioactively-labeled oligonucleotides would be used.
  • the gamma-labeled NPTs of this s invention can be used to monitor reaction by monitoring modified nucleotide by measuring separation of tagged gamma phosphate from intact nucleotide, where intact nucleotide produces minimal signal, but cleaved products produce detectable signals that increase or change over time and these changes indicate that the activity being monitored is occurring or has occurred.
  • the gamma-labeled NTPs of this invention can be used to perform kinase activity assays on multiple candidate substrates along with positive and negative controls, all in parallel.
  • One preferred method involves immobilizing the controls and candidate substrate polypeptides in an array on a substrate, such as a polypeptide array.
  • the kinase and labeled-NTP of this invention are allowed to contact the immobilized species under conditions to promote the kinase activity resulting in the transfer of the labeled phosphate of the labeled-NTP to the candidate substrate polypeptide.
  • Knowledge of the substrates that are phosphorylated and the corresponding sites of phosphorylation of the candidate substrates provide information about the recognition target of the assayed kinase.
  • T-NTPs can be designed or tailored for use by a specific kinase.
  • T-NTPs or T-L-NTPs of this invention are contacted with a polypeptide in the presence of a catalyst, preferably a protein kinase, to generate a phosphorylated, labeled polypeptide.
  • a catalyst preferably a protein kinase
  • the phosphorylated, labeled polypeptide can be cleaved, enabling determination of the site of phosphorylation.
  • a fusion protein encoding polynucleotide sequence including a protein encoding region encoding a protein of interest and at least one phophorylation site encoding sequence ligated to either 5′ end or 3′ end or both the 5′ and 3′ ends of the protein encoding region, where the phophorylation site encoding sequence encoded amino acid sequences that correspond to a kinase phosphorylation amino acid sequence.
  • the expressed fusion protein can then be contacted with a T-NTP or T-L-NTP of this invention and the kinase for phosphorylating the fused phosphorylation site(s) producing a specifically labeled fusion protein, where the label is designed to not significantly impact protein folding or native activity, but gives rise to site-specifically labeled target proteins. If multiple fluorophores are attached to the fusion protein, then the labels can be either the same or different colors.
  • Such labeled fusion proteins can be used to monitor the expression, activation, activity and deactivation of these fusion proteins in the expressed cells, cell cluster, tissue or organ.
  • the T-NTPs or T-L-NTPs of this invention can be used to create color-coded polynucleotide (DNA, RNA, or DNA/RNA) molecular weight markers for use in DNA, RNA or DNA/RNA analyses similar to the kaleidoscope markers used in protein molecular size standards.
  • color-coded polynucleotide markers allows for unambiguous identification of the molecular weight and/or size of each separated polynucleotide fragment.
  • the same process can be used to form polypeptide markers provided that the polypeptide sequence includes at least one site capable of being phosphorylated using a T-NTP or T-L-NTP of this invention.
  • restriction nucleotide fragments can be labeled through phosphate exchange reaction using PNK (e.g., T4-PNK) for example to form labeled restriction nucleotide fragments.
  • PNK e.g., T4-PNK
  • These labeled restriction nucleotide fragments can be used to visualize and/or identify the fragments and do not involve staining the fragments such as with ethidium bromide and the DNA is viewed with longer wavelength light, thereby minimizing DNA damage, so that the fragments are still active fragments for subsequent use.
  • Complete labeling is not essential for visualization (i.e., only sufficient labeling for visualization and/or identification), ensuring that pristine DNA fragments can be isolated, as needed, for downstream manipulation.
  • T-NTPs or T-L-NTPs and unreacted NTPs purification may be needed.
  • the unreacted NTPs and T-NTPs or T-L-NTPs can be separated from the labeled targets and unreacted targets using a sizing column (or similar strategy).
  • Purifying the labeled target can be done enzymatically using an enzyme that distinguishes between 5′-labeled target nucleotides and non-labeled target nucleotides through recognition of the 5′ end to specifically degrade the non-labeled nucleotide.
  • Lambda exonuclease and phosphodiesterase 2 are candidate 5′ to 3′ exonucleases.
  • HPLC purification may be used to isolate the labeled nucleotide target. Spin sizing columns or similar techniques can be used in the purification process.
  • the labeled nucleotides of this invention can be used as probes in ATP-utilizing or GTP-utilizing enzymatic processes to determine specific activity, kinetics, mechanisms useful for biochemist and enzymologist.
  • Enzymes could be NMP kinases, NDP kinases, sugar kinases, and/or etc (see attached list of APT-utilizing Enzymes or variants thereof). These reactions are usually coupled to a second NAD(H)-dependent enzyme for accurate specific activity determination.
  • the labeled nucleotides of this invention can be used to investigate the activity, kinetics and mechanism of action of sugar kinase mediated phosphorylations, a large class of biomolecules.
  • a suicide tag to the gamma-phosphate of an NTP. If the tagged gamma phosphate is transferred to a substrate such as a target protein, the protein kinase and the substrate are held in their associated state with sufficient strength for post reaction purification and/or identification, where term held means that the suicide tag interacts with both the kinase and the substrate with sufficient chemical and/or physical interactions to maintain the kinase and the substrate in their associated state.
  • the chemical and/or physical interactions can be hydrogen bonding, covalent bonding, ionic bonding, electrostatic attractions, or mixtures or combinations thereof. This is useful for identifying interacting partners (i.e., the specific protein(s) phoshorylated by a specific kinase).
  • the modified NTPs of this invention can be designed for use with proteins that require the unmodified NTP in order to bind and/or transform a substrate protein or polypeptide.
  • the modified NTP can be designed to interact with the protein and to induce a conformation change in the protein that would facilitate its binding to its substrate protein or polypeptide.
  • the interaction is irreversible locking the protein in its active conformation.
  • the modified NTP would lock the kinase in its active conformation, the conformation that permits binding and/or transformation of its substrate protein and/or polypeptide.
  • the modified NTP can be designed to lock the protein in a conformation with an altered affinity for a second substrate.
  • This activated protein can then either be attached to a support through a reactive group such as a His-affinity tag—Nickel resin, biotin—streptavidin, or antibody or the protein can be preattached and activated by the modified NTP by passing a solution containing the modified NTP over the substrate having preattached protein.
  • the substrate having the activated protein can then be used to screen a library of potential substrates by passing a solution of the substrates over the support.
  • the substrates will then be separated into bound and unbound substrates, unbound pass through over the support with little or no delay, while bound substrates stay attached to the activated protein.
  • the bound substrates can then be eluted off the support and identified. This procedure is similar to the identification of unknown proteins isolated by their affinity to a known protein via the yeast two hybrid system.
  • the modified NTPs can also be used to examine at the affinity of known interactions where the binding of one substrate is dependent on the binding of an NTP by using a modified NTP to trap the enzyme in a state that would have an increased affinity for a given substrate and monitoring the binding affinity between the two substrates (e.g., Biacore or gel filtration).
  • the present modified ATP are ideally suited for use in Yeast strain cell free lysate kinase utilization assays under Native Condition similar to the process described in Kolpdziej, P. A., Young, R. A. [35] Epitope Tagging and Protein Surveillance. Methods of Enzymology, vol. 194, pp 508-519.
  • the procedure includes the steps of: (1) inoculate the yeast strain in 10 ml YPD media and grow at 30° C. overnight until cells reach log phase growth ( ⁇ 1 ⁇ 10 7 cells @O.D. 600 ); (2) harvest 2 mL of the log phase culture in an Eppendorf microcentrifuge tube; (3) pellet the cells at RT at 3000 ⁇ g.
  • Decant supernatant (4) resuspend pellet in 200 mL of Buffer A at 4° C. which comprises 10% glycerol, 20 mM Hepes pH 7.9, 10 mM EDTA (maybe omitted, phosphatase inhibitor), 1 mM DTT, 0.5 mg/mL BSA, 100 mM Ammonium Sulfate, and 1 mM PMSF (other protease inhibitors may be added); (5) add about 50 ml of 425-600 micron glass beads; (6) vortex vigorously, 7 ⁇ 30s, alternate with 30s on ice; (7) centrifuge 5 min.
  • Buffer A which comprises 10% glycerol, 20 mM Hepes pH 7.9, 10 mM EDTA (maybe omitted, phosphatase inhibitor), 1 mM DTT, 0.5 mg/mL BSA, 100 mM Ammonium Sulfate, and 1 mM PMSF (other protease inhibitors may be added)
  • the present modified ATP can be used in rapid transformation protocol yeast strain assays similar to the procedure described in Geitz, R. D., Woods, R. A. (2002) Transformation of Yeast by the LiAc/ssCarrier DNA/PEG Method. Methods of Enzymology 350: 87-96.
  • the procedure includes the steps of: (1) inoculate the yeast strain in 10 ml YPD media and grow at 30° C. overnight until cells reach log phase growth ( ⁇ 1 ⁇ 10 7 cells @O.D. 600 ); (2) harvest 2 mL of the log phase culture in an Eppendorf microcentrifuge tube; (3) pellet the cells at RT at 3000 ⁇ g.
  • Decant supernatant (4) resuspend the cells by adding the following components in the order listed: 240 mL of PEG 4000 50% w/v; 36 mL of lithium acetate (LiAc) 1.0M; 36 mL of 1.1 mM of Dye-dATP, and 48 mL dH 2 O; (5) incubate the mixture in a water bath at 42° C.
  • LiAc lithium acetate
  • the modified nucleotide reagents can be introduced into any cell or organism for monitoring NTP dependent cellular or organ functions.
  • FIG. 1A a block diagram of a method for making the tagged NTPs of this invention, generally 100 , is shown to involve reacting a nucleotide triphosphate (NTP) comprising a Base, a Sugar and three phosphate moieties (P ⁇ , P ⁇ and P ⁇ ) in a first step 102 with a tag T to form a tagged NTP (T-NTP).
  • NTP nucleotide triphosphate
  • T-NTP a nucleotide triphosphate
  • FIG. 1B a block diagram of a method for making the tagged NTPs of this invention, generally 120 , is shown to include a nucleotide triphosphate (NTP) comprising a Base, a Sugar and three phosphate moieties (P ⁇ , P ⁇ and P ⁇ ) which is reacted in a first step 122 with a linker L to form a linker modified NTP (L-NTP), where the linker L is bonded to the gamma phosphate P ⁇ .
  • L-NTP linker modified NTP
  • the L-NTP is reacted in a second step 124 with a tag T to form a tagged NTP (T-L-NTP).
  • a method for preparing a tagged ATP is shown to include an ATP which is reacted in a first step 142 with an ethylene diamine linker EDA to form an ethylene diamine modified ATP (EDA-ATP). Then in a second step 144 , the EDA-ATP is reacted with a tag T via a second amino group of the EDA to form an tagged ATP (T-EDA-ATP), where the tag is preferably a fluorescent tag.
  • FIG. 2A a block diagram of a method for tagging a synthetic 5′ and 3′ hydroxy terminated oligonucleotide (SON) at either the 5′ end using a tagged nucleotide triphosphate (T-L-NTP) of this invention, generally 200 , is shown to involve reacting a T-L-NTP comprising a Base, a Sugar and three phosphate moieties (P ⁇ , P ⁇ and P ⁇ ) with a SON in a step 202 in the presence of a catalyst or enzyme.
  • the catalyst or enzyme adds the tagged phosphate (T-L-P) of the T-L-NTP to the 5′ end, This same general procedure works equally well with T-NTPs of this invention.
  • FIG. 2B a block diagram of a method for tagging a natural or synthetic 5′ phosphate and 3′ hydroxy terminated oligonucleotide (NON) at the 5′ end using a tagged nucleotide triphosphate (T-L-NTP) of this invention, generally 250 , is shown to involve reacting a T-L-NTP comprising a Base, a Sugar and three phosphate moieties (P ⁇ , P ⁇ and P ⁇ ) with a NON in a step 252 in the presence of a catalyst or enzyme. The catalyst or enzyme exchanges the 5′ phosphate of the NON with the tagged phosphate (T-L-P) of the T-L-NTP.
  • T-L-NTP tagged nucleotide triphosphate
  • FIG. 3 a block diagram of a method for tagged polypeptides using a tagged nucleotide triphosphate (T-L-NTP) of this invention, generally 300 , is shown to involve reacting a T-L-NTP comprising a Base, a Sugar and three phosphate moieties (P ⁇ , P ⁇ and P ⁇ ) with a polypeptide PN in a step 302 in the presence of a catalyst or enzyme.
  • the catalyst or enzyme adds the tagged phosphate (T-L-P) of the T.L-NTP to a site AA m+1 of the PP, where the exact site of phosphorylation depends on the catalyst or enzyme used in the reaction.
  • This same general procedure works equally well with T-NTPs of this invention.
  • linker is a main group element selected from the group consisting of N, O, P, S and combinations thereof and where R is a carbon-containing group having between 1 and about 20 main group atoms selected from the group consisting of B, C, N, O, P, S and combinations thereof, with the valency being completed by H.
  • the linker can be diamines, diphosphines, dithiols, or mixed amine, phosphine and thiols.
  • the linkers can also be carbon chains having leaving groups to promote direct carbon phosphate bonding and direct carbon to dye bonding.
  • linkers can have different lengths, charges, sizes, shapes, and polarities, and other physical properties, the exact linker for use in a particular application may depend on different variables. However, it is expected that any of the modified ATPs 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.
  • This example illustrates the preparation of a ATP-L1-ROX or ATP-EDA-ROX, where the EDA is attached to the ⁇ -phosphate of ATP as shown in the following reaction scheme:
  • 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 NH 4 HCO 3 —MeOH/H 2 O buffer system.
  • the product peak was collected and lyophilized to yield a white powder which was dissolved in 200 ⁇ L water and quantitated by spectrophotometry at 259 nm; 24 mM.
  • the yield of the ATP-EDA intermediate was 38%.
  • a 1 mL water solution of the pellet was subjected to HPLC purification on a Supelco C18 column using a TEAA-acetonitrile buffer system.
  • the product peak having a retention time 11 minutes, was collected and lyophilized to give a pellet.
  • the pellet was dissolved in 5 mM, 780 ⁇ L of HEPES buffer and quantified by 576 nm reading on a spectrometer. The measured concentration of the solution was 1.1 mM representing a yield of 86% of ATP-EDA-ROX.
  • MALDI-Mass 1124 (M-3+Na+K); 1102 (M+K), 1058 (M-H 2 O+K).
  • FIG. 4 An HPLC chromatogram of ATP-1-ROX synthesis reaction (576 nm) to monitor the mobility of molecules linked to the fluorophore or free dye is shown in FIG. 4 .
  • HPLC chromatogram of ATP-1-ROX synthesis reaction (259 nm) to monitor oligonucleotide mobility is shown in FIG. 5 .
  • UV spectrum of ATP-1-ROX is shown in FIG. 6 .
  • one of the amine protons is missing from each terminal nitrogen atom because the linkers bond to the ⁇ -phosphate of ATP and the fluorescent dye through the terminal nitrogen atoms of the diamine linkers.
  • the twelve ⁇ -phosphate-modified-ATPs are tabulated Table 1.
  • a ⁇ -phosphate-modified nucleotide or deoxynucleotide can be removed by treating the solution with an appropriate phosphatase.
  • an appropriate phosphatase for example, if the nucleotide is ATP, then calf intestinal phosphatase (CIAP) or shrimp alkaline phosphatase (SAP) can be used to specifically remove the unmodified ATP leaving the ⁇ -phosphate-modified-ATP intact.
  • ⁇ -phosphate-modified-ATPs free from their corresponding natural analog are prepared. The removal of the unmodified ATPs greatly enhances successful implementation of the methodology of this invention.
  • This invention provides an effective process using phosphatases (e.g., CIAP and SAP) to remove non-modified NTPs or dNTPs from ⁇ -phosphate-modified-NTPs and dNTPs.
  • phosphatases e.g., CIAP and SAP
  • the general procedure for purifying a ⁇ -phosphate-modified nucleotide involves treating 1 mM, 1 mL, 1 nmol of the crude ⁇ -phosphate-modified nucleotide with 1 U/mL, 1 ⁇ L, 1U of CIAP in 10 mL or 20 mL of a 1 ⁇ buffer comprising 50 mM Tris.HCl pH 9.3, 1 mM MgCl 2 , 0.1 mM ZnCl 2 1 mM spermidine, at room temperature for 20 minutes. The treated sample was then used in a subsequent reaction as described herein. It is also possible to remove CIAP by heat-shock and centrifugal filtration.
  • This example illustrates the purification of ATP-ROX with CIAP.
  • ATP 10 mM, 2 ⁇ L, 20 nmol
  • ATP-ROX 1.1 mM, 2 ⁇ L, 2.2 nmol
  • CIAP 1 U/ ⁇ L, 1 ⁇ L, 1U
  • 1 ⁇ buffer 10 ⁇ L
  • Controls were the same re action mixtures without enzyme.
  • aliquots (1 ⁇ L) from the samples were analyzed with appropriate TLC chromatography and fluorescence imaging or UV-shadowing. The result is shown below in FIG. 7 .
  • This example illustrates the end labeling of the 5′ end of an oligonucleotide using T4 PNK and ATP-ROX.
  • T4 PolyNucleotide Kinase is able to use our ATP-1-Dye molecules as substrates for the 5′ end-labeling of an oligonucleotide.
  • the ability to enzymatically add a fluorophore to the 5′ end of an oligonucleotide has tremendous applicability for a wide variety of molecular biological techniques.
  • the initial labeled phosphate transfer experiment was set-up as a standard end-labeling reaction whereby one microliter of a 1.1 mM solution of ATP-L1-ROX was used in a ten microliter reaction using 10U of T4 PNK to end-label 100 nanograms of an in-house “TOP oligonucleotide having the nucleotide sequence SEQ. ID NO. 2 of 5′ gg TAC TAA gCg gCC gCA Tg 3′. The reaction was incubated at 37° C.
  • FITC-TOP fluorescein labeled TOP oligonucleotide
  • a CTAP reaction was performed containing one microliter of 1.1 mM solution of crude ATP-L1-ROX, 1 microliter of CIAP (available from Promega), one microliter of 10 ⁇ CLAP buffer (available from Promega), and seven microliters of sterile milli-Q water. This reaction was incubated at 37° C. for 30 minutes. Five microliters of the CLAP treated ATP-L1-ROX was added to 1 microliter T4 PNK (available from Promega), 1 microliter of 10 ⁇ PNK buffer (available from Promega), 100 nanograms of the TOP oligonucleotide in a total volume of ten microliters. The reaction was incubated at 37° C. overnight. Results from this reaction are shown in FIGS. 8 and 9 clearly evidencing 5′ end labeling.
  • This example illustrates the purification of end labeling of the 5′ end of an oligonucleotide using T4 PNK and ATP-L1-ROX or ATP-EDA-ROX.
  • CENTRI-SEP Columns (Princeton Separations, Inc. Cat. #CS-900) were used to remove unwanted dye-ATP and dye breakdown from the 5′ dye-labeled oligonucleotide product.
  • a ten microliter reaction was performed with the 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://www.prinsep.com/html/products/centri_sep/single_column/protocoli, where the modification replaced the 750 ⁇ g for 2 minute centrifugation step with a 500 ⁇ g for 3 minutes centrifugation step. Additionally, ten microliters of sterile milli-Q water were added to the ten microliter end-labeling reaction to bring the final volume to twenty microliters.
  • the following figures display experimental results investigating T4 PNK's ability to utilize the set of fluorescently labeled ATP molecules in 5′-end-labeling reactions of oligonucleotides. All reactions have undergone column purification prior to electrophoresis on a 20% denaturing polyacrylamide gel to remove excess fluorescent-ATP and free dye which obscures the 5′ fluorescently end-labeled oligonucleotide product. For each experiment, the most intense (major) band was assigned a relative activity value of 1.0 and all other bands were normalized to this value.
  • the initial experiment was designed to investigate the time required to maximize T4 PNK's ability to 5′ end-label a 19-base oligonucleotide with ATP-L1-ROX as shown FIGS. 12A&B .
  • a significant improvement in 5′ end-labeling occurred when reactions were allowed to continue overnight or for about 18 hours. Since reactions that were incubated for longer than 18 hours (not shown) did not improve labeling, 18 hours is designated as the standard labeling reaction time.
  • FIG. 12A a timecourse investigating 5′ end-labeling of a 19-base oligonucleotide with APT-L1-ROX. Reactions were incubated at 37 degrees Celsius for 0.5, 1, 3, 6, and 18 hours. All reactions were column purified and electrophoresed on a 20% denaturing gel. The negative control ( ⁇ ) does not contain T4 PNK.
  • FIG. 12B a graphical representation of quantitated bands from gel imaging. Gels are scanned with a BIORAD Molecular Imager FX Pro at the fluorophores specific emission wavelength filter setting. BIORAD Quantity One Quantitation software is used to analyze bands. Band values are obtained by highlighting a band's area and then subtracting background value (equivalent gel image area without a band) to obtain an optical density value.
  • Reactions were assembled by adding 1 ⁇ g of a 19-base oligonucleotide (5′ GGTACTAAGCGGCCGCATG 3′), 1 ⁇ l of Promega 10 ⁇ Kinase buffer (700 mM Tris-HCl, pH 7.6, 100 mM MgCl2, and 50 mM DTT), 2 nmol ATP-Linker-Fluorophore (or ATP-Linker-Biotin), 12% Polyethylene glycol 8000, Promega T4 PNK (20U), and sterile milliQ water to a final volume of 10 ⁇ l.
  • a 19-base oligonucleotide 5′ GGTACTAAGCGGCCGCATG 3′
  • Promega 10 ⁇ Kinase buffer 700 mM Tris-HCl, pH 7.6, 100 mM MgCl2, and 50 mM DTT
  • 2 nmol ATP-Linker-Fluorophore or ATP-Linker-Biotin
  • reactions were incubated overnight (18 hours) at 37 degrees Celsius in a BioRad 96 microtube iCycler thermocycler with heated-lid and purified with a Centri-SEP column (Princeton Separations, Inc.) following the manufacture's protocol with the exceptions: (1) columns were centrifuged at 500 ⁇ g for 3 minutes (2) reaction volumes were adjusted to 20 ⁇ l with sterile MilliQ water prior to loading.
  • Reaction products can be directly loaded onto a 20% denaturing polyacrylamide gel for electrophoresis or purified through a CentriSEP spin-column to remove excess ATP-Linker-Fluorophore or ATP-Linker-Biotin that has not been utilized in the reaction.
  • Samples are electrophoresed at 50W, 50 degrees Celsius, for a period of approximately 1-1.5 hours and then the gel is scanned with a BIORAD Molecular Imager FX Pro at the fluorophores specific emission wavelength filter setting. Subsequent gel analysis is performed with BIORAD Quantity One Quantitation software.
  • T4 PNK needed for 5′ end-labeling with a fluorescent-ATP
  • two different concentrations (10U and 20U) of T4 PNK were tested with increasing concentrations of APT-L1-ROX as shown in FIGS. 13A-B .
  • the results demonstrate that 20 Units of T4 PNK increases the efficiency of 5′ end-labeling at all concentrations of APT-L1-ROX tested. No improvements were observed when greater than 20U of T4 PNK were tested (data not shown).
  • FIG. 13A a 5′ end-labeling of a 19-base oligonucleotide examining optimal T4 PNK activity (10U and 20U). Three concentrations of APT-L1-ROX (110, 220, and 550 micromolar) were tested. Reactions were incubated at 37 degrees Celsius for 18 hours, column purified, and electrophoresed on a 20% denaturing gel. Referring now to FIG. 13B , Graphical representation of quantitated bands from gel imaging.
  • High molecular weight polymers are reported to promote T4 PNK activity (i.e., transfer of the ⁇ 32 P of a radio-labeled ATP) by stabilizing the enzyme via macromolecular crowding.
  • polyethylene glycol (PEG 8000) was added to end-labeling reactions to determine if a similar affect is observed when fluorescently-labeled ATP is used as the label source as shown in FIG. 14 .
  • Increases in the amount of 5′ ROX-labeled oligonucleotide are observed as a result of increased PEG in the reaction. Further increases in PEG past 12% did not improve the sefficiency of the fluorescent labeling reaction.
  • FIG. 14 an effect following addition of polyethylene glycol 8000 (PEG 8000) on the amount of labeled oligonucleotide.
  • PEG 8000 polyethylene glycol 8000
  • 5′ end-labeling reactions containing different concentrations of PEG 8000, (4, 6, 8, 12, and 18%) were examined with a 19-base oligonucleotide and APT-L1-ROX.
  • the graph represents quantitated bands determined via gel imaging and analysis.
  • T4 PNK reveals that the active site resembles a shallow tunnel in which a gamma-phosphate of ATP can be positioned on one side of the tunnel for an in-line phosphoryl transfer to the 5′-OH of a nucleic acid substrate on the opposite side.
  • Two features of the labeled-ATP which may influence the phosphoryl transfer by T4 PNK are the chemical characteristics of the fluorophore and the linker attachment. Their structural size and/or rigidity may be critical to enzymatic activity, depending on potential steric constraints within the active site tunnel.
  • T4 PNK's end-labeling ability evidence which supports the differential effects that a DNA substrate has upon T4 PNK's end-labeling ability is the enzyme's decreased activity when confronted with recessed 5′ ends and nicks in double stranded DNA, and an apparent bias it exhibits towards preferentially phosphorylating a 5′ terminal guanosine base over other bases.
  • Other considerations include the effect that hydrophobicity and polarity may have upon catalysis. This is particularly important due to the hydrophobic nature of many of the fluorescent dyes.
  • T4 PNK's active site surface is almost entirely composed of charged or polar residues, however several hydrophobic residues are involved in formation of the walls.
  • FIGS. 15A and B Two sets of the fluorescently labeled-ATPs (ATP-Linker-ROX and ATP-Linker-Fluorescein) were examined shown in FIGS. 15A and B. Oligonucleotide labeling with the ROX-labeled ATPs show that T4 PNK's efficiency is higher when linker L1 joins the ATP and fluorophore, whereas linker L2 increases oligonucleotide labeling when fluorescein is used.
  • FIGS. 15A&B the differential effect of L1, L2, L3, and L4 on T4 PNK's utilization of fluorescently labeled ATP molecules in the 5′ end-labeling reaction.
  • the reactions examining each of the four linkers were tested with a 19-base oligonucleotide and the set of ( FIG. 15A ) ATP-L-ROX (220 and 440 micromolar) molecules and ( FIG. 15B ) ATP-L-Fluorescein molecules (220 and 440 micromolar). Reactions were incubated at 37° C. for 18 hours, column purified, and electrophoresed on a 20% denaturing gel, not shown. The graph represents quantitated bands from gel imaging and analysis.
  • DNA polymerase extension reactions were performed. Since polymerases are restricted to incorporation at the 3′ end, a 19-base oligonucleotide was 5′ end-labeled with APT-L1-ROX and then hybridized to a 20-base complementary oligonucleotide containing a single base overhang ‘A’ as shown in FIG. 16A . This overhanging ‘A’ was used by the polymerase as a template for the incorporation of an incoming base-labeled dUTP at the ROX-oligonucleotide's 3′ end as shown in FIG. 16B .
  • FIG. 16A a Schematic of the 5′ end-labeled ROX-oligonucleotide duplexed to a complementary 20-base oligonucleotide and used as primer-template for incorporation of a base-labeled dUTP.
  • FIG. 16B a gel electrophoresis of samples (Lane 1): . ROX-oligonucleotide with an incorporated base-labeled dUTP at its 3′ end (Lane 2): ROX-oligonucleotide without the base-labeled dUTP incorporated. Reaction products were detected using a ROX emission filter.
  • Oligonucleotides were annealed to form duplex by incubating primer and complementary template at equimolar amounts in a BioRad 96 microtube iCycler thermocycler with heated-lid starting at 96 degrees Celsius and slow-cooling to 20 degrees Celsius over a one hour period.
  • Primer extension reactions contain the duplexed molecules, 1 ⁇ l HIV reverse transcriptase (1 mg/ml), 1 ⁇ l of 10 ⁇ Promega Taq buffer (100 mM Tris-HCl (pH 9.0 at 25° C.), 500 mM KCl, 1.5 mM MgCl 2 , and 1% Triton X-100), and 200 ⁇ M dUTP-Alexa 488 (Invitrogen/Molecular Probes). The reaction was incubated in a 37° C. water bath for 2 hours.
  • FIG. 17A The experiment where a ROX-labeled oligonucleotide and unlabeled oligonucleotide were treated with lambda exonuclease for different periods of time is shown in FIG. 17A .
  • Intact ROX-oligonucleotide (Red) and unlabeled oligonucleotide (Green) are visible at timepoint 0.
  • the amount of each remaining after treatment with lambda exonuclease is compared as shown in FIG. 17B .
  • FIG. 17A&B depict examining lambda exonuclease activity on ROX-labeled versus unlabeled oligonucleotide.
  • FIG. 17A a 5′ end-labeled ROX-oligonucleotide and unlabeled oligonucleotide that have been treated with lambda exonuclease for 0 to 10 minutes at 37° C. Reactions were electrophoresed and oligonucleotide integrity monitored by ROX emission for the ROX-oligonucleotide (RED) and SyBR Gold staining for unlabeled oligonucleotide (GREEN); gel images overlayed.
  • RED ROX-oligonucleotide
  • GREEN SyBR Gold staining for unlabeled oligonucleotide
  • the two green bands observed at timepoint 0 for the unlabeled oligonucleotide represent intact 19-base and 18-base oligonucleotide (a byproduct of incomplete oligonucleotide synthesis, ⁇ 1).
  • a graph represents quantitated bands, the intact band at timepoint 0 min. for ROX-1-oligonucleotide and unlabeled oligonucleotide (19-base) have each been assigned the relative activity of 1.0, with all other bands normalized to their resp. control values.
  • Standard 5′ end-labeling reactions were performed with ATP-L1-ROX with the exception that after overnight incubation natural ATP was added to a final concentration of 1 mM and reactions were allowed to incubate for an additional 2 hours at 37 degrees Celsius. The reactions were then purified on CentriSep columns and the eluent was vacuum dried.
  • Treatment consisted of adding 5 ⁇ l of 10 ⁇ lambda exonuclease buffer (1 ⁇ buffer: 67 mM Glycine-KOH, 2.5 mM MgCl 2 , 50 ⁇ g/ml BSA, pH 9.4 at 25° C.), 5 ⁇ l (25U) of lambda exonuclease (NeW England Biolabs, MA), and then incubated for specified timepoints at 37 degrees Celsius. Timepoint aliquots were immediately removed to 5 ⁇ l stop solution (88% formamide, 1% bromophenol blue, 0.6M EDTA). Samples were electrophoresed on a 20% denaturing gel and the gel was scanned for ROX emission (red channel) and stained with SYBR Gold (Invitrogen/Molecular Probes) (green channel).
  • the inventors also identified an alternative to the lambda exonuclease treatment for clearing unlabeled, single-stranded DNA and RNA from our T4 PNK labeling reactions; effectively raising the specific activity of the labeling reaction.
  • the inventors have found that the phosphodiesterases 2 (PDE2) enzyme has several advantages over the Lambda exonuclease including: (1) no cold-phosphorylation of the unlabeled oligonucleotide is required, thus making pre-treatment of the reaction with natural ATP unnecessary, and (2) PDE2 will work on both DNA and RNA substrates.
  • APT-L1-biotin is comprised of a biotin attached by linker L1 to the gamma-phosphate of ATP.
  • linker L1 is comprised of a biotin attached by linker L1 to the gamma-phosphate of ATP.
  • the attached biotin-linker moiety is transferred with the gamma-phosphate to the 5′ end of the oligonucleotide by T4 PNK.
  • Detection of 5′ biotin-labeling of the oligonucleotide was observed by modifying an immunoblot analysis that is based on the strong interaction between biotin and streptavidin.
  • Column purified (CentriSEP) 5′ biotinylated oligonucleotide was covalently attached to Whatman DE81 filter paper disc by spotting and allowed to air dry. Filter discs were processed with a streptavidin-alkaline-phosphatase conjugate and subsequently developed with NBT/BCIP reagent. Displayed are three DE81 filter discs that have undergone color development as shown in FIG. 18 .
  • APT-L1-biotin any residual APT-L1-biotin not utilized in the reaction was removed by CentriSEP column purification, as indicated by the lack of a spot on the negative control disc.
  • the positive control center filter
  • the positive control has been spotted with a dilution series of a chemically synthesized biotin-labeled oligonucleotide (Integrated DNA Technologies, Inc.) and is used as a comparison for labeling.
  • FIG. 18 a 5′ end-labeling experiment of a 19-base oligonucleotide with APT-LI-biotin is shown.
  • Three DE81 filters that have been color-processed (Left disc) Negative control: reaction without T4 PNK, (Center disc) Positive control: Chemically synthesized biotin-labeled oligonucleotide (Integrated DNA Technologies, Inc.) spotted are 1, 0.1, and 0.01 pmol indicated below each spot, (Right)
  • APT-L1-biotin experiment reaction containing T4 PNK.
  • Reaction products are CentriSEP column purified and the eluted sample is vacuum dried and resuspended in 5 ⁇ l of sterile milliQ water. The reaction is spotted onto Whatman DE81 filter paper in 1 ⁇ l aliquots with air drying between spottings. Filters are incubated in 10 mL of 1 ⁇ phosphate buffered saline (PBS) solution for 10 minutes at room temperature with agitation. The filter is then incubated in 1 ⁇ PBS+0.1% bovine serum albumin (BSA) as a block for 30 minutes at room temperature with agitation.
  • PBS phosphate buffered saline
  • BSA bovine serum albumin
  • a reaction was performed as described previously (see Oligonucleotide 5′ end-labeling reaction: Method details) and a 2 microliter sample volume of the purified ROX-labeled oligonucleotide, 50 microMolar ATP-L1-ROX, and resuspension buffer were analyzed on a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Inc, USA) according to the manufacturer's recommendations. Absorbances were measured at 260 nm ( ⁇ max for nucleic acid) and 584 nm ( ⁇ max for ROX dye) to calculate the Base: Dye ratio.
  • the absorbance of the ATP-L1-ROX was measured at these wavelengths to correct for the dye contribution at the 260 nm reading, i.e., a correction factor (CF 260 ). Equations are given in Research and Design Methods section, see Determining the relative use of the labeled-ATP product in oligonucleotide labeling reactions.
  • a 20 base RNA oligonucleotide was 5′ end-labeled with ATP-2-Cy3 for the purpose of examining labeling via signal intensity of the hybridized oligonucleotide to a DNA microarray.
  • One microgram of the RNA oligonucleotide was used in a standard 5′ end-labeling reaction,(see Oligonucleotide 5′ end-labeling reaction: Method details) and then hybridized for 18 hours to the array.
  • the array was composed of oligos consisting of the perfect match (PM), single base mismatch (MM1), two mismatched bases (MM2), a single base deletion (Del1), two deletions (Del2), or background spot which contains no oligonucleotide (BGD).
  • the signal intensity profile and corresponding array cross-section illustrate that the Cy3-oligonucleotide is efficiently labeled and specifically hybridizes to the array. This demonstrates proof of principle for direct labeling of RNA and use of same in a microarray experiment, as shown in FIG. 19 , below.
  • FIG. 19 depicts 5′ end-labeling an RNA oligonucleotide with ATP-L2-Cy3 and hybridization to a DNA microarray.
  • Microarray layout consists of BGD (Background), Perfect Match (PM), Mismatch of 1 base (MM1), Mismatch of 2 bases (MM2), Deletion of 1 base (Del1), and Deletion of 2 bases (Del2).
  • the labeling techniques used in current microarray technology to determine information about the levels of numerous transcripts in parallel are not reproducible, primarily due to labeling and hybridization biases.
  • the present labeling technique will allow microarry researchers to directly label RNA and to characterize the labeling efficiency to determine if the labeled targets are adequate to detect their transcript of interest, prior to committing a costly microarray to a suboptimal experiment (due to insufficient labeling of the target).
  • the present technique transfers a labeled phosphate of an ⁇ -phosphate labeled ATP to the 5′OH (the 5′ end) of a nucleic acid such as DNA, RNA, RNA/DNA mixed biomolecules, ribozymes, or other biomolecules having a 5′OH DNA or RNA terminus.
  • a nucleic acid such as DNA, RNA, RNA/DNA mixed biomolecules, ribozymes, or other biomolecules having a 5′OH DNA or RNA terminus.
  • the mRNA is fragmented prior to labeling. For other biomolecules, fragmentation may or may not be necessary depending on the type of DNA or RNA containing biomolecule.
  • the fragmentation process preferably produces fragments having an average length of about 200 bases, the targeted size for cDNA used in microarray hybridization experiments.
  • Fragmentation conditions that produce the desired consistency in RNA length can be determined by first exposing total RNA and subsequently mRNA in an alkaline buffer having a pH 9 for increasing amounts of time, and examining the resulting size distribution of the fragmented RNA via denaturing polyacryamide gel electrophoresis. The fragmentation conditions is designed to produce similarly sized mRNA for use in the end-labeling reaction and minimize error.
  • the fragmented RNA are then end-labeled as described above.
  • the end labeling efficiency is then determined for high, medium and low abundance transcripts.
  • end labeling actin as an abundant RNA marker, glyceraldehydes-3-phosphate dehydrogenase(GAPDH) as a moderately abundant RNA marker and hypoxanthine-guanine phosphoribosyltransferase(HPRT) as a low abundance RNA marker.
  • GPDH glyceraldehydes-3-phosphate dehydrogenase
  • HPRT hypoxanthine-guanine phosphoribosyltransferase
  • the present invention also relates to a library of ⁇ -phosphate labeled ATPs or other NTP.
  • One preferred class of libraries comprise a plurality of ⁇ -phosphate labeled ATPs where the linker is the same and the fluorophore is different.
  • Another preferred class of libraries comprise a plurality of ⁇ -phosphate labeled ATPs where the fluorophore is the same and the linker is different.
  • Another preferred class of libraries comprise a plurality of ⁇ -phosphate labeled ATPs where the fluorophore and linkers are different.
  • TLC Thin Layer Chromatography
  • PDE phosphodiesterase
  • This example illustrates the preparation of ATP-L2-Cy5(TEA + ). Although this example illustrates the preparation of ATP-L2-Cy5(TEA + ), the preparation works equally well with L3 and L4.
  • TEAB buffer (1M, 1L) was made from triethylamine (139 mL) and dry ice ( ⁇ 200 g).
  • Dowex resin (H + ) 100 g is treated with water (1L), ethanol (300 mL), water (2 L), TEAB (1 M, 1L), water (5L), to prepare the Dowex resin (TEA + ) ( ⁇ 95 g).
  • ATP.Na 2 57 ⁇ mol
  • ATP.TEA by passing through Dowex resin (TEA + ) (2 g).
  • the collected fractions are tested with a UV lamp and combined solution is lyophilized. The pellet is dissolved in water and its concentration is measured on UV spectrometer (174 mM*300 ⁇ L, 52 ⁇ mol).
  • the supernatant was then passed through a red 22 ⁇ m syringe filter.
  • the sample was purified on HPLC (SAX column) with TEAB/H2O as elution buffer system ( ⁇ 200 mL of 1M TEAB). In some cases two such purifications were needed for this amount of material.
  • the collected product fraction was then lyophilized. The pellet was dissolved in HEPES buffer (5 mM, pH 8.5) and its concentration was measured on UV spectrometer (200 ⁇ L*48 mM, 9.6 ⁇ mol). Its purity was evaluated on silica TLC plate visualized by UV lamp and ninhydrin stain. Yield was 48%.
  • ATP-2 (0.8 ⁇ mol, 17 ⁇ L) in NaHCO 3 buffer (1M, pH 9, 40 ⁇ L) and Cy5-NHS (1 ⁇ mol) in DMF (42 ⁇ L) were mixed and the resulting mixture is set on the shaker to react for 7 hours.
  • Water 1.5 mL was added and the mixture is lyophilized.
  • a water solution (200 ⁇ L) of the pellet was passed through a 1*28 cm Sephadex G-25 column. The right fractions are collected and the combined solution was lyophilyzed.
  • the pellet is dissolved in TEAA (100 mM, 1.5 mL) and purified on HPLC (C18) with TEAA (200 mL, 100 mM)/CH 3 CN.
  • the product fraction is collected and lyophilized.
  • the pellet was dissolved in HEPES buffer (5 mM, pH8.5). Its concentration was determined on UV spectrometer at 646 nm (80 ⁇ L*2.8 mM, 0.22 ⁇ mol). Yield was 28%. If necessary, a phosphate assay was carried out as an independent concentration determination. Enzymatic assay was done and analyzed on TLC (silica or PEI cellulose). Mass Spectrometry was determined if necessary.
  • This example illustrates the preparation of ATP-L1-Cy5 (TEA + ). This reaction does not work efficiently for the linker L2, L3, and L4. Although ethylene diamine was used as the linker, the preparation is well suited for other alkenyldiamines.
  • ATPNa 2 (12.7 ⁇ mol) was reacted with L1 (110 ⁇ mol) in the presence of EDC (110 ⁇ mol) at room temperature for 3 hours and pH was maintained at ⁇ 5.7 over the time.
  • the reaction was monitored by silica TLC.
  • the solution was adjusted to pH ⁇ 7.5 and rotavaped.
  • the pellet was dissolved in water (1.5 mL) and the sample was purified on HPLC (C18) with TEAA (100 mM, 200 mL)/CH 3 CN (100 mL) as elution system. If needed, the sample was split into two halves for two purifications.
  • the product fraction was lyophilized and the pellet was dissolved in HEPES buffer (5 mM, pH 8.5). Concentration was determined on UV spectrometer (200 ⁇ L*24 mM, 4.8 ⁇ mol). Yield was 37%. Its purity was evaluated on silica TLC.
  • APT-L1 (1 ⁇ mol) in NaHCO 3 buffer (1 M, pH9, 50 ⁇ L) and Cy5-NHS (1.3 ⁇ mol) in DMF (100 ⁇ L) were mixed and the resulting mixture was set on the shaker and reacted for 6 hr.
  • Water (1.5 mL) was added and the mixture is lyophilized.
  • a water solution (200 ⁇ L) of the pellet was passed through a 1*28 cm Sephadex G-25 column. The right fractions were collected and the combined solution was lyophilyzed.
  • the pellet was dissolved in TEAA (100 mM, 1.2 mL) and purified on HPLC (C18) with TEAA (100 mM, 200 mL)/CH 3 CN (100 mL) as elution system. The product fraction was collected and lyophilized. The pellet was dissolved in HEPES buffer (5 mM, pH8.5). Its concentration was determined on UV spec at 646 nm (1.1 mM*470 ⁇ L). Yield is 52%. Enzymatic assay was done and analyzed on TLC. MS was determined if necessary.

Abstract

Labeled nucleotide triphosphates are disclosed having a label bonded to the gamma phosphate of the nucleotide triphosphate. Methods for using the gamma phosphate labeled nucleotide are also disclosed where the gamma phosphate labeled nucleotide are used to attach the labeled gamma phosphate in a catalyzed (enzyme or man-made catalyst) reaction to a target biomolecule or to exchange a phosphate on a target biomolecule with a labeled gamme phosphate. Preferred target biomolecules are DNAs, RNAs, DNA/RNAs, PNA, polypeptide (e.g., proteins enzymes, protein, assemblages, etc.), sugars and polysaccharides or mixed biomolecules having two or more of DNAs, RNAs, DNA/RNAs, polypeptide, sugars and polysaccharides moieties.

Description

    RELATED APPLICATIONS
  • The present invention claims provisional priority to U.S. Provisional Patent Application Ser. No. 60/527,909, filed 8 Dec. 2003 and is a Continuation-in-part of U.S. patent application Ser. No. 09/901,782 filed 9 Jul. 2001, which claim provisional priority to U.S. Provisional Patent Application Ser. No. 60/216,594, filed 7 Jul. 2000, all of which are incorporated herein by reference.
    • BACKGROUND OF THE INVENTION
  • 1. Field of the Invention
  • The present invention relates to labeled nucleotides (NTPs), to method for making labeled NTPs and to method for using labeled NTPs such as a method of transferring the label to a target molecule.
  • More particularly, the present invention relates to gamma (γ) phosphate labeled nucleotides (NTPs), where the label includes a detectable tag and an optional linker interposed between the tag and the γ phosphate, to method for making γ phosphate labeled NTPs and to method for transferring the γ phosphate and the label to a target molecule including an oligonucleotide, a polynucleotide, a polypeptide, a protein, a monosaccharide, a polysaccharide, or mixtures or combinations thereof.
  • 2. Description of the Related Art
  • Many labeling procedures have been developed over the years for labeling nucleotide and polypeptide sequences with both radio and non-radio labels. These methods are routinely used to aid our understanding of molecular, cellular and intercellular dynamics and interaction. However, there are few relatively simple and versatile reagents and methods for attaching non-radio labels to both nucleotide sequence (DNA, RNA, DNA-RNA, etc.), peptide sequences (polypeptides, proteins, enzymes, macro-assembly, etc.) or mixtures or combinations thereof (ribozymes, peptide-nucleotide mixed sequences, etc.).
  • Fluorescence-based technologies are rapidly emerging as the methods of choice for nucleic acid labeling and detection. A variety of molecular biology techniques have benefitted from fluorescent innovations. For example, DNA Dye-Terminator Sequencing has been revolutionized by the creation of individually identifiable color-coded bases thereby making system automation possible. Assays such as Fluorescent In Situ Hybridization (FISH) and Quantitative/Real-time PCR (qPCR) also capitalized on the ability to monitor multiple fluorophores simultaneously. Furthermore, these new assays also make extensive use of the unique data provided by fluorophore-fluorophore interactions.
  • Of vital importance is the added benefit provided to personal health and safety by offering a robust alternative to the scientific community's dependence on radioactive labels. Specifically, radio-isotopic labeling entails a variety of negative aspects which include: (1) health hazards related to exposure, (2) extra licensing requirements and permits, (3) contained storage requirements, (4) additional requirements for waste removal, and (5) the limited lifetime of a radioactive probe. Fluorescent technologies eliminate both health risks and regulatory paperwork and provide a greater degree of flexibility in experimental design.
  • In addition to fluorescence, another important nucleic acid labeling method is the attachment of biotin to DNA or RNA. A variety of nucleic acid and protein capture applications are developed that exploit the highly specific interaction between biotin and streptavidin.
  • As an example, magnetic column separations where biotinylated oligonucleotides are captured by streptavidin coated microbeads enable a straightforward way to separate biotinylated from non-biotinylated molecules as described herein. This system permits the capture of DNA molecules, RNA molecules, DNA and RNA-binding proteins, and sequence specific transcripts.
  • The expanding need for higher throughput technologies suggests that the demand for both fluorescently labeled-ATPs and biotinylated-ATPs will increase. Thus, there is a need in the art for reagents to label both oligonucleotide and polynucleotides and polypeptide, proteins, enzymes, monosaccharides, polysaccharides, or mixtures or combinations thereof with labels having a readily detectable property.
    • DEFINITIONS
  • The term “tag” or “label” means an atom or molecule that has a detectable property and is capable of being attached to a γ phosphate of a nucleotide triphosphate.
  • The term “detectable property” means a physical or chemical property of a tag that is capable of independent detection and/or monitoring by an analytical technique after being attached to a target bio-molecule, i.e., the property is capable of being detected in the presence of the system under analysis. The property can be light emission after excitation, quenching of a known emission sites, electron spin, radio activity (electron emission, positron emission, alpha particle emission, etc.), nuclear spin, color, absorbance, near JR absorbance, UV absorbance, far UV absorbance, etc.
  • The term “analytical technique” means an analytical chemical or physical instrument for detecting and/or monitoring the property. Such instruments are based on spectroscopic analytical methods such as electron spin resonance spectrometry, nuclear magnetic resonance (NMR) spectrometry, UV and visible light spectrometry, far IR, IR and near IR spectrometry, X-ray spectrometry, etc.
  • The term “base” means any natural or synthetic purine or pyrimidine or nucleotide analogs (e.g., 7-deaza-deoxyguanine) that is capable of forming nucleotides and sequences thereof, including, without limitation, adenine (A), cytosine (C), guanine (G), inosine (I), thymine (T), uracil (U), pseudouridine (Y), xanthine (X), Orotidine (O), 5-bromouridine (B), thiouridine (S), 5,6-dihydrouridine (D) or the like. The natural or synthetic purines or pyrimidines may includes tags or may have been modified to have a particular detectable property such as enrichment with an NMR active nuclei.
  • The term “bonded to” means that chemical and/or physical interactions sufficient to maintain the label or tag at a given site of a target molecule. The chemical and/or physical interactions include, without limitation, covalent bonding (preferred), ionic bonding, hydrogen bonding, apolar bonding, attractive electrostatic interactions, dipole interactions, or any other electrical or quantum mechanical interaction sufficient in toto to maintain the polymerizing agent in a desired region of the substrate.
  • The term “nucleoside” means a base bonded to a sugar such as a five carbon sugar e.g., ribose.
  • The term “nucleotide” means nucleoside bonded to at least one phosphate.
  • The term NMP means a nucleotide monophosphate.
  • The term NDP means a nucleotide diphosphate.
  • The term NTP means a nucleotide triphosphate.
  • The term AMP means adenosine monophosphate.
  • The term ADP means adenosine diphosphate.
  • The term ATP means adenosine triphosphate.
  • The term TMP means thymidine nucleotide monophosphate.
  • The term TDP means thymidine nucleotide diphosphate.
  • The term TTP means thymidine nucleotide triphosphate.
  • The term CMP means cytidine nucleotide monophosphate.
  • The term CDP means cytidine nucleotide diphosphate.
  • The term CTP means cytidine nucleotide triphosphate.
  • The term GMP means guanine nucleotide monophosphate.
  • The term GDP means guanine nucleotide diphosphate.
  • The term GTP means guanine nucleotide triphosphate.
  • The term PNA means a peptide nucleic acid.
  • The term T-NTP means an NTP having an atomic and/or molecular tag bonded to the gamma phosphate of the NTP.
  • The term T-P means a phosphate (P) bonded to a tag (T).
  • The term L-NTP means an NTP having a linking reagent or linker bonded at its first end to the gamma phosphate of the NTP.
  • The term T-L-NTP means an L-NTP having a tag or label bonded to a second end of the linker.
  • The term T-L-P means a phosphate (P) bonded to the first end of the linker (L) and a tag (T) bonded to the second end of the linker (L).
  • The term ON means an oligonucleotide—a short sequence of nucleotides generally less than about 100 nucleotides, preferably less than about 50 nucleotides.
  • The term PN means a polynucleotide or nucleic acid—a long sequence of nucleotides generally over about 100 nucleotides.
  • The term PP means a polypeptide sequence, including at least two amino acids joined together via a peptide bond such as proteins, enzymes, protein assemblages, or the like.
  • The term PRN means a protein, which includes enzymes and protein assemblages.
  • The term AA means an amino acid either natural or synthetic and capable of forming peptide bonds.
  • The term T-ON means an oligonucleotide having an T-P bonded to the ON at either its 5′ or 3′ end.
  • The term T-L-ON means an oligonucleotide having an T-L-P bonded to the ON at either its 5′ or 3′ end.
  • The term T-PN means a polynucleotide having an T-P bonded to the PN at either its 5′ or 3′ end.
  • The term T-L-PN means a polynucleotide having an T-L-P bonded to the PN at either its 5′ or 3′ end.
  • The term T-P-PP means a polypeptide having an T-P bonded thereto.
  • The term T-L-P-PP means a polypeptide having an T-L-P bonded thereto.
  • The term T-P-PPN means a polypeptide having an T-P bonded thereto.
  • The term T-L-P-PPN means a polypeptide having an T-L-P bonded thereto.
  • The term PNPP means a biomolecule including both nucleosides, nucleotides, oligonucleotide or a polynucleotide and an amino acid, polypeptide or protein.
  • The term T-P-PNPP means an PNPP having an T-P bonded thereto.
  • The term T-L-P-PNPP means an PNPP having an T-L-P bonded thereto.
  • The term “MES buffer” means morpholinoethanesulfonic acid buffer.
  • The term TLC means thin layer chromatography.
    • SUMMARY OF THE INVENTION
  • The present invention provides labeled nucleotides (T-NTPs or T-L-NTPs), where the label (T-P or T-L-P) is readily transferable to nucleotide sequences, amino acid sequences, saccharides or compositions comprising a nucleotide, an amino acid and/or a sugar.
  • The present invention also provides a method for making labeled nucleotides, where the label includes an atomic and/or molecular tag bonded to the gamma phosphate of the nucleotide (T-NTP5) comprising the steps of contacting an NTP with an atomic and/or molecular tag precursor to form the T-NTP.
  • The present invention also provides a method for making labeled nucleotides, where the label includes an atomic and/or molecular tag bonded to one end of a linker which is bonded to the gamma phosphate of the nucleotide (T-L-NTPs) comprising the steps of contacting an NTP with a linker precursor to form a linker modified NTP (L-NTP). The L-NTP is then contacted with an atomic and/or molecular tag to form the T-L-NTP.
  • The present invention provides a method for labeling oligonucleotides including the step of contacting a T-NTP or T-L-NTP with an oligonucleotide (ON) in the presence of a catalyst to form a tagged oligonucleotide (T-ON or T-L-ON).
  • The present invention also provides a method for labeling polypeptide (PP) including the step of contacting a T-NTP or T-L-NTP with the PP in the presence of a catalyst to form a tagged polypeptide (T-P-PP or T-L-P-PP).
  • The present invention provides a method for labeling proteins (PRN) including the step of contacting a T-NTP or T-L-NTP with the PRN in the presence of a catalyst to form a tagged protein (T-P-PRN or T-L-P-PRN).
  • The present invention provides a method for labeling a biomolecule including both nucleosides, nucleotides, oligonucleotide or a polynucleotide and an amino acid, polypeptide or protein (PNPP) including the step of contacting a T-NTP or T-L-NTP with the PNPP in the presence of a catalyst to form a tagged biomolecule (T-P-PNPP or T-L-P-PNPP).
  • The present invention provides a set of ATP-linker-fluorescent dye molecules and ATP-linker-biotin molecules. The ATP molecules differ in the linker interposed between the ATP γ-phosphate and the fluorescent dye or biotin. The linkers differ in such properties as chain length, bulk or size, rigidity, and/or polarity. The synthesis of the precursor ATP-linker molecules provides intermediates that are used for attachment of a variety of individual fluorescent dyes, biotin or other binding molecules. Specific commercially available dyes were investigated to determine the efficiency of the transfer reaction in applications that have commercial potential. Such commercially available dyes includes; (1) Fluorescein [excitation λ: 495 nm, emission λ: 520 nm], (2) Cy3 [excitation λ: 550 nm, emission λ: 570 nm], (3) TAMRA [excitation λ: 555 nm, emission λ: 580 nm], (4) ROX [excitation λ: 578 nm, emission λ: 604 nm], and (5) Cy5 [excitation λ: 649 nm, emission λ: 670 nm]. Of course, the processes of this invention are capable of using a wide variety of γ-phosphate-labeled nucleotides such as ATP.
  • The present invention also provides for the use of T4 PNK or other phosphatase or kinase enzymes to 5′ end-label oligonucleotides with a variety of fluorophore, biotin or other binding molecules using labeled ATP molecules.
  • The present invention also provides a screening assay to examine each of the ATP-Linker-Fluorophore and ATP-Linker-Biotin molecules in 5′ end-labeling reactions with T4 PNK. The assay is based on the protocol for radio-isotopic 5′ end-labeling of oligonucleotides by T4 PNK. Fluorescent reactions are separated by PAGE and analysis is performed with a fluorescent gel scanning imager and software. Biotin reactions are examined via covalent attachment of biotinylated oligonucleotide to DE81 filter paper, incubation in streptavidin-alkaline phosphatase conjugate, and color development in nitroblue tetrazolium chloride (NBT)/5-bromo-4-chloro-3-indolyl-phosphate (BCIP) solution. Parameters that affect labeling efficiency include: (1) the incubation time, (2) labeling bias due to 5′ base sequence of the oligonucleotide, (3) concentration (ATP-Linker-Moiety) substrate, (4) T4 PNK amount and (5) oligonucleotide length.
  • Once a labeled-ATP is synthesized, it will then undergo quality control measures which entail: (1) TLC analysis to determine labeled-ATP synthesis integrity, (2) Spectrophotometric analysis to calculate labeling efficiency and, (3) Fluorometric analysis to verify the quantum yields of the ATP-labeled molecule and the fluorescently-labeled oligonucleotide.
  • The labeling efficiency are calculated by measuring the base:dye ratio on a NanoDrop spectrophotometer. This method uses Molecular Probes to calculate the labeling efficiency of their ULYSIS Nucleic Acid Labeling Kit (MP21650). The NanoDrop spectrophotometer is used to measure the absorbance of the nucleic acid-dye conjugate at 260 nm (λ260) and at a λmax for the dye (λdye). A measurement is also taken using the buffer alone at 260 nm and λmax and these numbers are subtracted from the raw sample absorbances values. To correct for the dye contribution at the 260 nm reading a correction factor is introduced (CF260). The correction factor is given by:

  • CF 260 =A 260 for the free dye/A max for the free dye
  • By applying the correction factor to the following equation, an accurate absorbance measurement can be obtained:

  • A base =A 260=(A dye ×CF 260)
  • Finally, the base:dye ratio is given by:

  • base:dye ratio=(A base×Ådye)/(A dye×εbase)
  • where ε is an extinction coefficient of the dye and is unique for each dye.
  • The present invention provides a method for screening kinases including the step of providing one immobilized substrate or a plurality of immobilized substrates on a support. The immobilized substrate(s) is then contacted with a solution including a kinase and a γ-phosphate labeled NTP for a first time and at a first temperature sufficient to determine whether a transfer of the labeled phosphate from the γ-phosphate labeled NTP to the substrate mediated by the kinase. Next, the support is washed for a second time and at a second temperature sufficient to remove the kinase and unreacted labeled NTP. After washing, the support is analyzed to determine whether the substrate have been labeled with the labeled phosphate from the γ-phosphate labeled NTP.
  • The present invention provides a method for screening kinase substrate candidates including the step of providing an immobilized kinase on a support. The support is then contacted with a solution including one or more kinase substrate candidates and a labeled NTP for the kinase. The substrate candidates are then analyzed for the presence or absence of the label.
  • The present invention provides a method including the step of contacting a solution comprising non-modified nucleotides or deoxynucleotides and modified nucleotides or deoxynucleotides, where the enzyme selectively degrades the non-modified nucleotides or deoxynucleotides.
  • The present invention provides a method for monitoring an NTP dependent reaction including the step of supplying to a system in which an NTP dependent reaction occurs, a γ-phosphate labeled nucleotide (NTP) and monitoring the label during the reaction.
    • DESCRIPTION OF THE DRAWINGS
  • The invention can be better understood with reference to the following detailed description together with the appended illustrative drawings in which like elements are numbered the same:
  • FIG. 1A depicts a general scheme for preparing gamma phosphate tagged NTPs;
  • FIG. 1B depicts a general scheme for preparing gamma phosphate tagged NTPs with a linker interposed between the gamma phosphate and the tag;
  • FIG. 1C a general scheme for preparing a gamma phosphate tagged ATP with a linker interposed between the gamma phosphate and the tag;
  • FIG. 2A a general scheme for preparing 3′ and/or 5′ tagged oligonucleotides using the tagged NTPs of this invention;
  • FIG. 2B another general scheme for preparing 3′ and/or 5′ tagged oligonucleotides using the tagged NTPs of this invention or for exchanging an untagged phosphate group for a tagged phosphate group;
  • FIG. 3 a general scheme for preparing phosphorylated polypeptide or proteins using the tagged NTPs of this invention;
  • FIG. 4 depicts an HPLC chromatogram of the reaction product ATP-EDA-ROX at 576 nm;
  • FIG. 5 depicts an HPLC chromatogram of the reaction product ATP-EDA-ROX at 259 nm;
  • FIG. 6 depicts a UV spectrum of ATP-EDA-ROX;
  • FIG. 7A depict TLC monitoring of reactions of ATP and ATP-EDA-Rox with CLAP;
  • FIG. 8 depicts PAGE monitoring of T4 PNK 5′ end labeling of a TOP oligonucleotide using ATP-EDA-ROX;
  • FIG. 9 depicts gel plates of T4 PNK 5′ end labeling of a TOP oligonucleotide using ATP-EDA-ROX;
  • FIG. 10 depicts a plot of CNT vs. pmol for ROX-T-Top;
  • FIG. 11 depicts a plot of CNT vs. ng Top oligonucleotide and different T4 PNK concentrations;
  • FIGS. 12A&B depict plots of T4 PNK 5′ end-labeling timecourse using ATP-L1-ROX;
  • FIGS. 13A&B depict plots of T4 PNK concentration effects on 5′ end-labeling using ATP-L1-ROX;
  • FIG. 14 depicts plots of T4 PNK 5′ end-labeling t using ATP-L1-ROX in the presence of PEG 8000;
  • FIGS. 15A&B depict plots of linker effects on T4 PNK 5′ end-labeling using ROX labeled ATPs;
  • FIGS. 16A&B depict extension reactions of a ROX labeled oligonucleotide;
  • FIGS. 17A&B depict the relatively activity of an exonuclease against a 5′ end-labeled oligonucleotide and an un-labeled oligonucleotide;
  • FIG. 18 depicts three developed filter paper disks showing T4 PNK 5′ end-labeling using ATP-L1-biotin compared to a negative control and a synthetic biotin labeled oligonucleotide; and
  • FIG. 19 depicts 5′ end-labeling an RNA oligonucleotide with ATP-L2-Cy3 and hybridization to a DNA microarray.
    •  DETAILED DESCRIPTION OF THE INVENTION
  • The inventors have found a versatile, inexpensive and efficient technique for labeling nucleic acids, polypeptides and/or biomolecules including both nucleic acids and amino acids with atomic and/or molecular tags having a detectable property and reagents for accomplishing the tagging reaction. The techniques involves the preparation of labeled NTPs capable of transferring their label to a target nucleotide, polypeptide, saccharide, and/or a biomolecule including one or combinations of a nucleoside, nucleotide, oligonucleotide or a polynucleotide and an amino acid, polypeptide or protein, combinations of a nucleoside, nucleotide, oligonucleotide or a polynucleotide and a monosaccharide or polysaccharide and combinations of an amino acid, polypeptide or protein and a monosaccharide or polysaccharide. The inventors have found that novel fluorescently-tagged ATP molecules and biotin-tagged ATP molecules can be prepare and used by T4 Polynucleotide Kinase (T4 PNK) in gamma-phosphate transfer reactions to the 5′ end of oligonucleotides, giving the end-user the ability to quickly label an oligonucleotide with a desired fluorophore or a binding molecule such as biotin.
  • The present invention relates to the modification of the gamma-phosphate of a nucleotide, preferably ATP and GTP, to form gamma-phosphate labeled nucleotides, which can subsequently be used to transfer the labeled gamma phosphate moiety to a target substrate such as DNA, RNA, RNA/DNA, protein, polypeptide, sugars, polysaccharides or biomolecules including DNA, RNA, polypeptides, sugars or polysaccharides. The label can include an atomic and/or molecular tag having a separately detectable property such as a fluorescent tag, a biotinylated tag, electrochemical tag, lanthanide or actinide series containing tag, radical tag or paramagnetic tag, nmr tag (13C, 15N, or other isotopically enriched atom or molecular tags) or any other tag capable of detection. The label can also include a linker interposed molecularly between the gamma-phosphate and the tag, which may influence the efficiency of the transfer reaction, and this efficiency may be specific for the transfer reaction under study. Additionally, the transfer reaction may be influenced by the identity of the tag.
  • The present invention broadly relates to a composition for efficiently labeling oligonucleotide, polynucleotides, polypeptide, proteins and/or biomolecules including both a nucleoside, nucleotide, oligonucleotide, and/or polynucleotide and a polypeptide and/or protein, where the composition includes an NTP, a linker and a tag, where the linker is bonded at one end to a gamma phosphate of the NTP and at the other end to the tag.
  • The present invention broadly relates to a method for efficiently labeling oligonucleotide, polynucleotides, polypeptide, proteins and/or biomolecules including both a nucleoside, nucleotide, oligonucleotide, and/or polynucleotide and a polypeptide and/or protein, where the method includes the step of contacting oligonucleotide, polynucleotides, polypeptide, proteins and/or biomolecules including both a nucleoside, nucleotide, oligonucleotide, and/or polynucleotide and a polypeptide and/or protein with a labeled NTP (T-L-NTP) to form a labeled oligonucleotide, polynucleotides, polypeptide, proteins and/or biomolecules including both a nucleoside, nucleotide, oligonucleotide, and/or polynucleotide and a polypeptide and/or protein in the presence of a catalyst, where the contacting transfers the gamma phosphate, linker and tag to the oligonucleotide, polynucleotides, polypeptide, proteins and/or biomolecules including both a nucleoside, nucleotide, oligonucleotide, and/or polynucleotide and a polypeptide and/or protein.
  • For decades T4 polynucleotide kinase (T4 PNK) has been essential for phosphorylating, either radioactively or non-radioactively, the 5′-end of oligonucleotides for subsequent use in a variety of molecular biology applications. T4 PNK has two distinct functions: (1) transfer of the γ-phosphate of adenosine triphosphate (ATP) or other nucleoside triphosphates to the 5′ hydroxyl end of a polynucleotide and (2) 3′-phosphatase activity that is independent of ATP and able to hydrolyze 2′,3′-cyclic phosphodiesters.
  • The dual functionality of the enzyme can be explained by its physiological role within the T4 bacteriophage life cycle. Upon infection by T4, some strains of Escherichia coli have the capability to initiate a suicide defense mechanism that causes the specific cleavage of bacterial lysine tRNA (tRNAlys) and results in the abrogation of protein synthesis. In response, the phage initiates tRNAlys repair via the bacteriophage encoded T4 PNK and T4 RNA ligase. T4 PNK specifically phosphorylates the 5′-hydroxyl group and simultaneously reprocesses the 3′ end by opening the 2′,3′-cyclic phosphate and then removing the 3′-phosphate. This results in a suitable substrate for T4 RNA ligase which can then repair the tRNAlys lesion and allow continued phage propagation.
  • Both of T4 PNK's actions, phosphorylation and dephosphorylation, have been exploited by molecular biologists for the purpose of radio-labeling nucleic acids for use as hybridization probes, sequencing primers, transcript mapping, and for the cold phosphorylation of DNA ends for cloning. However, only limited attempts have been made to develop non-radioactive, gamma-labeled ATP substrates that could be used for 5′ end-labeling nucleic acids.
  • Currently, an oligonucleotide is typically labeled during its chemical synthesis and the label may be directed to the 3′ end, 5′ end, or at an internal position. Additionally, a label may be added post-synthesis by polymerase incorporation of a base-labeled dNTP onto the 3′ end of a duplexed molecule or via terminal deoxy transfer activity (TdT). At present, chemical attachment of a dye during the process of oligonucleotide synthesis or via amino-chemistry after synthesis are the only methods used to add a fluorescent moiety to the oligonuleotides 5′-end. The necessity of chemically labeling the 5′-end can be explained by the inherent directionality of DNA synthesis by a polymerase. To initiate DNA synthesis, a polymerase must be able to access the 3′-end of the DNA at the primer-template junction. Fluorescent dyes that have been attached at the 3′-nucleotide's base, sugar, or alpha phosphate can severely alter the conformation of the DNA, subsequently inhibiting or dramatically reducing DNA synthesis efficiency. Explanations for the affect that fluorophore presence has on DNA structural perturbations involve the notable size of the fluorescent dye and its hydrophobic nature.
  • Although fluorescence is rapidly emerging as the technology of choice, it can be cost prohibitive. For example, a standard 25 base oligonucleotide that is fluorescently 5′-end-labeled using current methods can range in price from $98.75 to upwards of $600 at the 250 nmole scale, depending on the choice of fluorophore, its place of attachment, and the level of purity desired. The time needed for the labeled oligonucleotide order to arrive can range between 3 to 10 days, depending on the complexity of the synthesis. Additionally, inefficiencies in the coupling of the fluorophore to the oligonucleotide may result in a large fraction of the product being unlabeled, and require its re-synthesis resulting in further delay.
  • The Labeled ATPs of this invention are ideally suited for used in the following assays and detection procedures: absorbance assays, fluorescence intensity assays, fluorescence polarization assays, time resolved fluorescence assays, fluorescence resonance energy transfer (FRET) assays, or other assays.
  • The present invention also relates to a kit adapted to 5′ end-label an target oligonucleotide with a fluorophore or biotin or other binding molecules. The reagents of this invention can be used with either previously synthesized or newly synthesized oligonucleotides in labeling reactions and tailor experiments on the fly by selecting an appropriate fluorescently labeled ATP or biotin labeled ATP.
    • Suitable Reagents Listings
  • Suitable atomic tags for use in this invention include, without limitation, any atomic element amenable to attachment to a specific site in a target or dNTP, especially Europium shift agents, NMR active atoms or the like.
  • Suitable molecular tags for use in this invention include, without limitation, any molecule amenable to attachment to a specific site of a target PN or PP, such as fluorescent molecules, quenching molecules, Europium shift agents, NMR active molecules, Raman active molecules, near IR active molecules, or the like.
  • Suitable NMR tags include any active NMR nuclei. Exemplary examples of NMR active nuclei include, without limitation, 1H, 13C, 15N, 19F, 29Si, 57Fe, 103Rh, etc.
  • Suitable molecular tags for use in this invention include, without limitation, any molecule amenable to attachment to a specific site in a target or dNTP, especially fluorescent dyes or molecules that quench the fluorescence of the fluorescent dyes, paramagnetic molecules such as those disclosed in U.S. Pat. Nos: 6,458,758; 6,436,640; 6,410,255; 6,316,198; 6,303,315; 5,840,701; 5,833,601; 5,824,781; 5,817,632; 5,807,831; 5,804,561; 5,741,893; 5,725,8395; 706,805; and 5,494,030, incorporated herein by reference, electrochemical probes or tags, or other similar molecular tags or probes. Fluorescent dyes include, without limitation, such as d-Rhodamine acceptor dyes including Cy5, dichloro[R110], dichloro[R6G], dichloro[TAMRA], dichloro[ROX] or the like, fluorescein donor dye including fluorescein, 6-FAM, or the like; Acridine including Acridine orange, Acridine yellow, Proflavin, pH 7, or the like; Aromatic Hydrocarbon including 2-Methylbenzoxazole, Ethyl p-dimethylaminobenzoate, Phenol, Pyrrole, benzene, toluene, or the like; Arylmethine Dyes including Auramine O, Crystal violet, H2O, Crystal violet, glycerol, Malachite Green or the like; Coumarin dyes including 7-Methoxycoumarin-4-acetic acid, Coumarin 1, Coumarin 30, Coumarin 314, Coumarin 343, Coumarin 6 or the like; Cyanine Dye including 1,1′-diethyl-2,2′-cyanine iodide, Cryptocyanine, Indocarbocyanine (C3)dye, Indodicarbocyanine (C5)dye, Indotricarbocyanine (C7)dye, Oxacarbocyanine (C3)dye, Oxadicarbocyanine (C5)dye, Oxatricarbocyanine (C7)dye, Pinacyanol iodide, Stains all, Thiacarbocyanine (C3)dye, ethanol, Thiacarbocyanine (C3)dye, n-propanol, Thiadicarbocyanine (C5)dye, Thiatricarbocyanine (C7)dye, or the like; Dipyrrin dyes including N,N′-Difluoroboryl-1,9-dimethyl-5-(4-iodophenyl)-dipyrrin, N,N′-Difluoroboryl-1,9-dimethyl-5-[(4-(2-trimethylsilylethynyl), N,N′-Difluoroboryl-1,9-dimethyl-5-phenydipyrrin, or the like; Merocyanines including 4-(dicyanomethylene)-2-methyl-6-(p-dimethylaminostyryl)-4H-pyran (DCM), acetonitrile, 4-(dicyanomethylene)-2-methyl-6-(p-dimethylaminostyryl)-4H-pyran (DCM), methanol, 4-Dimethylamino-4′-nitrostilbene, Merocyanine 540, or the like; Miscellaneous Dye including 4′,6-Diamidino-2-phenylindole (DAPI), 4′,6-Diamidino-2-phenylindole (DAPI), dimethylsulfoxide, 7-Benzylamino-4-nitrobenz-2-oxa-1,3-diazole, Dansyl glycine, H2O, Dansyl glycine, dioxane, Hoechst 33258, DMF, Hoechst 33258, H2O, Lucifer yellow CH, Piroxicam, Quinine sulfate, 0.05 M H2SO4, Quinine sulfate, 0.5 M H2SO4, Squarylium dye III, or the like; Oligophenylenes including 2,5-Diphenyloxazole (PPO), Biphenyl, POPOP, p-Quaterphenyl, p-Terphenyl, or the like; Oxazines including Cresyl violet perchlorate, Nile Blue, methanol, Nile Red, Nile blue, ethanol, Oxazine 1, Oxazine 170, or the like; Polycyclic Aromatic Hydrocarbons including 9,10-Bis(phenylethynyl)anthracene, 9,10-Diphenylanthracene, Anthracene, Naphthalene, Perylene, Pyrene, or the like; polyene/polyynes including 1,2-diphenylacetylene, 1,4-diphenylbutadiene, 1,4-diphenylbutadiyne, 1,6-Diphenylhexatriene, Beta-carotene, Stilbene, or the like; Redox-active Chromophores including Anthraquinone, Azobenzene, Benzoquinone, Ferrocene, Riboflavin, Tris(2,2′-bipyridypruthenium(II), Tetrapyrrole, Bilirubin, Chlorophyll a, diethyl ether, Chlorophyll a, methanol, Chlorophyll b, Diprotonated-tetraphenylporphyrin, Hematin, Magnesium octaethylporphyrin, Magnesium octaethylporphyrin (MgOEP), Magnesium phthalocyanine (MgPc), PrOH, Magnesium phthalocyanine (MgPc), pyridine, Magnesium tetramesitylporphyrin (MgTMP), Magnesium tetraphenylporphyrin (MgTPP), Octaethylporphyrin, Phthalocyanine (Pc), Porphin, Rox, TAMRA, Tetra-t-butylazaporphine, Tetra-t-butylnaphthalocyanine, Tetrakis(2,6-dichlorophenyl)porphyrin, Tetrakis(o-aminophenyl)porphyrin, Tetramesitylporphyrin (TMP), Tetraphenylporphyrin (TPP), Vitamin B12, Zinc octaethylporphyrin (ZnOEP), Zinc phthalocyanine (ZnPc), pyridine, Zinc tetramesitylporphyrin (ZnTMP), Zinc tetramesitylporphyrin radical cation, Zinc tetraphenylporphyrin (ZnTPP), or the like; Xanthenes including Eosin Y, Fluorescein, basic ethanol, Fluorescein, ethanol, Rhodamine 123, Rhodamine 6G, Rhodamine B, Rose bengal, Sulforhodamine 101, or the like; or mixtures or combination thereof or synthetic derivatives thereof or FRET fluorophore-quencher pairs including DLO-FB1 (5′-FAM/3′-BHQ-1) DLO-TEB1 (5′-TET/3′-BHQ-1), DLO-JB1 (5′-JOE/3′-BHQ-1), DLO-1-HB1 (5′-HEX/3′-BHQ-1), DLO-C3B2 (5′-Cy3/3′-BHQ-2), DLO-TAB2 (5′-TAMRA/3′-BHQ-2), DLO-RB2 (5′-ROX/3′-BHQ-2), DLO-C5B3 (5′-Cy5/3′-BHQ-3), DLO-C55B3 (5′-Cy5.5/3′-BHQ-3), MBO-FB1(5′-FAM/3′-BHQ-1), MBO-TEB1 (5′-TET/3′-BHQ-1), MBO-JB1 (5-JOE/3′-BHQ-1), MBO-HB1 (5′-HEX/3′-BHQ-1), MBO-C3B2 (5′-Cy3/3′-BHQ-2), MBO-TAB2 (5′-TAMRA/3′-BHQ-2), MBO-RB2 (5′-ROX/3′-BHQ-2); MBO-C5B3 (5′-Cy5/3′-BHQ-3), MBO-C55B3 (5′-Cy5.5/3′-BHQ-3) or similar FRET pairs available from Biosearch Technologies, Inc. of Novato, Calif., tags with nmr active groups, tags with spectral features that can be easily identified such as IR, near IR, far IR, visible UV, far UV or the like.
  • Suitable phosphorylation catalysts or enzymes are any naturally occurring, human modified or synthetic molecule or molecular assembly that can phosphorylate 5′ and/or 3′ hydroxy terminated oligonucleotides or polynucleotides, phosphorylate peptide or proteins or that can exchange an existing phosphate with a labeled phosphate of this invention. Exemplary examples include kinases. Preferred kinases include, without limitation, T4 Polynucleotide Kinase, Abl (mouse), Abl, Abl (T315I), ALK, AMPK (rat), Arg (mouse), Aurora-A, Axl, Blk (mouse), Bmx, BTK, CaMKII (rat), CaMKIV, CDK1/cyclinB, CDK2/cyclinA, CDK2/cyclinE, CDK3/cyclinE, CDK5/p35, CDK6/cyclinD3, CDK7/cyclinH/MAT1, CHK1, CHK2, CK1 (yeast), CK1δ, CK2, c-RAF, CSK, cSRC, EGFR, EphB2, EphB4, Fes, FGFR3, Flt3, Fms, Fyn, GSK3α, GSK3β, IGF-1R, IKKα, IKKβ, IR, JNK1α1, JNK2α2, JNK3, Lck, Lyn, Lyn (mouse), MAPK1, MAPK2, MAPK2 (mouse), MAPKAP-K2, MEK1, Met, MKK4 (mouse), MKK6, MKK7β, MSK1, MST2, NEK2, p70S6K, PAR-1Bα, PDGFRα, PDGFRβ, PDK1, PAK2, PKA (bovine), PKA, PKBα, PKBβ, PKBγ, PKCα, PKCβII, PKCγ, PKCδ, PKCε, PKCη, PKCl, PKCμ, PKCθ, PKCζ, PKD2, PRAK, PRK2, ROCK-II, ROCK-II (mouse), Ros, Rsk1, Rsk1 (rat), Rsk2, Rsk3, SAPK2α, SAPK2β, SAPK3, SAPK4, SGK, Syk, Tie2, TrkB, Yes, ZAP, or mixtures or combinations thereof or the like.
  • Suitable enzymes that can be used to transfer the labeled γ-phosphate of the γ-phosphate labeled nucleotides of this invention or for which the γ-phosphate labeled nucleotides of this invention can be used in monitoring the enzyme activity include, without limitation, ID 1.2.1.30 Aryl-aldehyde dehydrogenase (NADP+) (CA: An aromatic aldehyde+NADP(+)+AMP+diphosphate+H(2)O=an aromatic acid+NADPH+ATP); ID 1.3.99.15 Benzoyl-CoA reductase. (CA: Benzoyl-CoA+reduced acceptor+2 ATP=cyclohexa-1,5-diene-1-carbonyl-CoA+acceptor+2 ADP+2 phosphate); ID 1.13.12.7 Photinus-luciferin 4-monooxygenase (ATP-hydrolyzing) (CA: Photinus luciferin+O(2)+ATP=oxidized Photinus luciferin+CO(2)+H(2)O+AMP+diphosphate+light); ID 1.18.6.1 Nitrogenase. (CA: 8 reduced ferredoxin+8 H(+)+N(2)+16 ATP=8 oxidized ferredoxin+2 NH(3)+16 ADP+16 phosphate); ID 1.19.6.1 Nitrogenase (flavodoxin). (CA: 8 reduced flavodoxin(HQ)+8 H(+)+N(2)+16 ATP=8 oxidized flavodoxin(SQ)+2 NH(3)+16 ADP+16 phosphate); ID 2.3.3.8 ATP citrate synthase. (CA: ADP+phosphate+acetyl-CoA+oxaloacetate=ATP+citrate+CoA); ID 2.4.2.17 ATP phosphoribosyltransferase. (CA: 1-(5-phospho-D-ribosyl)-ATP+diphosphate=ATP+5-phospho-alpha-D-ribose 1-diphosphate); ID 2.5.1.6 Methionine adenosyltransferase. (CA: ATP+L-methionine+H(2)O=phosphate+diphosphate+S-adenosyl-L-methionine); ID 2.5.1.17 Cob(I)alamin adenosyltransferase. (CA: ATP+cob(I)alamin+H(2)O=phosphate+diphosphate+adenosylcobalamin); ID 2.6.99.1 dATP(dGTP)—DNA purine transferase. (CA: dATP+depurinated DNA=ribose triphosphate+DNA); ID 2.7.1.1 Hexokinase. (CA: ATP+D-hexose=ADP+D-hexose 6-phosphate); ID 2.7.1.2 Glucokinase. (CA: ATP+D-glucose=ADP+D-glucose 6-phosphate); ID 2.7.1.3 Ketohexokinase. (CA: ATP+D-fructose=ADP+D-fructose 1-phosphate); ID 2.7.1.4 Fructokinase. (CA: ATP+D-fructose=ADP+D-fructose 6-phosphate); ID 2.7.1.5 Rhamnulokinase. (CA: ATP+L-rhamnulose=ADP+L-rhamnulose 1-phosphate); ID 2.7.1.6 Galactokinase. (CA: ATP+D-galactose=ADP+D-galactose 1-phosphate); ID 2.7.1.7 Mannokinase. (CA: ATP+D-mannose=ADP+D-mannose 6-phosphate); ID 2.7.1.8 Glucosamine kinase. (CA: ATP+glucosamine=ADP+glucosamine phosphate); ID 2.7.1.10 Phosphoglucokinase. (CA: ATP+D-fructose 1-phosphate=ADP+D-fructose 1,6-bisphosphate); ID 2.7.1.11 6-phosphofructokinase. (CA: ATP+D-fructose 6-phosphate=ADP+D-fructose 1,6-bisphosphate); ID 2.7.1.12 Gluconokinase. (CA: ATP+D-gluconate=ADP+6-phospho-D-gluconate); ID 2.7.1.13 Dehydogluconokinase. (CA: ATP+2-dehydro-D-gluconate=ADP+6-phospho-2-dehydro-D-gluconate); ID 2.7.1.14 Sedoheptulokinase. (CA: ATP+sedoheptulose=ADP+sedoheptulose 7-phosphate); ID 2.7.1.15 Ribokinase. (CA: ATP+D-ribose=ADP+D-ribose 5-phosphate); ID 2.7.1.16 L-ribulokinase. (CA: ATP+L-ribulose=ADP+L-ribulose 5-phosphate); ID 2.7.1.17 Xylulokinase. (CA: ATP+D-xylulose=ADP+D-xylulose 5-phosphate); ID 2.7.1.18 Phosphoribokinase. (CA: ATP+D-ribose 5-phosphate=ADP+D-ribose 1,5-bisphosphate); ID 2.7.1.19 Phosphoribulokinase. (CA: ATP+D-ribulose 5-phosphate=ADP+D-ribulose 1,5-bisphosphate); ID 2.7.1.20 Adenosine kinase. (CA: ATP+adenosine=ADP+AMP); ID 2.7.1.21 Thymidine kinase. (CA: ATP+thymidine=ADP+thymidine 5′-phosphate); ID 2.7.1.22 Ribosylnicotinamide kinase. (CA: ATP+N-ribosylnicotinamide=ADP+nicotinamide ribonucleotide); ID 2.7.1.23 NAD(+) kinase. (CA: ATP+NAD(+)=ADP+NADP(+)); ID 2.7.1.24 Dephospho-CoA kinase. (CA: ATP+dephospho-CoA=ADP+CoA); ID 2.7.1.25 Adenylylsulfate kinase. (CA: ATP+adenylylsulfate=ADP+3′-phosphoadenylylsulfate); ID 2.7.1.26 Riboflavin kinase. (CA: ATP+riboflavin=ADP+FMN); ID 2.7.1.27 Erythritol kinase. (CA: ATP+erythritol=ADP+D-erythritol 4-phosphate); ID 2.7.1.28 Triokinase. (CA: ATP+D-glyceraldehyde=ADP+D-glyceraldehyde 3-phosphate); ID 2.7.1.29 Glycerone kinase. (CA: ATP+glycerone=ADP+glycerone phosphate); ID 2.7.1.30 Glycerol kinase. (CA: ATP+glycerol=ADP+glycerol 3-phosphate); ID 2.7.1.31 Glycerate kinase. (CA: ATP+(R)-glycerate=ADP+3-phospho-(R)-glycerate); ID 2.7.1.32 Choline kinase. (CA: ATP+choline=ADP+O-phosphocholine); ID 2.7.1.33 Pantothenate kinase. (CA: ATP+pantothenate=ADP+D-4′-phosphopantothenate); ID 2.7.1.34 Pantetheine kinase. (CA: ATP+pantetheine=ADP+pantetheine 4′-phosphate); ID 2.7.1.35 Pyridoxal kinase. (CA: ATP+pyridoxal=ADP+pyridoxal 5′-phosphate); ID 2.7.1.36 Mevalonate kinase. (CA: ATP+(R)-mevalonate=ADP+(R)-5-phosphomevalonate); ID 2.7.1.37 Protein kinase. (CA: ATP+a protein=ADP+a phosphoprotein); ID 2.7.1.38 Phosphorylase kinase. (CA: 4 ATP+2 phosphorylase B=4 ADP+phosphorylase A); ID 2.7.1.39 Homoserine kinase. (CA: ATP+L-homoserine=ADP+O-phospho-L-homoserine); ID 2.7.1.40 Pyruvate kinase. (CA: ATP+pyruvate=ADP+phosphoenolpyruvate); ID 2.7.1.43 Glucuronokinase. (CA: ATP+D-glucuronate=ADP+1-phospho-alpha-D-glucuronate); ID 2.7.1.44 Galacturonokinase. (CA: ATP+D-galacturonate=ADP+1-phospho-alpha-D-galacturonate); ID 2.7.1.45 2-dehydro-3-deoxygluconokinase. (CA: ATP+2-dehydro-3-deoxy-D-gluconate=ADP+6-phospho-2-dehydro-3-deoxy-D-gluconate); ID 2.7.1.46 L-arabinokinase. (CA: ATP+L-arabinose=ADP+L-arabinose 1-phosphate); ID 2.7.1.47 D-ribulokinase. (CA: ATP+D-ribulose=ADP+D-ribulose 5-phosphate); ID 2.7.1.48 Uridine kinase. (CA: ATP+uridine=ADP+UMP); ID 2.7.1.49 Hydroxymethylpyrimidine kinase. (CA: ATP+4-amino-2-methyl-5-hydroxymethylpyrimidine=ADP+4-amino-2-methyl-5-phosphomethylpyrimidine); ID 2.7.1.50 Hydroxyethylthiazole kinase. (CA: ATP+4-methyl-5-(2-hydroxyethyl)-thiazole=ADP+4-methyl-5-(2-phosphoethyl)-thiazole); ID 2.7.1.51 L-fuculokinase. (CA: ATP+L-fuculose=ADP+L-fuculose 1-phosphate); ID 2.7.1.52 Fucokinase. (CA: ATP+6-deoxy-L-galactose=ADP+6-deoxy-L-galactose 1-phosphate); ID 2.7.1.53 L-xylulokinase. (CA: ATP+L-xylulose=ADP+L-xylulose 5-phosphate); ID 2.7.1.54 D-arabinokinase. (CA: ATP+D-arabinose=ADP+D-arabinose 5-phosphate); ID 2.7.1.55 Allose kinase. (CA: ATP+D-allose=ADP+D-allose 6-phosphate); ID 2.7.1.56 1-phosphofructokinase. (CA: ATP+D-fructose 1-phosphate=ADP+D-fructose 1,6-bisphosphate); ID 2.7.1.58 2-dehydro-3-deoxygalactonokinase. (CA: ATP+2-dehydro-3-deoxy-D-galactonate=ADP+2-dehydro-3-deoxy-D-galactonate 6-phosphate); ID 2.7.1.59 N-acetylglucosamine kinase. (CA: ATP+N-acetyl-D-glucosamine=ADP+N-acetyl-D-glucosamine 6-phosphate); ID 2.7.1.60 N-acylmannosamine kinase. (CA: ATP+N-acyl-D-mannosamine=ADP+N-acyl-D-mannosamine 6-phosphate); ID 2.7.1.64 Inositol 3-kinase. (CA: ATP+myo-inositol=ADP+1D-myo-inositol 3-phosphate); ID 2.7.1.65 Scyllo-inosamine kinase. (CA: ATP+1-amino-1-deoxy-scyllo-inositol=ADP+1-amino-1-deoxy-scyllo-inositol 4-phosphate); ID 2.7.1.66 Undecaprenol kinase. (CA: ATP+undecaprenol=ADP+undecaprenyl phosphate); ID 2.7.1.67 1-phosphatidylinositol 4-kinase. (CA: ATP+1-phosphatidyl-1D-myo-inositol=ADP+1-phosphatidyl-1D-myo-inositol 4-phosphate); ID 2.7.1.68 1-phosphatidylinositol-4-phosphate 5-kinase. (CA: ATP+1-phosphatidyl-1D-myo-inositol 4-phosphate=ADP+1-phosphatidyl-1D-myo-inositol 4,5-bisphosphate); ID 2.7.1.70 Protamine kinase. (CA: ATP+[protamine]=ADP+[protamine]O-phospho-L-serine); ID 2.7.1.71 Shikimate kinase. (CA: ATP+shikimate=ADP+shikimate 3-phosphate); ID 2.7.1.72 Streptomycin 6-kinase. (CA: ATP+streptomycin=ADP+streptomycin 6-phosphate); ID 2.7.1.73 Inosine kinase. (CA: ATP+inosine=ADP+IMP); ID 2.7.1.76 Deoxyadenosine kinase. (CA: ATP+deoxyadenosine=ADP+dAMP); ID 2.7.1.78 Polynucleotide 5′-hydroxyl-kinase. (CA: ATP+5′-dephospho-DNA=ADP+5′-phospho-DNA); ID 2.7.1.82 Ethanolamine kinase. (CA: ATP+ethanolamine=ADP+O-phosphoethanolamine); ID 2.7.1.83 Pseudouridine kinase. (CA: ATP+pseudouridine=ADP+pseudouridine 5′-phosphate); ID 2.7.1.84 Alkylglycerone kinase. (CA: ATP+O-alkylglycerone=ADP+O-alkylglycerone phosphate); ID 2.7.1.85 Beta-glucoside kinase. (CA: ATP+cellobiose=ADP+6-phospho-beta-D-glucosyl-(1,4)-D-glucose); ID 2.7.1.86 NADH kinase. (CA: ATP+NADH=ADP+NADPH); ID 2.7.1.87 Streptomycin 3″-kinase. (CA: ATP+streptomycin=ADP+streptomycin 3″-phosphate); ID 2.7.1.88 Dihydrostreptomycin-6-phosphate 3′-alpha-kinase. (CA: ATP+dihydrostreptomycin 6-phosphate=ADP+dihydrostreptomycin-3′-alpha-6-bisphosphate); ID 2.7.1.89 Thiamine kinase. (CA: ATP+thiamine=ADP+thiamine phosphate); ID 2.7.1.91 Sphinganine kinase. (CA: ATP+sphinganine=ADP+sphinganine 1-phosphate); ID 2.7.1.92 5-dehydro-2-deoxygluconokinase. (CA: ATP+5-dehydro-2-deoxy-D-gluconate=ADP+6-phospho-5-dehydro-2-deoxy-D-gluconate); ID 2.7.1.93 Alkylglycerol kinase. (CA: ATP+1-O-alkyl-sn-glycerol=ADP+1-O-alkyl-sn-glycerol 3-phosphate); ID 2.7.1.94 Acylglycerol kinase. (CA: ATP+acylglycerol=ADP+acyl-sn-glycerol 3-phosphate); ID 2.7.1.95 Kanamycin kinase. (CA: ATP+kanamycin=ADP+kanamycin 3′-phosphate); ID 2.7.1.99 [Pyruvate dehydrogenase(lipoamide)]kinase. (CA: ATP+[pyruvate dehydrogenase (lipoamide)]=ADP+[pyruvate dehydrogenase (lipoamide)]phosphate); ID 2.7.1.100 5-methylthioribose kinase. (CA: ATP+S(5)-methyl-5-thio-D-ribose=ADP+S(5)-methyl-5-thio-D-ribose 1-phosphate); ID 2.7.1.101 Tagatose kinase. (CA: ATP+D-tagatose=ADP+D-tagatose 6-phosphate); ID 2.7.1.102 Hamamelose kinase. (CA: ATP+D-hamamelose=ADP+D-hamamelose 2′-phosphate); ID 2.7.1.103 Viomycin kinase. (CA: ATP+viomycin=ADP+O-phosphoviomycin); ID 2.7.1.105 6-phosphofructo-2-kinase. (CA: ATP+D-fructose 6-phosphate=ADP+D-fructose 2,6-bisphosphate); ID 2.7.1.107 Diacylglycerol kinase. (CA: ATP+1,2-diacylglycerol=ADP+1,2-diacylglycerol 3-phosphate); ID 2.7.1.109 [Hydroxymethylglutaryl-CoA reductase(NADPH)]kinase. (CA: ATP+[3-hydroxy-3-methylglutaryl-CoA reductase (NADPH)]=ADP+[3-hydroxy-3-methylglutaryl-CoA reductase (NADPH)]phosphate); ID 2.7.1.110 Dephospho-[reductase kinase]kinase. (CA: ATP+dephospho[[3-hydroxy-3-methylglutaryl-CoA reductase (NADPH)]kinase]=ADP+[[3-hydroxy-3-methylglutaryl-CoA reductase (NADPH)]kinase]); ID 2.7.1.112 Protein-tyrosine kinase. (CA: ATP+a protein tyrosine=ADP+protein tyrosine phosphate); ID 2.7.1.113 Deoxyguanosine kinase. (CA: ATP+deoxyguanosine=ADP+dGMP); ID 2.7.1.115 [3-methyl-2-oxobutanoate dehydrogenase (lipoamide)]kinase. (CA: ATP+[3-methyl-2-oxobutanoate dehydrogenase (lipoamide)]=ADP+[3-methyl-2-oxobutanoate dehydrogenase (lipoamide)]phosphate); ID 2.7.1.116 [Isocitrate dehydrogenase (NADP+)]kinase. (CA: ATP+[isocitrate dehydrogenase (NADP+)]=ADP+[isocitrate dehydrogenase (NADP+)]phosphate); ID 2.7.1.117 [Myosin light-chain]kinase. (CA: ATP+[myosin light-chain]=ADP+[myosin light-chain]phosphate); ID 2.7.1.119 Hygromycin-B kinase. (CA: ATP+hygromycin B=ADP+7″-O-phosphohygromycin B); ID 2.7.1.120 Caldesmon kinase. (CA: ATP+[caldesmon]=ADP+[caldesmon]phosphate); ID 2.7.1.122 Xylitol kinase. (CA: ATP+xylitol=ADP+xylitol 5-phosphate); ID 2.7.1.123 Calcium/calmodulin-dependent protein kinase. (CA: ATP+protein=ADP+O-phosphoprotein); ID 2.7.1.124 Tyrosine 3-monooxygenase kinase. (CA: ATP+[tyrosine-3-monooxygenase]=ADP+[tyrosine-3-monooxygenase]phosphate); ID 2.7.1.125 Rhodopsin kinase. (CA: ATP+[rhodopsin]=ADP+[rhodopsin]phosphate); ID 2.7.1.126 [Beta-adrenergic-receptor]kinase. (CA: ATP+[beta-adrenergic receptor]=ADP+[beta-adrenergic receptor]phosphate); ID 2.7.1.127 Inositol-trisphosphate 3-kinase. (CA: ATP+1D-myo- inositol 1,4,5-trisphosphate=ADP+1D-myo- inositol 1,3,4,5-tetrakisphosphate); ID 2.7.1.128 [Acetyl-CoA carboxylase]kinase. (CA: ATP+[acetyl-CoA carboxylase]=ADP+[acetyl-CoA carboxylase]phosphate); ID 2.7.1.129 [Myosin heavy-chain]kinase. (CA: ATP+[myosin heavy-chain]=ADP+[myosin heavy-chain]phosphate); ID 2.7.1.130 Tetraacyldisaccharide 4′-kinase. (CA: ATP+2,3-bis(3-hydroxytetradecanoyl)-D-glucosarninyl-(beta-D-1,6)-2,3-bis(3-hydroxytetradecanoyl)-D-glucosaminyl beta-phosphate=ADP+2,3,2′,3′-tetrakis(3-hydroxytetradecanoyl)-D-glucosaminyl-1,6-beta-D- glucosamine 1,4′-bisphosphate); ID 2.7.1.131 [Low-density lipoprotein receptor]kinase. (CA: ATP+[low-density lipoprotein receptor]L-serine=ADP+[low-density lipoprotein receptor]O-phospho-L-serine); ID 2.7.1.132 Tropomyosin kinase. (CA: ATP+[tropomyosin]=ADP+[tropomyosin]O-phospho-L-serine); ID 2.7.1.134 Inositol-tetrakisphosphate 1-kinase. (CA: ATP+1D-myo- inositol 3,4,5,6-tetrakisphosphate=ADP+1D-myo- inositol 1,3,4,5,6-pentakisphosphate); ID 2.7.1.135 [Tau protein]kinase. (CA: ATP+[tau protein]=ADP+[tau protein]O-phospho-L-serine); ID 2.7.1.136 Macrolide 2′-kinase. (CA: ATP+oleandomycin=ADP+oleandomycin 2′-O-phosphate); ID 2.7.1.137 Phosphatidylinositol 3-kinase. (CA: ATP+1-phosphatidyl-1D-myo-inositol=ADP+1-phosphatidyl-1D-myo-inositol 3-phosphate); ID 2.7.1.138 Ceramide kinase. (CA: ATP+ceramide=ADP+ceramide 1-phosphate); ID 2.7.1.140 1D-myo-inositol-tetrakisphosphate 5-kinase. (CA: ATP+1D-myo- inositol 1,3,4,6-tetrakisphosphate=ADP+1D-myo- inositol 1,3,4,5,6-pentakisphosphate); ID 2.7.1.141 [RNA-polymerase]-subunit kinase. (CA: ATP+[DNA-directed RNA polymerase]=ADP+phospho-[DNA-directed RNA polymerase]); ID 2.7.1.144 Tagatose-6-phosphate kinase. (CA: ATP+D-tagatose 6-phosphate=ADP+D-tagatose 1,6-bisphosphate); ID 2.7.1.145 Deoxynucleoside kinase. (CA: ATP+2′-deoxynucleoside=ADP+2′-deoxynucleoside 5′-phosphate); ID 2.7.1.148 4-(cytidine 5′-diphospho)-2-C-methyl-D-erythritol kinase. (CA: ATP+4-(cytidine 5′-diphospho)-2-C-methyl-D-erythritol=ADP+2-phospho-4-(cytidine 5′-diphospho)-2-C-methyl-D-erythritol); ID 2.7.1.149 1-phosphatidylinositol-5-phosphate 4-kinase. (CA: ATP+1-phosphatidyl-1D-myo-inositol 5-phosphate=ADP+1-phosphatidyl-1D-myo-inositol 4,5-bisphosphate); ID 2.7.1.150 1-phosphatidylinositol-3-phosphate 5-kinase. (CA: ATP+1-phosphatidyl-1D-myo-inositol 3-phosphate=ADP+1-phosphatidyl-1D-myo-inositol 3,5-bisphosphate); ID 2.7.1.151 Inositol-polyphosphate multikinase. (CA: ATP+1D-myo- inositol 1,4,5-trisphosphate=ADP+1D-myo- inositol 1,4,5,6-tetrakisphosphate=ATP+1D-myo- inositol 1,4,5,6-tetrakisphosphate=ADP+1D-myo- inositol 1,3,4,5,6-pentakisphosphate); ID 2.7.1.152 Inositol-hexakisphosphate kinase. (CA: ATP+myo-inositol hexakisphosphate=ADP+diphospho-myo-inositol pentakisphosphate (isomeric configuration unknown)); ID 2.7.1.153 Phosphatidylinositol-4,5-bisphosphate 3-kinase. (CA: ATP+1-phosphatidyl-1D-myo-inositol 4,5-bisphosphate=ADP+1-phosphatidyl-1D-myo- inositol 3,4,5-trisphosphate); ID 2.7.1.154 Phosphatidylinositol-4-phosphate 3-kinase. (CA: ATP+1-phosphatidyl-1D-myo-inositol 4-phosphate=ADP+1-phosphatidyl-1D-myo-inositol 3,4-bisphosphate); ID 2.7.2.1 Acetate kinase. (CA: ATP+acetate=ADP+acetyl phosphate); ID 2.7.2.2 Carbamate kinase. (CA: ATP+NH(3)+CO(2)=ADP+carbamoyl phosphate); ID 2.7.2.3 Phosphoglycerate kinase. (CA: ATP+3-phospho-D-glycerate=ADP+3-phospho-D-glyceroyl phosphate); ID 2.7.2.4 Aspartate kinase. (CA: ATP+L-aspartate=ADP+4-phospho-L-aspartate); ID 2.7.2.6 Formate kinase. (CA: ATP+formate=ADP+formyl phosphate); ID 2.7.2.7 Butyrate kinase. (CA: ATP+2-butanoate=ADP+butanoyl phosphate); ID 2.7.2.8 Acetylglutamate kinase. (CA: ATP+N-acetyl-L-glutamate=ADP+N-acetyl-L-glutamate 5-phosphate); ID 2.7.2.11 Glutamate 5-kinase. (CA: ATP+L-glutamate=ADP+L-glutamate 5-phosphate); ID 2.7.2.13 Glutamate 1-kinase. (CA: ATP+L-glutamate=ADP+alpha-L-glutamyl phosphate); ID 2.7.2.14 Branched-chain-fatty-acid kinase. (CA: ATP+2-methylpropanoate=ADP+2-methylpropanoyl phosphate); ID 2.7.3.1 Guanidoacetate kinase. (CA: ATP+guanidoacetate=ADP+phosphoguanidoacetate); ID 2.7.3.2 Creatine kinase. (CA: ATP+creatine=ADP+phosphocreatine);ID 2.7.3.3 Arginine kinase. (CA: ATP+L-arginine=ADP+N-phospho-L-arginine); ID 2.7.3.4 Taurocyamine kinase. (CA: ATP+taurocyamine=ADP+N-phosphotaurocyamine); ID 2.7.3.5 Lombricine kinase. (CA: ATP+lombricine=ADP+N-phospholombricine); ID 2.7.3.6 Hypotaurocyamine kinase. (CA: ATP+hypotaurocyamine=ADP+N(omega)-phosphohypotaurocyamine); ID 2.7.3.7 Opheline kinase. (CA: ATP+guanidinoethyl methyl phosphate=ADP+N′-phosphoguanidinoethyl methyl phosphate); ID 2.7.3.8 Ammonia kinase. (CA: ATP+NH(3)=ADP+phosphoramide); ID 2.7.3.10 Agmatine kinase. (CA: ATP+agmatine=ADP+N(4)-phosphoagmatine); ID 2.7.3.11 Protein-histidine pros-kinase. (CA: ATP+protein L-histidine=ADP+protein N(pi)-phospho-L-histidine); ID 2.7.3.12 Protein-histidine tele-kinase. (CA: ATP+protein L-histidine=ADP+protein N(tau)-phospho-L-histidine); ID 2.7.4.1 Polyphosphate kinase. (CA: ATP+{phosphate}(N)=ADP+{phosphate}(N+1)); ID 2.7.4.2 Phosphomevalonate kinase. (CA: ATP+(R)-5-phosphomevalonate=ADP+(R)-5-diphosphomevalonate); ID 2.7.4.3 Adenylate kinase. (CA: ATP+AMP=ADP+ADP); ID 2.7.4.4 Nucleoside-phosphate kinase. (CA: ATP+nucleoside phosphate=ADP+nucleoside diphosphate); ID 2.7.4.6 Nucleoside-diphosphate kinase. (CA: ATP+nucleoside diphosphate=ADP+nucleoside triphosphate); ID 2.7.4.7 Phosphomethylpyrimidine kinase. (CA: ATP+4-amino-2-methyl-5-phosphomethylpyrimidine=ADP+4-amino-2-methyl-5-diphosphomethylpyrimidine); ID 2.7.4.8 Guanylate kinase. (CA: ATP+GMP=ADP+GDP); ID 2.7.4.9 Thymidylate kinase. (CA: ATP+thymidine 5′-phosphate=ADP+thymidine 5′-diphosphate); ID 2.7.4.11 (Deoxy)adenylate kinase. (CA: ATP+dAMP=ADP+dADP); ID 2.7.4.12 T2-induced deoxynucleotide kinase. (CA: ATP+dGMP (or dTMP)=ADP+dGDP (or dTDP)); ID 2.7.4.13 (Deoxy)nucleoside-phosphate kinase. (CA: ATP+deoxynucleoside phosphate=ADP+deoxynucleoside diphosphate); ID 2.7.4.14 Cytidylate kinase. (CA: ATP+(d)CMP=ADP+(d)CDP); ID 2.7.4.15 Thiamine-diphosphate kinase. (CA: ATP+thiamine diphosphate=ADP+thiamine triphosphate); ID 2.7.4.16 Thiamine-phosphate kinase. (CA: ATP+thiamine phosphate=ADP+thiamine diphosphate); ID 2.7.4.18 Farnesyl-diphosphate kinase. (CA: ATP+farnesyl diphosphate=ADP+farnesyl triphosphate); ID 2.7.4.19 5-methyldeoxycytidine-5′-phosphate kinase. (CA: ATP+5-methyldeoxycytidine 5′-phosphate=ADP+5-methyldeoxycytidine diphosphate); ID 2.7.6.1 Ribose-phosphate diphosphokinase. (CA: ATP+D-ribose 5-phosphate=AMP+5-phospho-alpha-D-ribose 1-diphosphate); ID 2.7.6.2 Thiamine diphosphokinase. (CA: ATP+thiamine=AMP+thiamine diphosphate); ID 2.7.6.3 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine diphosphokinase. (CA: ATP+2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine=AMP+2-amino-7,8-dihydro-4-hydroxy-6-(diphosphooxymethyl)pteridine); ID 2.7.6.4 Nucleotide diphosphokinase. (CA: ATP+nucleoside 5′-phosphate=AMP+5′-phosphonucleoside 3′-diphosphate); ID 2.7.6.5 GTP diphosphokinase. (CA: ATP+GTP=AMP+guanosine 3′-diphosphate 5′-triphosphate); ID 2.7.7.1 Nicotinamide-nucleotide adenylyltransferase. (CA: ATP+nicotinamide ribonucleotide=diphosphate+NAD(+)); ID 2.7.7.2 FMN adenylyltransferase. (CA: ATP+FMN=diphosphate+FAD); ID 2.7.7.3 Pantetheine-phosphate adenylyltransferase. (CA: ATP+pantetheine 4′-phosphate=diphosphate+3′-dephospho-CoA); ID 2.7.7.4 Sulfate adenylyltransferase. (CA: ATP+sulfate=diphosphate+adenylylsulfate); ID 2.7.7.18 Nicotinate-nucleotide adenylyltransferase. (CA: ATP+nicotinate ribonucleotide=diphosphate+deamido-NAD(+)); ID 2.7.7.19 Polynucleotide adenylyltransferase. (CA: N ATP+{nucleotide}(M)=N diphosphate+{nucleotide}(M+N)); ID 2.7.7.25 tRNA adenylyltransferase. (CA: ATP+{tRNA}(N)=diphosphate+{tRNA}(N+1)); ID 2.7.7.27 Glucose-1-phosphate adenylyltransferase. (CA: ATP+alpha-D-glucose 1-phosphate=diphosphate+ADP-glucose); ID 2.7.7.42 Glutamate-ammonia-ligase adenylyltransferase. (CA: ATP+[L-glutamate:ammonia ligase (ADP-forming)]=diphosphate+adenylyl-[L-glutamate:ammonia ligase (ADP-forming)]); ID 2.7.7.47 Streptomycin 3″-adenylyltransferase. (CA: ATP+streptomycin=diphosphate+3″-adenylylstreptomycin); ID 2.7.7.53 ATP adenylyltransferase. (CA:ADP+ATP=phosphate+P(1),P(4)-bis(5′-adenosyl)tetraphosphate); ID 2.7.7.54 Phenylalanine adenylyltransferase. (CA: ATP+L-phenylalanine=diphosphate+N-adenylyl-L-phenylalanine); ID 2.7.7.55 Anthranilate adenylyltransferase. (CA: ATP+anthranilate=diphosphate+N-adenylylanthranilate); ID 2.7.7.58 (2,3-dihydroxybenzoyl)adenylate synthase. (CA: ATP+2,3-dihydroxybenzoate=diphosphate+(2,3-dihydroxybenzoyl)-adenylate); ID 2.7.8.25 Triphosphoribosyl-dephospho-CoA synthase. (CA: ATP+3-dephospho-CoA=2′-(5″-triphosphoribosyl)-3′-dephospho-CoA+adenine); ID 2.7.9.1 Pyruvate,phosphate dikinase. (CA: ATP+pyruvate+phosphate=AMP+phosphoenolpyruvate+diphosphate); ID 2.7.9.2 Pyruvate, water dikinase. (CA: ATP+pyruvate+H(2O)O=AMP+phosphoenolpyruvate+phosphate); ID 2.7.9.3 Selenide,water dikinase. (CA: ATP+selenide+H(2)O=AMP+selenophosphate+phosphate); ID 2.7.9.4 Alpha-glucan,water dikinase. (CA: ATP+alpha-glucan+H(2)O=AMP+phospho-alpha-glucan+phosphate); ID 3.1.11.5 Exodeoxyribonuclease V. (CA: Exonucleolytic cleavage (in the presence of ATP) in either 5′- to 3′- or 3′- to 5′-direction to yield 5′-phosphooligonucleotides); ID 3.1.21.3 Type I site-specific deoxyribonuclease. (CA: Endonucleolytic cleavage of DNA to give random double-stranded fragments with terminal 5′-phosphate; ATP is simultaneously hydrolyzed); ID 3.4.21.53 Endopeptidase La. (CA: Hydrolysis of large proteins such as globin, casein and denaturated serum albumin, in presence of ATP); ID 3.4.21.92 Endopeptidase Clp. (CA: Hydrolysis of proteins to small peptides in the presence of ATP and magnesium. Alpha-casein is the usual test substrate. In the absence of ATP, only oligopeptides shorter than five residues are cleaved (such as succinyl-Leu-Tyr-|-NHMEC; and Leu-Tyr-Leu-|-Tyr-Trp, in which the cleavage of the -Tyr-|-Leu- and -Tyr-|-Trp-bond also occurs)); ID 3.5.2.9 5-oxoprolinase (ATP-hydrolyzing). (CA: ATP+5-oxo-L-proline+2 H(2)O=ADP+phosphate+L-glutamate); ID 3.5.2.14 N-methylhydantoinase (ATP-hydrolyzing). (CA: ATP+N-methylimidazolidine-2,4-dione+2 H(2)O=ADP+phosphate+N-carbamoylsarcosine); ID 3.5.4.18 ATP deaminase. (CA: ATP+H(2)O=ITP+NH(3)); ID 3.6.1.3 Adenosinetriphosphatase. (CA: ATP+H(2)O=ADP+phosphate); ID 3.6.1.5 Apyrase. (CA: ATP+2 H(2)O=AMP+2 phosphate); ID 3.6.1.8 ATP diphosphatase. (CA: ATP+H(2)O=AMP+diphosphate); ID 3.6.1.14 Adenosine-tetraphosphatase. (CA: Adenosine 5′-tetraphosphate+H(2)O=ATP+phosphate); ID 3.6.1.31 Phosphoribosyl-ATP diphosphatase. (CA: 1-(5-phosphoribosyl)-ATP+H(2)O=1-(5-phosphoribosyl)-AMP+diphosphate); ID 3.6.3.1 Magnesium-ATPase. (CA: ATP+H(2)O=ADP+phosphate); ID 3.6.3.2 Magnesium-importing ATPase. (CA: ATP+H(2)O+Mg(2+)(Out)=ADP+phosphate+Mg(2+)(In)); ID 3.6.3.3 Cadmium-exporting ATPase. (CA: ATP+H(2)O+Cd(2+)(In)=ADP+phosphate+Cd(2+)(Out)); ID 3.6.3.4 Copper-exporting ATPase. (CA: ATP+H(2)O+Cu(2+)(In)=ADP+phosphate+Cu(2+)(Out)); ID 3.6.3.5 Zinc-exporting ATPase. (CA: ATP+H(2)O+Zn(2+)(In)=ADP+phosphate+Zn(2+)(Out)); ID 3.6.3.6 Proton-exporting ATPase. (CA: ATP+H(2)O+H(+)(In)=ADP+phosphate+H(+)(Out)); ID 3.6.3.7 Sodium-exporting ATPase. (CA: ATP+H(2)O+Na(+)(In)=ADP+phosphate+Na(+)(Out)); ID 3.6.3.8 Calcium-transporting ATPase. (CA: ATP+H(2)O+Ca(2+)(Cis)=ADP+phosphate+Ca(2+)(Trans)); ID 3.6.3.9 Sodium/potassium-exchanging ATPase. (CA: ATP+H(2)O+Na(+)(In)+K(+)(Out)=ADP+phosphate+Na(+)(Out)+K(+)(In)); ID 3.6.3.10 Hydrogen/potassium-exchanging ATPase. (CA: ATP+H(2)O+H(+)(In)+K(+)(Out)=ADP+phosphate+H(+)(Out)+K(+)(In)); ID 3.6.3.11 Chloride-transporting ATPase. (CA: ATP+H(2)O+Cl(−)(Out)=ADP+phosphate+Cl(−)(In)); ID 3.6.3.12 Potassium-transporting ATPase. (CA: ATP+H(2)O+K(+)(Out)=ADP+phosphate+K(+)(In)); ID 3.6.3.14 H(+)-transporting two-sector ATPase. (CA: ATP+H2O)O+H(+)(In)=ADP+phosphate+H(+)(Out)); ID 3.6.3.15 Sodium-transporting two-sector ATPase. (CA: ATP+H(2)O=ADP+phosphate); ID 3.6.3.16 Arsenite-transporting ATPase. (CA: ATP+H(2)O+arsenite(In)=ADP+phosphate+arsenite(Out)); ID 3.6.3.17 Monosaccharide-transporting ATPase. (CA: ATP+H(2)O+monosaccharide(Out)=ADP+phosphate+monosaccharide(In)); ID 3.6.3.18 Oligosaccharide-transporting ATPase. (CA: ATP+H(2)O+oligosaccharide(Out)=ADP+phosphate+oligosaccharide(In)); ID 3.6.3.19 Maltose-transporting ATPase. (CA: ATP+H(2)O+maltose(Out)=ADP+phosphate+maltose(In)); ID 3.6.3.20 Glycerol-3-phosphate-transporting ATPase. (CA: ATP+H(2)O+glycerol-3-phosphate(Out)=ADP+phosphate+glycerol-3-phosphate(In)); ID 3.6.3.21 Polar-amino-acid-transporting ATPase. (CA: ATP+H(2)O+polar amino acid(Out)=ADP+phosphate+polar amino acid(In)); ID 3.6.3.22 Nonpolar-amino-acid-transporting. ATPase. (CA: ATP+H(2)O+nonpolar amino acid(Out)=ADP+phosphate+nonpolar amino acid(In)); ID 3.6.3.23 Oligopeptide-transporting ATPase. (CA: ATP+H(2)O+oligopeptide(Out)=ADP+phosphate+oligopeptide(In)); ID 3.6.3.24 Nickel-transporting ATPase. (CA: ATP+H(2)O+Ni(2+)(Out)=ADP+phosphate+Ni(2+)(In)); ID 3.6.3.25 Sulfate-transporting ATPase. (CA: ATP+H(2)O+sulfate(Out)=ADP+phosphate+sulfate(In)); ID 3.6.3.26 Nitrate-transporting ATPase. (CA: ATP+H(2)O+nitrate(Out)=ADP+phosphate+nitrate(In)); ID 3.6.3.27 Phosphate-transporting ATPase. (CA: ATP+H(2)O+phosphate(Out)=ADP+phosphate+phosphate(In)); ID 3.6.3.28 Phosphonate-transporting ATPase. (CA: ATP+H(2)O+phosphonate(Out)=ADP+phosphate+phosphonate(In)); ID 3.6.3.29 Molybdate-transporting ATPase. (CA: ATP+H(2)O+molybdate(Out)=ADP+phosphate+molybdate(In)); ID 3.6.3.30 Fe(3+)-transporting ATPase. (CA: ATP+H(2)O+Fe(3+)(Out)=ADP+phosphate+Fe(3+)(In)); ID 3.6.3.31 Polyamine-transporting ATPase. (CA: ATP+H(2)O+polyamine(Out)=ADP+phosphate+polyamine(In)); ID 3.6.3.32 Quaternary-amine-transporting ATPase. (CA: ATP+H(2)O+quaternary amine(Out)=ADP+phosphate+quaternary amine(In)); ID 3.6.3.33 Vitamin B12-transporting ATPase. (CA: ATP+H(2)O+vitamin B12(Out)=ADP+phosphate+vitamin B12(In)); ID 3.6.3.34 Iron-chelate-transporting ATPase. (CA: ATP+H(2)O+iron chelate(Out)=ADP+phosphate+iron chelate(In)); ID 3.6.3.35 Manganese-transporting ATPase. (CA: ATP+H(2)O+Mn(2+)(Out)=ADP+phosphate+Mn(2+)(In)); ID 3.6.3.36 Taurine-transporting ATPase. (CA: ATP+H(2)O+taurine(Out)=ADP+phosphate+taurine(In)); ID 3.6.3.37 Guanine-transporting ATPase. (CA: ATP+H(2)O+gtianine(Out)=ADP+phosphate+guanine(In)); ID 3.6.3.38 Capsular-polysaccharide-transporting ATPase. (CA: ATP+H(2)O+capsular polysaccharide(In)=ADP+phosphate+capsular polysaccharide(Out)); ID 3.6.3.39 Lipopolysaccharide-transporting ATPase. (CA: ATP+H(2)O+lipopolysaccharide(In)=ADP+phosphate+lipopolysaccharide(Out)); ID 3.6.3.40 Teichoic-acid-transporting ATPase. (CA: ATP+H(2)O+teichoic acid(In)=ADP+phosphate+teichoic acid(Out)); ID 3.6.3.41 Heme-transporting ATPase. (CA: ATP+H(2)O+heme(In)=ADP+phosphate+heme(Out)); ID 3.6.3.42 Beta-glucan-transporting ATPase. (CA: ATP+H(2)O+b-glucan(In)=ADP+phosphate+b-glucan(Out)); ID 3.6.3.43 Peptide-transporting ATPase. (CA: ATP+H(2)O+peptide(In)=ADP+phosphate+peptide(Out)); ID 3.6.3.44 Xenobiotic-transporting ATPase. (CA: ATP+H(2)O+xenobiotic(In)=ADP+phosphate+xenobiotic(Out)); ID 3.6.3.45 Steroid-transporting ATPase. (CA: ATP+H(2)O+steroid(In)=ADP+phosphate+steroid(Out)); ID 3.6.3.46 Cadmium-transporting ATPase. (CA: ATP+H(2)O=ADP+phosphate); ID 3.6.3.47 Fatty-acyl-CoA-transporting ATPase. (CA: ATP+H(2)O+fatty acyl CoA(cis)=ADP+phosphate+fatty acyl CoA(trans)); ID 3.6.3.48 Alpha-factor-transporting ATPase. (CA: ATP+H(2)O+alpha-factor(In)=ADP+phosphate+alpha-factor(Out)); ID 3.6.3.49 Channel-conductance-controlling ATPase. (CA: ATP+H(2)O=ADP+phosphate); ID 3.6.3.50 Protein-secreting ATPase. (CA: ATP+H(2)O=ADP+phosphate); ID 3.6.3.51 Mitochondrial protein-transporting ATPase. (CA: ATP+H(2)O=ADP+phosphate); ID 3.6.3.52 Chloroplast protein-transporting ATPase. (CA: ATP+H(2)O=ADP+phosphate); ID 3.6.3.53 Ag(+)-exporting ATPase. (CA: ATP+H(2)O+Ag(+)(In)=ADP+phosphate+Ag(+)(Out)); ID 3.6.4.1 Myosin ATPase. (CA: ATP+H(2)O=ADP+phosphate); ID 3.6.4.2 Dynein ATPase. (CA: ATP+H(2)O=ADP+phosphate); ID 3.6.4.3 Microtubule-severing ATPase. (CA: ATP+H(2)O=ADP+phosphate); ID 3.6.4.4 Plus-end-directed kinesin ATPase. (CA: ATP+H(2)O=ADP+phosphate); ID 3.6.4.5 Minus-end-directed kinesin ATPase. (CA: ATP+H(2)O=ADP+phosphate); ID 3.6.4.6 Vesicle-fusing ATPase. (CA: ATP+H2)O=ADP+phosphate); ID 3.6.4.7 Peroxisome-assembly ATPase. (CA: ATP+H(2)O=ADP+phosphate); ID 3.6.4.8 Proteasome ATPase. (CA: ATP+H(2)O=ADP+phosphate); ID 3.6.4.9 Chaperonin ATPase. (CA: ATP+H(2)O=ADP+phosphate); ID 3.6.4.10 Non-chaperonin molecular chaperone ATPase. (CA: ATP+H(2)O=ADP+phosphate); ID 3.6.4.11 Nucleoplasmin ATPase. (CA: ATP+H(2)O=ADP+phosphate); ID 4.1.1.33 Diphosphomevalonate decarboxylase. (CA: ATP+(R)-5-diphosphomevalonate=ADP+phosphate+isopentenyl diphosphate+CO(2)); ID 4.1.1.49 Phosphoenolpyruvate carboxykinase (ATP). (CA: ATP+oxaloacetate=ADP+phosphoenolpyruvate+CO(2)); ID 4.2.1.93 ATP-dependent H(4)NAD(P)OH dehydratase. (CA: ATP+(6S)-6-beta-hydroxy-1,4,5,6-tetrahydronicotinamide-adenine dinucleotide=ADP+phosphate+NADH); ID 4.6.1.1 Adenylate cyclase. (CA: ATP=3′,5′-cyclic AMP+diphosphate); ID 5.1.1.11 Phenylalanine racemase (ATP-hydrolyzing). (CA: ATP+L-phenylalanine=AMP+diphosphate+D-phenylalanine); ID 5.99.1.2 DNA topoisomerase. (CA: ATP-independent breakage of single-stranded DNA, followed by passage and rejoining); ID 5.99.1.3 DNA topoisomerase (ATP-hydrolyzing). (CA: ATP-dependent breakage, passage and rejoining of double-stranded DNA); ID 6.1.1.1 Tyrosine—tRNA ligase. (CA: ATP+L-tyrosine+tRNA(Tyr)=AMP+diphosphate+L-tyrosyl-tRNA(Tyr)); ID 6.1.1.2 Tryptophan—tRNA ligase. (CA: ATP+L-tryptophan+tRNA(Trp)=AMP+diphosphate+L-tryptophanyl-tRNA(Trp)); ID 6.1.1.3 Threonine—tRNA ligase. (CA: ATP+L-threonine+tRNA(Thr)=AMP+diphosphate+L-threonyl-tRNA(Thr)); ID 6.1.1.4 Leucine—tRNA ligase. (CA: ATP+L-leucine+tRNA(Leu)=AMP+diphosphate+L-leucyl-tRNA(Leu)); ID 6.1.1.5 Isoleucine—tRNA ligase. (CA: ATP+L-isoleucine+tRNA(Ile)=AMP+diphosphate+L-isoleucyl-tRNA(Ile)); ID 6.1.1.6 Lysine—tRNA ligase. (CA: ATP+L-lysine+tRNA(Lys)=AMP+diphosphate+L-lysyl-tRNA(Lys)); ID 6.1.1.7 Alanine—tRNA ligase. (CA: ATP+L-alanine+tRNA(Ala)=AMP+diphosphate+L-alanyl-tRNA(Ala)); ID 6.1.1.9 Valine—tRNA ligase. (CA: ATP+L-valine+tRNA(Val)=AMP+diphosphate+L-valyl-tRNA(Val)); ID 6.1.1.10 Methionine—tRNA ligase. (CA: ATP+L-methionine+tRNA(Met)=AMP+diphosphate+L-methionyl-tRNA(Met)); ID 6.1.1.11 Serine—tRNA ligase. (CA: ATP+L-serine+tRNA(Ser)=AMP+diphosphate+L-seryl-tRNA(Ser)); ID 6.1.1.12 Aspartate—tRNA ligase. (CA: ATP+L-aspartate+tRNA(Asp)=AMP+diphosphate+L-aspartyl-tRNA(Asp)); ID 6.1.1.13 D-alanine—poly(phosphoribitol)ligase. (CA: ATP+D-alanine+poly(ribitol phosphate)=AMP+diphosphate+O-D-alanyl-poly(ribitol phosphate)); ID 6.1.1.14 Glycine—tRNA ligase. (CA: ATP+glycine+tRNA(Gly)=AMP+diphosphate+glycyl-tRNA(Gly)); ID 6.1.1.15 Proline—tRNA ligase. (CA: ATP+L-proline+tRNA(Pro)=AMP+diphosphate+L-prolyl-tRNA(Pro)); ID 6.1.1.16 Cysteine—tRNA ligase. (CA: ATP+L-cysteine+tRNA(Cys)=AMP+diphosphate+L-cysteinyl-tRNA(Cys)); ID 6.1.1.17 Glutamate—tRNA ligase. (CA: ATP+L-glutamate+tRNA(Glu)=AMP+diphosphate+L-glutamyl-tRNA(Glu)); ID 6.1.1.18 Glutamine—tRNA ligase. (CA: ATP+L-glutamine+tRNA(Gln)=AMP+diphosphate+L-glutaminyl-tRNA(Gln)); ID 6.1.1.19 Arginine—tRNA ligase. (CA: ATP+L-arginine+tRNA(Arg)=AMP+diphosphate+L-arginyl-tRNA(Arg)); ID 6.1.1.20 Phenylalanine—tRNA ligase. (CA: ATP+L-phenylalanine+tRNA(Phe)=AMP+diphosphate+L-phenylalanyl-tRNA(Phe)); ID 6.1.1.21 Histidine—tRNA ligase. (CA: ATP+L-histidine+tRNA(His)=AMP+diphosphate+L-histidyl-tRNA(His)); ID 6.1.1.22 Asparagine—tRNA ligase. (CA: ATP+L-asparagine+tRNA(Asn)=AMP+diphosphate+L-asparaginyl-tRNA(Asn)); ID 6.1.1.23 Aspartate—tRNA(Asn) ligase. (CA: ATP+L-aspartate+tRNA(Asx)=AMP+diphosphate+Aspartyl-tRNA(Asx)); ID 6.1.1.24 Glutamate—tRNA(Gln) ligase. (CA: ATP+L-glutamate+tRNA(Glx)=AMP+diphosphate+Glutamyl-tRNA(Glx)); ID 6.1.1.25 Lysine—tRNA(Pyl) ligase. (CA: ATP+L-lysine+tRNA(Pyl)=AMP+diphosphate+L-lysyl-tRNA(Pyl)); ID 6.2.1.1 Acetate—CoA ligase. (CA: ATP+acetate+CoA=AMP+diphosphate+acetyl-CoA); ID 6.2.1.2 Butyrate—CoA ligase. (CA: ATP+an acid+CoA=AMP+diphosphate+an acyl-CoA); ID 6.2.1.3 Long-chain-fatty-acid—CoA ligase. (CA: ATP+a long-chain carboxylic acid+CoA=AMP+diphosphate+an acyl-CoA); ID 6.2.1.5 Succinate—CoA ligase (ADP-forming). (CA: ATP+succinate+CoA=ADP+succinyl-CoA+phosphate); ID 6.2.1.6 Glutarate—CoA ligase. (CA: ATP+glutarate+CoA=ADP+phosphate+glutaryl-CoA); ID 6.2.1.7 Cholate—CoA ligase. (CA: ATP+cholate+CoA=AMP+diphosphate+choloyl-CoA); ID 6.2.1.8 Oxalate—CoA ligase. (CA: ATP+oxalate+CoA=AMP+diphosphate+oxalyl-CoA); ID 6.2.1.9 Malate—CoA ligase. (CA: ATP+malate+CoA=ADP+phosphate+malyl-CoA); ID 6.2.1.11 Biotin—CoA ligase. (CA: ATP+biotin+CoA=AMP+diphosphate+biotinyl-CoA); ID 6.2.1.12 4-coumarate—CoA ligase. (CA: ATP+4-coumarate+CoA=AMP+diphosphate+4-coumaroyl-CoA); ID 6.2.1.13 Acetate—CoA ligase (ADP-forming). (CA: ATP+acetate+CoA=ADP+phosphate+acetyl-CoA); ID 6.2.1.14 6-carboxyhexanoate—CoA ligase. (CA: ATP+6-carboxyhexanoate+CoA=AMP+diphosphate+6-carboxyhexanoyl-CoA); ID 6.2.1.15 Arachidonate—CoA ligase. (CA: ATP+arachidonate CoA=AMP+diphosphate+arachidonoyl-CoA); ID 6.2.1.16 Acetoacetate—CoA ligase. (CA: ATP+acetoacetate+CoA=AMP+diphosphate+acetoacetyl-CoA); ID 6.2.1.17 Propionate—CoA ligase. (CA: ATP+propanoate+CoA=AMP+diphosphate+propanoyl-CoA); ID 6.2.1.18 Citrate—CoA ligase. (CA: ATP+citrate+CoA=ADP+phosphate+(3S)-citryl-CoA); ID 6.2.1.19 Long-chain-fatty-acid—luciferin-component ligase. (CA: ATP+an acid+protein=AMP+diphosphate+an acyl-protein thiolester); ID 6.2.1.20 Long-chain-fatty-acid—acyl-carrier protein ligase. (CA: ATP+an acid+[acyl-carrier protein]=AMP+diphosphate+acyl-[acyl-carrier protein]); ID 6.2.1.22 [Citrate (pro-3S)-lyase]ligase. (CA: ATP+acetate+[citrate (pro-3S)-lyase](thiol form)=AMP+diphosphate+[citrate (pro-3S)-lyase](acetyl form)); ID 6.2.1.23 Dicarboxylate—CoA ligase. (CA: ATP+an omega-dicarboxylic acid=AMP+diphosphate+an omega-carboxyacyl-CoA); ID 6.2.1.24 Phytanate—CoA ligase. (CA: ATP+phytanate+CoA=AMP+diphosphate+phytanoyl-CoA); ID 6.2.1.25 Benzoate—CoA ligase. (CA: ATP+benzoate+CoA=AMP+diphosphate+benzoyl-CoA); ID 6.2.1.26 O-succinylbenzoate—CoA ligase. (CA: ATP+O-succinylbenzoate+CoA=AMP+diphosphate+O-succinylbenzoyl-CoA); ID 6.2.1.27 4-hydroxybenzoate—CoA ligase. (CA: ATP+4-hydroxybenzoate+CoA=AMP+diphosphate+4-hydroxybenzoyl-CoA); ID 6.2.1.28 3-alpha,7-alpha-dihydroxy-5-beta-cholestanate—CoA ligase. (CA: ATP+3-alpha,7-alpha-dihydroxy-5-beta-cholestanate+CoA=AMP+diphosphate+3-alpha, 7-alpha-dihydroxy-5-beta-cholestanoyl-CoA); ID 6.2.1.29 3-alpha,7-alpha,12-alpha-trihydroxy-5-beta-cholestanate—CoA ligase. (CA: ATP+3-alpha,7-alpha,12-alpha-trihydroxy-5-beta-cholestanate+CoA=AMP+diphosphate+3-alpha,7-alpha,12-alpha-trihydroxy-5-beta-cholestanoyl-CoA); ID 6.2.1.30 Phenylacetate—CoA ligase. (CA: ATP+phenylacetate+CoA=AMP+diphosphate+phenylacetyl-CoA); ID 6.2.1.31 2-furoate—CoA ligase. (CA: ATP+2-furoate+CoA=AMP+diphosphate+2-furoyl-CoA); ID 6.2.1.32 Anthranilate—CoA ligase. (CA: ATP+anthranilate+CoA=AMP+diphosphate+anthranilyl-CoA); ID 6.2.1.33 4-chlorobenzoate-CoA ligase. (CA: 4-chlorobenzoate+CoA+ATP=4-chlorobenzoyl-CoA+AMP+diphosphate); ID 6.2.1.34 Trans-feruloyl-CoA synthase. (CA: Ferulic acid+CoA+ATP=trans-feruloyl-CoA+products of ATP breakdown); ID 6.3.1.1 Aspartate—ammonia ligase. (CA: ATP+L-aspartate+NH(3)=AMP+diphosphate+L-asparagine); ID 6.3.1.2 Glutamate—ammonia ligase. (CA: ATP+L-glutamate+NH(3)=ADP+phosphate+L-glutamine); ID 6.3.1.4 Aspartate—ammonia ligase (ADP-forming). (CA: ATP+L-aspartate+NH(3)=ADP+phosphate+L-asparagine); ID 6.3.1.5 NAD(+) synthase. (CA: ATP+deamido-NAD(+)+NH(3)=AMP+diphosphate+NAD(+)); ID 6.3.1.6 Glutamate—ethylamine ligase. (CA: ATP+L-glutamate+ethylamine=ADP+phosphate+N(5)-ethyl-L-glutamine); ID 6.3.1.7 4-methyleneglutamate—ammonia ligase. (CA: ATP+4-methylene-L-glutamate+NH(3)=AMP+diphosphate+4-methylene-L-glutamine); ID 6.3.1.8 Glutathionylspermidine synthase. (CA: Gamma-L-glutamyl-L-cysteinyl-glycine+spermidine+ATP=N(1)-(gamma-L-glutamyl-L-cysteinyl-glycyl)-spermidine+ADP+phosphate); ID 6.3.1.9 Trypanothione synthase. (CA: Gamma-L-glutamyl-L-cysteinyl-glycine+N(1)-(gamma-L-glutamyl-L-cysteinyl-glycyl)-spermidine+ATP=N(1),N(8)-bis-(gamma-L-glutamyl-L-cysteinyl-glycyl)-spermidine+ADP+phosphate); ID 6.3.2.1 Pantoate—beta-alanine ligase. (CA: ATP+(R)-pantoate+beta-alanine=AMP+diphosphate+(R)-pantothenate); ID 6.3.2.2 Glutamate—cysteine ligase. (CA: ATP+L-glutamate+L-cysteine=ADP+phosphate+gamma-L-glutamyl-L-cysteine); ID 6.3.2.3 Glutathione synthase. (CA: ATP+gamma-L-glutamyl-L-cysteine+glycine=ADP+phosphate+glutathione); ID 6.3.2.4 D-alanine-D-alanine ligase. (CA: ATP+2 D-alanine=ADP+phosphate+D-alanyl-D-alanine); ID 6.3.2.6 Phosphoribosylaminoimidazole-succinocarboxamide synthase. (CA: ATP+5-amino-1-(5-phospho-D-ribosyl)imidazole-4-carboxylate+L-aspartate=ADP+phosphate+(S)-2-[5-amino-1-(5-phospho-D-ribosyl)imidazole-4-carboxamido]succinate); ID 6.3.2.7 UDP-N-acetylmuramoyl-L-alanyl-D-glutamate—L-lysine ligase. (CA: ATP+UDP-N-acetylmuramoyl-L-alanyl-D-glutamate+L-lysine=ADP+phosphate+UDP-N-acetylmuramoyl-L-alanyl-D-glutamyl-L-lysine); ID 6.3.2.8 UDP-N-acetylmuramate—L-alanine ligase. (CA: ATP+UDP-N-acetylmuramate+L-alanine=ADP+phosphate+UDP-N-acetylmuramoyl-L-alanine); ID 6.3.2.9 UDP-N-acetylmuramoylalanine—D-glutamate ligase. (CA: ATP+UDP-N-acetylmuramoyl-L-alanine+glutamate=ADP+phosphate+UDP-N-acetylmuramoyl-L-alanyl-D-glutamate); ID 6.3.2.10 UDP-N-acetylmuramoyl-tripeptide—D-alanyl-D-alanine ligase. (CA: ATP+UDP-N-acetylmuramoyl-L-alanyl-gamma-D-glutamyl-L-lysine+D-alanyl-D-alanine=ADP+phosphate+UDP-N-acetylmuramoyl-L-alanyl-gamma-D-glutamyl-L-lysyl-D-alanyl-D-alanine); ID 6.3.2.11 Carnosine synthase. (CA: ATP+L-histidine+beta-alanine=AMP+diphosphate+camosine); ID 6.3.2.12 Dihydrofolate synthase. (CA: ATP+dihydropterate+L-glutamate=ADP+phosphate+dihydrofolate); ID 6.3.2.13 UDP-N-acetylmuramoylalanyl-D-glutamate-2,6-diaminopimelate ligase. (CA: ATP+UDP-N-acetylmuramoyl-L-alanyl-D-glutamate+meso-2,6-diaminoheptanedioate=ADP+phosphate+UDP-N,acetylmuramoyl-L-alanyl-D-gamma-glutamyl-meso-2,6-diamino-heptanedioate); ID 6.3.2.14 2,3-dihydroxybenzoate—serine ligase. (CA: ATP+2,3-dihydroxybenzoate+L-serine=products of ATP breakdown+N-(2,3-dihydroxybenzoyl)-L-serine); ID 6.3.2.16 D-alanine—alanyl-poly(glycerolphosphate) ligase. (CA: ATP+D-alanine+alanyl-poly(glycerolphosphate)=ADP+phosphate+D-alanyl-alanyl-poly(glycerolphosphate)); ID 6.3.2.17 Folylpolyglutamate synthase. (CA: ATP+{tetrahydrofolyl-[Glu]}(N)+L-glutamate=ADP+phosphate+{tetrahydrofolyl-[Glu]}(N+1)); ID 6.3.2.18 Gamma-glutamylhistamine synthase. (CA: ATP+L-glutamate+histamine=products of ATP breakdown+N(alpha)-gamma-L-glutamylhistamine); ID 6.3.2.19 Ubiquitin—protein ligase. (CA: ATP+ubiquitin+protein lysine=AMP+diphosphate+protein N-ubiquityllysine); ID 6.3.2.20 Indoleacetate—lysine ligase. (CA: ATP+indole-3-acetate+L-lysine=ADP+N(6)-[(indole-3-yl)acetyl]-L-lysine); ID 6.3.2.21 Ubiquitin—calmodulin ligase. (CA: N ATP+calmodulin+N ubiquitin=N AMP+N diphosphate+(ubiquitin}(N)-calmodulin); ID 6.3.2.22 Diphthine—ammonia ligase. (CA: ATP+diphthine+NH(3)=ADP+phosphate+diphthamide); ID 6.3.2.23 Homoglutathione synthase. (CA: ATP+gamma-L-glutaniyl-L-cysteine+beta-alanine=ADP+phosphate+gamma-L-glutamyl-L-cysteinyl-beta-alanine); ID 6.3.2.24 Tyrosine—arginine ligase. (CA: ATP+L-tyrosine+L-arginine=AMP+diphosphate+L-tyrosyl-L-arginine); ID 6.3.2.25 Tubulin—tyrosine ligase. (CA: ATP+detyrosinated alpha-tubulin+L-tyrosine=alpha-tubulin+ADP+phosphate); ID 6.3.2.26 N-(5-amino-5-carboxypentanoyl)-L-cysteinyl-D-valine synthase. (CA: L-2-aminohexanedioate+L-cysteine+L-valine+3 ATP=N-[L-5-amino-5-carboxypentanoyl]-L-cysteinyl-D-valine+3 AMP+3 diphosphate); ID 6.3.2.27 Aerobactin synthase. (CA: 4 ATP+citrate+N(6)-acetyl-N(6)-hydroxylysine=4 ADP+4 phosphate+aerobactin); ID 6.3.3.1 Phosphoribosylformylglycinamidine cyclo-ligase. (CA: ATP+2-(formamido)-N(1)-(5-phospho-D-ribosyl)acetamidine=ADP+phosphate+5-amino-1-(5-phospho-D-ribosyl)imidazole); ID 6.3.3.2 5-formyltetrahydrofolate cyclo-ligase. (CA: ATP+5-formyltetrahydrofolate=ADP+phosphate+5,10-methenyltetrahydrofolate); ID 6.3.3.3 Dethiobiotin synthase. (CA: ATP+7,8-diaminononanoate+CO(2)=ADP+phosphate+dethiobiotin); ID 6.3.4.1 GMP synthase. (CA: ATP+xanthosine 5′-phosphate+NH(3)=AMP+diphosphate+GMP); ID 6.3.4.2 CTP synthase. (CA: ATP+UTP+NH(3)=ADP+phosphate+CTP); ID 6.3.4.3 Formate—tetrahydrofolate ligase. (CA: ATP+formate+tetrahydrofolate=ADP+phosphate+10-formyltetrahydrofolate); ID 6.3.4.5 Argininosuccinate synthase. (CA: ATP+L-citrulline+L-aspartate=AMP+diphosphate+L-argininosuccinate); ID 6.3.4.6 Urea carboxylase. (CA: ATP+urea+CO(2)=ADP+phosphate+urea-l-carboxylate); ID 6.3.4.7 Ribose-5-phosphate—ammonia ligase. (CA: ATP+D-ribose 5-phosphate+NH(3)=ADP+phosphate+5-phosphoribosylamine); ID 6.3.4.8 Imidazoleacetate—phosphoribosyldiphosphate ligase. (CA: ATP+imidazole-4-acetate+5-phosphoribosyl diphosphate=ADP+phosphate+1-(5-phosphoribosyl)imidazole-4-acetate+diphosphate); ID 6.3.4.9 Biotin—[methylmalonyl-CoA-carboxyltransferase]ligase. (CA: ATP+biotin+apo-[methylmalonyl-CoA:pyruvate carboxyltransferase]=AMP+diphosphate+[methylmalonyl-CoA:pyruvate carboxyltransferase]); IE) 6.3.4.10 Biotin—[propionyl-CoA-carboxylase (ATP-hydrolyzing)]ligase. (CA: ATP+biotin+apo-[propanoyl-CoA:carbon-dioxide ligase (ADP-forming))=AMP+diphosphate+[propanoyl-CoA:carbon-dioxide ligase (ADP-forming)]); ID 6.3.4.11 Biotin-[methylcrotonoyl-CoA-carboxylase]ligase. (CA: ATP+biotin+apo-[3-methylcrotonoyl-CoA:carbon-clioxide ligase (ADP-forming)]=AMP+diphosphate+[3-methylcrotonoyl-CoA:carbon-dioxide ligase (ADP-forming)]); ID 6.3.4.12 Glutamate—methylamine ligase. (CA: ATP+L-glutamate+methylamine=ADP+phosphate+N(5)-methyl-L-glutamine); ID 6.3.4.13 Phosphoribosylamine—glycine ligase. (CA: ATP+5-phospho-D-ribosylamine+glycine=ADP+phosphate+N(1)-(5-phospho-D-ribosyl)glycinamide); ID 6.3.4.14 Biotin carboxylase. (CA: ATP+biotin-carboxyl-carrier protein+CO(2)=ADP+phosphate+carboxybiotin-carboxyl-carrier protein); ID 6.3.4.15 Biotin—[acetyl-CoA-carboxylase] ligase. (CA: ATP+biotin+apo-[acetyl-CoA:carbon-dioxide ligase (ADP forming)]=AMP+diphosphate+[acetyl-CoA:carbon-dioxide ligase (ADP forming)]); ID 6.3.4.16 Carbamoyl-phosphate synthase (ammonia). (CA: 2 ATP+NH(3)+CO(2)+H(2)O=2 ADP+phosphate+carbamoyl phosphate); ID 6.3.4.17 Formate-dihydrofolate ligase. (CA: ATP+formate+dihydrofolate=ADP+phosphate+10-formyldihydrofolate); ID 6.3.5.1 NAD(+) synthase (glutamine-hydrolyzing). (CA: ATP+deamido-NAD (+)+L-glutamine+H(2)O=AMP+diphosphate+NAD(+)+L-glutamate); ID 6.3.5.2 GMP synthase (glutamine-hydrolyzing). (CA: ATP+xanthosine 5′-phosphate+L-glutamine+H(2)O=AMP+diphosphate+GMP+L-glutamate); ID 6.3.5.3 Phosphoribosylformylglycinamidine synthase. (CA: ATP+N(2)-formyl-N(1)-(5-phospho-D-ribosyl)glycinamide+L-glutamine+H(2)O=ADP+phosphate+2-(formamido)-N(1)-(5-phospho-D-ribosyl)acetamidine+L-glutamate); ID 6.3.5.4 Asparagine synthase (glutamine-hydrolyzing). (CA: ATP+L-aspartate+L-glutamine=AMP+diphosphate+L-asparagine+L-glutamate); ID 6.3.5.5 Carbamoyl-phosphate synthase (glutamine-hydrolyzing). (CA: 2 ATP+L-glutamine+CO(2)+H(2)O=2 ADP+phosphate+L-glutamate+carbamoyl phosphate); ID 6.3.5.6 Asparaginyl-tRNA synthase (glutamine-hydrolyzing). (CA: ATP+Aspartyl-tRNA(Asn)+L-glutamine=ADP+phosphate+Asparaginyl-tRNA(Asn)+L-glutamate); ID 6.3.5.7 Glutaminyl-tRNA synthase (glutamine-hydrolyzing). (CA: ATP+Glutamyl-tRNA(Gln)+L-glutamine=ADP+phosphate+Glutaminyl-tRNA(Gln)+L-glutamate); ID 6.4.1.1 Pyruvate carboxylase. (CA: ATP+pyruvate+HCO(3)(−)=ADP+phosphate+oxaloacetate); ID 6.4.1.2 Acetyl-CoA carboxylase. (CA: ATP+acetyl-CoA+HCO(3)(−)=ADP+phosphate+malonyl-CoA); ID 6.4.1.3 Propionyl-CoA carboxylase. (CA: ATP+propanoyl-CoA+HCO(3)(−)=ADP+phosphate+(S)-methylmalonyl-CoA); ID 6.4.1.4 Methylcrotonyl-CoA carboxylase. (CA: ATP+3-methylcrotonyl-CoA+HCO(3)(−)=ADP+phosphate+3-methylglutaconyl-CoA); ID 6.4.1.5 Geranoyl-CoA carboxylase. (CA: ATP+geranoyl-CoA+HCO(3)(−)=ADP+phosphate+3-(4-methylpent-3-en-1-yl)pent-2-enedioyl-CoA); ID 6.4.1.6 Acetone carboxylase. (CA: Acetone+CO(2)+ATP+2 H(2)O=acetoacetate+AMP+2 phosphate); ID 6.5.1.1 DNA ligase (ATP). (CA: ATP+{deoxyribonucleotide}(N)+{deoxyribonucleotide}(M)=AMP+diphosphate+{deoxyribonucleotide}(N+M)); and ID 6.5.1.3 RNA ligase (ATP). (CA: ATP+{ribonucleotide}(N)+{ribonucleotide}(M)=AMP+diphosphate+ribonucleotide}(N+M)).
  • Suitable linkers include, without limitation, α,ω-diamines, α,ω-disphosphines, α-amine-ω-phosphine, α-phosphine-ω-thiols or mixture or combinations thereof. Preferred α,ω-diamines are compounds of the general formula H2N—R—NH2, where R is selected from the group consisting of a linear or branched alkenyl group, an arenyl group, a linear or branched ara-alkenyl group, a linear or branched alka-arenyl group and hetero atom analogs thereof. The hetero atom analogs comprises the recited groups where: (a) one or more carbon atoms are replaced by a hetero atom selected from the group consisting of O, S, P, and mixtures thereof; (b) one or more carbon atoms are replaced by a hetero atom-containing groups selected from the group consisting of —C(O)NH— (amido group), —NHC(O)NH— (uryl group), or the like; (c) one or more hydrogen atoms are are replaced by a hetero atom selected from the group consisting of O, S, P, and mixtures thereof; (d) one or more hydrogen atoms are replaced by a hetero atom-containing groups selected from the group consisting of —C(O)NH— (amido group), —NHC(O)NH— (uryl group), or the like; (e) and mixture or combinations thereof. Preferred hetero atom analogs include compounds of the general formula —CH2(CH2)n[ECH2(CH2)n]mECH2(CH2)— where E is O, S, or P, n is an integer having a value between 0 and 10 and m is an integer having a value between 0 and 10. Preferred alkenyl group include —(CH2)k— where k is an integer having a value between 1 and 16. Preferred arenyl groups includes phenylene, divalent naphthylene, divalent antracene, or similer polycondensed aromatics or mixtures or combinations thereof, where divalent means that the amine, phosphine or thiol groups are attached at two different sites of the aromatic molecules. Preferred alka-arenyl groups include dialkyenyl benzenes, dialkylenyl naphthalenes, dialkenyl antracenes or similar condensed fused aromatics with two dialkenyl groups. Preferred ara-alkenyl group include phenyl substituted alkenyl group or the like.
    • Articulation of Reagent Usages Labeling the 5′ End of Oligonucleotides
  • The labeled-5′ phosphate of the gamma phosphate labeled nucleotide (T-NTP or T-L-NTP) is transferred by T4 polynucleotide kinase (PNK) to the unlabeled, 5′ end of an oligonucleotide. Alternatively, phosphate exchange reaction methodology can be used to transfer the labeled phosphate of the T-NTP or T-L-NTP to the 5′ end of an oligonucleotide. Thus, the T-NTPs or T-L-NTPs of this invention are used to catalytically or enzymatically label oligonucleotides. The enzymatically or catalytically labeled oligonucleotides of this invention are well-suited as probes taking the place of a chemically-synthesized fluorescent probes, or in any application where one needs to track an oligonucleotide (for example, a 32P-labeled probe). Multiple fluorophores can be added into a single reaction to an oligonucleotide solution, where each fluorophore has a different color and each resulting color coded oligonucleotide can be monitored in parallel. The enzymatically labeled oligonucleotides of this invention can also be used to prepare probes such as TaqMan probes, molecular beacons, etc. Because this reaction allows for existing oligonucleotide or polynucleotide to be labeled after preparation, whereas in the past, labeled oligonucleotide and polynucleotides had to be specially synthesized, the present invention allows labeling of pre-existing unlabeled oligonucleotides or polynucleotides. Thus, researchers can rapidly attach a fluorescent label or other type of label to an on-hand oligonucleotide. Using the technology of this invention provides researchers with a minimal time (generally overnight) method for labeling existing oligonucleotides as compared to the delays encountered when a fluorescently labeled oligonucleotide is ordered, a typically 3 to 10 day delay. Enzymatically labeled oligonucleotides can be used in any application in which chemically-labeled fluorescent or radioactively-labeled oligonucleotides would be used.
    • Reaction Monitoring
  • The gamma-labeled NPTs of this s invention can be used to monitor reaction by monitoring modified nucleotide by measuring separation of tagged gamma phosphate from intact nucleotide, where intact nucleotide produces minimal signal, but cleaved products produce detectable signals that increase or change over time and these changes indicate that the activity being monitored is occurring or has occurred.
    • High Throughput Screening of Kinase Substrate Specificity
  • The gamma-labeled NTPs of this invention can be used to perform kinase activity assays on multiple candidate substrates along with positive and negative controls, all in parallel. One preferred method involves immobilizing the controls and candidate substrate polypeptides in an array on a substrate, such as a polypeptide array. The kinase and labeled-NTP of this invention are allowed to contact the immobilized species under conditions to promote the kinase activity resulting in the transfer of the labeled phosphate of the labeled-NTP to the candidate substrate polypeptide. Knowledge of the substrates that are phosphorylated and the corresponding sites of phosphorylation of the candidate substrates provide information about the recognition target of the assayed kinase.
  • It is possible that different kinases will preferentially interact with specific fluorophores and/or linkers providing a measure of specificity to the reaction. Thus, T-NTPs can be designed or tailored for use by a specific kinase.
    • Mapping Phosphorylation Site Within a Protein
  • The T-NTPs or T-L-NTPs of this invention are contacted with a polypeptide in the presence of a catalyst, preferably a protein kinase, to generate a phosphorylated, labeled polypeptide. The phosphorylated, labeled polypeptide can be cleaved, enabling determination of the site of phosphorylation.
    • Incorporation of Kinase Modifiable Site in Recombinant Proteins
  • A fusion protein encoding polynucleotide sequence including a protein encoding region encoding a protein of interest and at least one phophorylation site encoding sequence ligated to either 5′ end or 3′ end or both the 5′ and 3′ ends of the protein encoding region, where the phophorylation site encoding sequence encoded amino acid sequences that correspond to a kinase phosphorylation amino acid sequence. The expressed fusion protein can then be contacted with a T-NTP or T-L-NTP of this invention and the kinase for phosphorylating the fused phosphorylation site(s) producing a specifically labeled fusion protein, where the label is designed to not significantly impact protein folding or native activity, but gives rise to site-specifically labeled target proteins. If multiple fluorophores are attached to the fusion protein, then the labels can be either the same or different colors. Such labeled fusion proteins can be used to monitor the expression, activation, activity and deactivation of these fusion proteins in the expressed cells, cell cluster, tissue or organ.
    • Color Coded Nucleotide and Polypeptide Markers
  • Because the T-P moiety of the T-NTP or the T-L-P moiety of the T-L-NTPs of this invention are readily transferred to the 5′ and/or 3′ ends of polynucleotides, the T-NTPs or T-L-NTPs of this invention can be used to create color-coded polynucleotide (DNA, RNA, or DNA/RNA) molecular weight markers for use in DNA, RNA or DNA/RNA analyses similar to the kaleidoscope markers used in protein molecular size standards. The use of color-coded polynucleotide markers allows for unambiguous identification of the molecular weight and/or size of each separated polynucleotide fragment. The same process can be used to form polypeptide markers provided that the polypeptide sequence includes at least one site capable of being phosphorylated using a T-NTP or T-L-NTP of this invention.
    • Labeling Restriction Fragments
  • Using the T-NTPs or T-L-NTPs of this invention, restriction nucleotide fragments can be labeled through phosphate exchange reaction using PNK (e.g., T4-PNK) for example to form labeled restriction nucleotide fragments. These labeled restriction nucleotide fragments can be used to visualize and/or identify the fragments and do not involve staining the fragments such as with ethidium bromide and the DNA is viewed with longer wavelength light, thereby minimizing DNA damage, so that the fragments are still active fragments for subsequent use. Complete labeling is not essential for visualization (i.e., only sufficient labeling for visualization and/or identification), ensuring that pristine DNA fragments can be isolated, as needed, for downstream manipulation.
    • Purifying Labeled Oligonucleotides
  • Because the labeling reaction produces labeled material as well as unlabeled material, T-NTPs or T-L-NTPs and unreacted NTPs, purification may be needed. The unreacted NTPs and T-NTPs or T-L-NTPs can be separated from the labeled targets and unreacted targets using a sizing column (or similar strategy). Purifying the labeled target can be done enzymatically using an enzyme that distinguishes between 5′-labeled target nucleotides and non-labeled target nucleotides through recognition of the 5′ end to specifically degrade the non-labeled nucleotide. Lambda exonuclease and phosphodiesterase 2 (PDE2) are candidate 5′ to 3′ exonucleases. Alternatively, HPLC purification may be used to isolate the labeled nucleotide target. Spin sizing columns or similar techniques can be used in the purification process.
    • Quick Assay of a Wide Range of ATP-utilizing Enzymes
  • The labeled nucleotides of this invention,' especially labeled ATP and GTP, can be used as probes in ATP-utilizing or GTP-utilizing enzymatic processes to determine specific activity, kinetics, mechanisms useful for biochemist and enzymologist. Enzymes could be NMP kinases, NDP kinases, sugar kinases, and/or etc (see attached list of APT-utilizing Enzymes or variants thereof). These reactions are usually coupled to a second NAD(H)-dependent enzyme for accurate specific activity determination. The labeled nucleotides of this invention can be used to investigate the activity, kinetics and mechanism of action of sugar kinase mediated phosphorylations, a large class of biomolecules.
    • Suicide Tags
  • Attach a suicide tag to the gamma-phosphate of an NTP. If the tagged gamma phosphate is transferred to a substrate such as a target protein, the protein kinase and the substrate are held in their associated state with sufficient strength for post reaction purification and/or identification, where term held means that the suicide tag interacts with both the kinase and the substrate with sufficient chemical and/or physical interactions to maintain the kinase and the substrate in their associated state. The chemical and/or physical interactions can be hydrogen bonding, covalent bonding, ionic bonding, electrostatic attractions, or mixtures or combinations thereof. This is useful for identifying interacting partners (i.e., the specific protein(s) phoshorylated by a specific kinase).
    • Kinase Substrate Screening Using Modified NTPs
  • The modified NTPs of this invention can be designed for use with proteins that require the unmodified NTP in order to bind and/or transform a substrate protein or polypeptide. The modified NTP can be designed to interact with the protein and to induce a conformation change in the protein that would facilitate its binding to its substrate protein or polypeptide. Preferably, the interaction is irreversible locking the protein in its active conformation. For example, if the protein which depends on the unmodified NTP for activity is a kinase, then the modified NTP would lock the kinase in its active conformation, the conformation that permits binding and/or transformation of its substrate protein and/or polypeptide. Thus, the modified NTP can be designed to lock the protein in a conformation with an altered affinity for a second substrate. This activated protein can then either be attached to a support through a reactive group such as a His-affinity tag—Nickel resin, biotin—streptavidin, or antibody or the protein can be preattached and activated by the modified NTP by passing a solution containing the modified NTP over the substrate having preattached protein. The substrate having the activated protein, can then be used to screen a library of potential substrates by passing a solution of the substrates over the support. The substrates will then be separated into bound and unbound substrates, unbound pass through over the support with little or no delay, while bound substrates stay attached to the activated protein. The bound substrates can then be eluted off the support and identified. This procedure is similar to the identification of unknown proteins isolated by their affinity to a known protein via the yeast two hybrid system.
  • The modified NTPs can also be used to examine at the affinity of known interactions where the binding of one substrate is dependent on the binding of an NTP by using a modified NTP to trap the enzyme in a state that would have an increased affinity for a given substrate and monitoring the binding affinity between the two substrates (e.g., Biacore or gel filtration).
    • Dye-ATP Yeast Strain Cell Free Lysate Kinase Utilization Assay
  • The present modified ATP are ideally suited for use in Yeast strain cell free lysate kinase utilization assays under Native Condition similar to the process described in Kolpdziej, P. A., Young, R. A. [35] Epitope Tagging and Protein Surveillance. Methods of Enzymology, vol. 194, pp 508-519. The procedure includes the steps of: (1) inoculate the yeast strain in 10 ml YPD media and grow at 30° C. overnight until cells reach log phase growth (˜1×107 cells @O.D.600); (2) harvest 2 mL of the log phase culture in an Eppendorf microcentrifuge tube; (3) pellet the cells at RT at 3000×g. Decant supernatant; (4) resuspend pellet in 200 mL of Buffer A at 4° C. which comprises 10% glycerol, 20 mM Hepes pH 7.9, 10 mM EDTA (maybe omitted, phosphatase inhibitor), 1 mM DTT, 0.5 mg/mL BSA, 100 mM Ammonium Sulfate, and 1 mM PMSF (other protease inhibitors may be added); (5) add about 50 ml of 425-600 micron glass beads; (6) vortex vigorously, 7×30s, alternate with 30s on ice; (7) centrifuge 5 min. at 3000×g; (8) aliquot supernatant to a new Eppendorf microcentrifuge tube; (9) centrifuge 5 min. at 3000×g; (10) aliquot supernatant to a new Eppendorf microcentrifuge tube; (11) add dye-dATP to a final working concentration of 100 mM; (12) incubate at 30° C. for a predetermined time-course not to exceed 24 h; (13) take 10 mL aliquots at the specified time points; (14) add 10 mL of 2× denaturing gel loading buffer. (place at −20° C.); (15) load the 20 mL time points (step 13+14) onto a 10% SDS-PAGE gel with appropriate markers and electrophorese at 200V until dye front is ˜1 cm from the bottom; (16) visualize/document phosphorylation by laser-light imaging at the specified dye excitation wavelength. (DO THIS STEP FIRST, DO NOT STAIN); (17) stain gel using a local coomassie protocol to visualize and document protein band pattern; and (18) overlay and compare laser-light image vs. Coomassie stain.
  • Dye-ATP (modified) Rapid Transformation Protocol for Yeast Strains
  • The present modified ATP can be used in rapid transformation protocol yeast strain assays similar to the procedure described in Geitz, R. D., Woods, R. A. (2002) Transformation of Yeast by the LiAc/ssCarrier DNA/PEG Method. Methods of Enzymology 350: 87-96. The procedure includes the steps of: (1) inoculate the yeast strain in 10 ml YPD media and grow at 30° C. overnight until cells reach log phase growth (˜1×107 cells @O.D.600); (2) harvest 2 mL of the log phase culture in an Eppendorf microcentrifuge tube; (3) pellet the cells at RT at 3000×g. Decant supernatant; (4) resuspend the cells by adding the following components in the order listed: 240 mL of PEG 4000 50% w/v; 36 mL of lithium acetate (LiAc) 1.0M; 36 mL of 1.1 mM of Dye-dATP, and 48 mL dH2O; (5) incubate the mixture in a water bath at 42° C. for 45 minutes to 1 hour; (6) prepare a 1:40 dilution in deionized H2O; (7) spot 5 mL of the 1:40 diluent oil to a clean glass slide; (8) spread to disseminate the cells on the slide (do not cover); and (9) visualize transformation efficiency via laser-light microscopy at the dye specific excitation wavelength.
  • The modified nucleotide reagents can be introduced into any cell or organism for monitoring NTP dependent cellular or organ functions.
    • General and Specific Methods for T-L-NTP Preparation
  • Referring now to FIG. 1A, a block diagram of a method for making the tagged NTPs of this invention, generally 100, is shown to involve reacting a nucleotide triphosphate (NTP) comprising a Base, a Sugar and three phosphate moieties (Pα, Pβ and Pγ) in a first step 102 with a tag T to form a tagged NTP (T-NTP).
  • Referring now to FIG. 1B, a block diagram of a method for making the tagged NTPs of this invention, generally 120, is shown to include a nucleotide triphosphate (NTP) comprising a Base, a Sugar and three phosphate moieties (Pα, Pβ and Pγ) which is reacted in a first step 122 with a linker L to form a linker modified NTP (L-NTP), where the linker L is bonded to the gamma phosphate Pγ. After the linker L is bonded to the NTP to form the L-NTP, the L-NTP is reacted in a second step 124 with a tag T to form a tagged NTP (T-L-NTP).
  • Referring now to FIG. 1C, a method for preparing a tagged ATP, generally 140, is shown to include an ATP which is reacted in a first step 142 with an ethylene diamine linker EDA to form an ethylene diamine modified ATP (EDA-ATP). Then in a second step 144, the EDA-ATP is reacted with a tag T via a second amino group of the EDA to form an tagged ATP (T-EDA-ATP), where the tag is preferably a fluorescent tag.
  • General Method for Labeling ONs or PNs with T-L-NTPs
  • Referring now to FIG. 2A, a block diagram of a method for tagging a synthetic 5′ and 3′ hydroxy terminated oligonucleotide (SON) at either the 5′ end using a tagged nucleotide triphosphate (T-L-NTP) of this invention, generally 200, is shown to involve reacting a T-L-NTP comprising a Base, a Sugar and three phosphate moieties (Pα, Pβ and Pγ) with a SON in a step 202 in the presence of a catalyst or enzyme. The catalyst or enzyme adds the tagged phosphate (T-L-P) of the T-L-NTP to the 5′ end, This same general procedure works equally well with T-NTPs of this invention.
  • Referring now to FIG. 2B, a block diagram of a method for tagging a natural or synthetic 5′ phosphate and 3′ hydroxy terminated oligonucleotide (NON) at the 5′ end using a tagged nucleotide triphosphate (T-L-NTP) of this invention, generally 250, is shown to involve reacting a T-L-NTP comprising a Base, a Sugar and three phosphate moieties (Pα, Pβ and Pγ) with a NON in a step 252 in the presence of a catalyst or enzyme. The catalyst or enzyme exchanges the 5′ phosphate of the NON with the tagged phosphate (T-L-P) of the T-L-NTP.
  • General Method for Labeling PPs or PRNs with T-L-NTPs
  • Referring now to FIG. 3, a block diagram of a method for tagged polypeptides using a tagged nucleotide triphosphate (T-L-NTP) of this invention, generally 300, is shown to involve reacting a T-L-NTP comprising a Base, a Sugar and three phosphate moieties (Pα, Pβ and Pγ) with a polypeptide PN in a step 302 in the presence of a catalyst or enzyme. The catalyst or enzyme adds the tagged phosphate (T-L-P) of the T.L-NTP to a site AA m+1 of the PP, where the exact site of phosphorylation depends on the catalyst or enzyme used in the reaction. This same general procedure works equally well with T-NTPs of this invention.
    • Experimental Section General Synthetic Procedure for Preparing γ-Phosphate-Modified ATP
  • The following synthetic scheme was used to prepare a variety of γ-Phosphate-Modified ATPs:
  • Figure US20100317005A1-20101216-C00001
  • where E is a main group element selected from the group consisting of N, O, P, S and combinations thereof and where R is a carbon-containing group having between 1 and about 20 main group atoms selected from the group consisting of B, C, N, O, P, S and combinations thereof, with the valency being completed by H. A more detailed explanation of the linker is set forth in the Reagent Listing Section. As stated previously, the linker can be diamines, diphosphines, dithiols, or mixed amine, phosphine and thiols. The linkers can also be carbon chains having leaving groups to promote direct carbon phosphate bonding and direct carbon to dye bonding. Because the linkers can have different lengths, charges, sizes, shapes, and polarities, and other physical properties, the exact linker for use in a particular application may depend on different variables. However, it is expected that any of the modified ATPs 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.
    • Example 1
  • This example illustrates the preparation of a ATP-L1-ROX or ATP-EDA-ROX, where the EDA is attached to the γ-phosphate of ATP as shown in the following reaction scheme:
  • Figure US20100317005A1-20101216-C00002
  • Preparation of ATP-L1 or ATP-EDA
  • A mixture of 7 mg, 12.7 μmol of ATP and 10 mg, 52 μmol of DEC in 0.1M, pH 5.7, 1.5 mL of MES buffer was stirred at room temperature for 10 min. 7 mg, 53 μmol of ethylene diamine hydrochloride (EDA.HCl) in 0.1M, pH 5.7, 2 mL of MES buffer was added and the mixture was stirred for 2-3 hr. The pH was maintained between 5.65-5.75 during this time. The reaction was monitored on Thin Layer Chromatography (TLC). The reaction mixture was then lyophilized and 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 NH4HCO3—MeOH/H2O buffer system. The product peak was collected and lyophilized to yield a white powder which was dissolved in 200 μL water and quantitated by spectrophotometry at 259 nm; 24 mM. The yield of the ATP-EDA intermediate was 38%.
  • Preparation of ATP-L1-ROX or ATP-EDA-ROX
  • 42 μL, 1 μmol of the ATP-EDA intermediate of Example 1 was dissolved in 1M, pH 9.0, 70 μL of NaHCO3 buffer and 2.5 mg, 4 μmol of ROX-NHS was dissolved in 100 μL of dry DMF. These two solutions were mixed and set on a shaker overnight. The reaction was monitored via HPLC on a C18 column using a TEAA-acetonitrile buffer system. After lyophilization, the pellet was dissolved in 0.3 mL of water and the solution was passed through a Sephadex G-25 column (1* 15 cm). The first eluted fraction was lyophilized. A 1 mL water solution of the pellet was subjected to HPLC purification on a Supelco C18 column using a TEAA-acetonitrile buffer system. The product peak, having a retention time 11 minutes, was collected and lyophilized to give a pellet. The pellet was dissolved in 5 mM, 780 μL of HEPES buffer and quantified by 576 nm reading on a spectrometer. The measured concentration of the solution was 1.1 mM representing a yield of 86% of ATP-EDA-ROX. MALDI-Mass: 1124 (M-3+Na+K); 1102 (M+K), 1058 (M-H2O+K). 1.1 mM, 1 μL of the ATP-EDA-ROX compound was treated with 65 U/mL, 1 μL of phosphodiesterase (PDE) in 10 μL of a 1× buffer comprising 110 mM Tris.HCl pH 8.9, 110 mM NaCl and 15 mM MgCl2 at room temperature for 20 minutes and then analyzed with TLC. The products were characterized as AMP and pyrophosphate (Ppi)-EDA-ROX by comparison with authentic samples.
  • An HPLC chromatogram of ATP-1-ROX synthesis reaction (576 nm) to monitor the mobility of molecules linked to the fluorophore or free dye is shown in FIG. 4. HPLC chromatogram of ATP-1-ROX synthesis reaction (259 nm) to monitor oligonucleotide mobility is shown in FIG. 5. UV spectrum of ATP-1-ROX is shown in FIG. 6.
    • Examples 2-10
  • Using the general scheme, a set of twelve γ-phosphate-modified-ATPs using four different linker were synthesized. The four linkers designated L1 or ethylene diamine (EDA), L2, L3 and L4 and having the structures shown below, were prepared and tested for activity in 5′ labeling of nucleotides:
  • Figure US20100317005A1-20101216-C00003
  • In these structures, one of the amine protons is missing from each terminal nitrogen atom because the linkers bond to the γ-phosphate of ATP and the fluorescent dye through the terminal nitrogen atoms of the diamine linkers. The twelve γ-phosphate-modified-ATPs are tabulated Table 1.
  • TABLE 1
    Prepared ATPs and Their Excitation and Emission Properties
    Modified ATPs Excitation (λ) Emission (λ)
    ATP-L1-Fluorescein 495 526
    ATP-L2-Fluorescein 495 518
    ATP-L3-Fluorescein 495 520
    ATP-L4-Fluorescein 495 521
    ATP-L1-Tamra 540 582
    ATP-L1-Rox 581 612
    ATP-L2-Rox 581 609
    ATP-L3-Rox 581 607
    ATP-L4-Rox 581 609
    ATP-L1-Cy3 550 562
    ATP-L1-Cy5 646 663
    ATP-L1-biotin n/a n/a

    Removal of Unlabeled Nucleotides from Labeled Nucleotides
  • Once a γ-phosphate-modified nucleotide or deoxynucleotide was prepared, then unlabeled nucleotide or deoxynucleotide can be removed by treating the solution with an appropriate phosphatase. For example, if the nucleotide is ATP, then calf intestinal phosphatase (CIAP) or shrimp alkaline phosphatase (SAP) can be used to specifically remove the unmodified ATP leaving the γ-phosphate-modified-ATP intact. In many applications, γ-phosphate-modified-ATPs free from their corresponding natural analog are prepared. The removal of the unmodified ATPs greatly enhances successful implementation of the methodology of this invention. This invention provides an effective process using phosphatases (e.g., CIAP and SAP) to remove non-modified NTPs or dNTPs from γ-phosphate-modified-NTPs and dNTPs.
  • The general procedure for purifying a γ-phosphate-modified nucleotide involves treating 1 mM, 1 mL, 1 nmol of the crude γ-phosphate-modified nucleotide with 1 U/mL, 1 μL, 1U of CIAP in 10 mL or 20 mL of a 1× buffer comprising 50 mM Tris.HCl pH 9.3, 1 mM MgCl2, 0.1 mM ZnCl 2 1 mM spermidine, at room temperature for 20 minutes. The treated sample was then used in a subsequent reaction as described herein. It is also possible to remove CIAP by heat-shock and centrifugal filtration.
    • Example 11
  • This example illustrates the purification of ATP-ROX with CIAP.
  • ATP (10 mM, 2 μL, 20 nmol) and ATP-ROX (1.1 mM, 2 μL, 2.2 nmol) were treated with CIAP (1 U/μL, 1 μL, 1U) in 1× buffer (10 μL) at room temperature for 20 minutes. Controls were the same re action mixtures without enzyme. After 25 min, aliquots (1 μL) from the samples were analyzed with appropriate TLC chromatography and fluorescence imaging or UV-shadowing. The result is shown below in FIG. 7.
    • Example 12
  • This example illustrates the end labeling of the 5′ end of an oligonucleotide using T4 PNK and ATP-ROX.
    • Objective
  • To determine if T4 PolyNucleotide Kinase is able to use our ATP-1-Dye molecules as substrates for the 5′ end-labeling of an oligonucleotide.
    • Utility
  • The ability to enzymatically add a fluorophore to the 5′ end of an oligonucleotide has tremendous applicability for a wide variety of molecular biological techniques. The initial labeled phosphate transfer experiment was set-up as a standard end-labeling reaction whereby one microliter of a 1.1 mM solution of ATP-L1-ROX was used in a ten microliter reaction using 10U of T4 PNK to end-label 100 nanograms of an in-house “TOP oligonucleotide having the nucleotide sequence SEQ. ID NO. 2 of 5′ gg TAC TAA gCg gCC gCA Tg 3′. The reaction was incubated at 37° C. for 30 minutes, loading dye was added, and the sample was loaded onto a 20% denaturing polyacrylamide gel along with a chemically synthesized (MWG) 5′ fluorescein labeled TOP oligonucleotide (FITC-TOP) as a size marker. Scanning the gel with Texas Red channel revealed no labeled oligonucleotide in the reaction lane which corresponded to 19mer TOP. FITC scanning of the gel clearly reveals the FITC-TOP oligonucleotide, indicating that the end-labeling reaction did not work.
  • The negative result prompted us to consider that our ATP-l1-ROX molecule contained unlabeled ATP that carried over from synthesis. Unlabeled ATP is thought to out-compete the ATP-L1-ROX molecule for T4 PNK binding and thereby reduce the yield of 5′ ROX-labeled oligonucleotide. To insure purity, the ATP-L1-ROX product was treated with CLAP to remove unlabeled ATP. Additionally, to circumvent a potentially slower reaction rate of T4 PNK with ATP-1-ROX substrate, an overnight incubation at 37° C. was conducted. A CTAP reaction was performed containing one microliter of 1.1 mM solution of crude ATP-L1-ROX, 1 microliter of CIAP (available from Promega), one microliter of 10× CLAP buffer (available from Promega), and seven microliters of sterile milli-Q water. This reaction was incubated at 37° C. for 30 minutes. Five microliters of the CLAP treated ATP-L1-ROX was added to 1 microliter T4 PNK (available from Promega), 1 microliter of 10× PNK buffer (available from Promega), 100 nanograms of the TOP oligonucleotide in a total volume of ten microliters. The reaction was incubated at 37° C. overnight. Results from this reaction are shown in FIGS. 8 and 9 clearly evidencing 5′ end labeling.
  • In follow up reactions, the inventors determined that more efficient 5′ end-labeling was achieved by increasing the amount of T4 PNK to two microliters (20U) and the amount of oligonucleotide to one microgram as shown in FIGS. 10 and 11.
    • Example 13
  • This example illustrates the purification of end labeling of the 5′ end of an oligonucleotide using T4 PNK and ATP-L1-ROX or ATP-EDA-ROX.
  • CENTRI-SEP Columns (Princeton Separations, Inc. Cat. #CS-900) were used to remove unwanted dye-ATP and dye breakdown from the 5′ dye-labeled oligonucleotide product. A ten microliter reaction was performed with the 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://www.prinsep.com/html/products/centri_sep/single_column/protocoli, where the modification replaced the 750×g for 2 minute centrifugation step with a 500×g for 3 minutes centrifugation step. Additionally, ten microliters of sterile milli-Q water were added to the ten microliter end-labeling reaction to bring the final volume to twenty microliters.
    • Removal of Dye Terminators Prior to Sequencing
  • Several methods have been identified for removing labeled-ATP and free dye from the labeled oligonucleotide (DNA, RNA, RNA/DNA) products of this invention. These methods include phenol-chloroform extraction, phenol-chloroform extraction and ethanol precipitation, and widely used spin-column removal processes (e.g., Qiagen, CentriSep, etc.).
    • Examining ATP-L1-ROX in 5′ End-labeling Oligonucleotide Reactions
  • The following figures display experimental results investigating T4 PNK's ability to utilize the set of fluorescently labeled ATP molecules in 5′-end-labeling reactions of oligonucleotides. All reactions have undergone column purification prior to electrophoresis on a 20% denaturing polyacrylamide gel to remove excess fluorescent-ATP and free dye which obscures the 5′ fluorescently end-labeled oligonucleotide product. For each experiment, the most intense (major) band was assigned a relative activity value of 1.0 and all other bands were normalized to this value.
    • Reaction Time
  • The initial experiment was designed to investigate the time required to maximize T4 PNK's ability to 5′ end-label a 19-base oligonucleotide with ATP-L1-ROX as shown FIGS. 12A&B. A significant improvement in 5′ end-labeling occurred when reactions were allowed to continue overnight or for about 18 hours. Since reactions that were incubated for longer than 18 hours (not shown) did not improve labeling, 18 hours is designated as the standard labeling reaction time.
    • Timecourse Experiment Examining 5′ End-labeling Efficiency
  • Referring now to FIG. 12A, a timecourse investigating 5′ end-labeling of a 19-base oligonucleotide with APT-L1-ROX. Reactions were incubated at 37 degrees Celsius for 0.5, 1, 3, 6, and 18 hours. All reactions were column purified and electrophoresed on a 20% denaturing gel. The negative control (−) does not contain T4 PNK. Referring now to FIG. 12B, a graphical representation of quantitated bands from gel imaging. Gels are scanned with a BIORAD Molecular Imager FX Pro at the fluorophores specific emission wavelength filter setting. BIORAD Quantity One Quantitation software is used to analyze bands. Band values are obtained by highlighting a band's area and then subtracting background value (equivalent gel image area without a band) to obtain an optical density value.
    • Oligonucleotide 5′ End-labeling Reaction: Method Details
  • Reactions were assembled by adding 1 μg of a 19-base oligonucleotide (5′ GGTACTAAGCGGCCGCATG 3′), 1 μl of Promega 10× Kinase buffer (700 mM Tris-HCl, pH 7.6, 100 mM MgCl2, and 50 mM DTT), 2 nmol ATP-Linker-Fluorophore (or ATP-Linker-Biotin), 12% Polyethylene glycol 8000, Promega T4 PNK (20U), and sterile milliQ water to a final volume of 10 μl. Reactions were incubated overnight (18 hours) at 37 degrees Celsius in a BioRad 96 microtube iCycler thermocycler with heated-lid and purified with a Centri-SEP column (Princeton Separations, Inc.) following the manufacture's protocol with the exceptions: (1) columns were centrifuged at 500× g for 3 minutes (2) reaction volumes were adjusted to 20 μl with sterile MilliQ water prior to loading.
    • Product Analysis of 5′ Fluorescently-labeled Oligonucleotides: Method Details
  • Reaction products can be directly loaded onto a 20% denaturing polyacrylamide gel for electrophoresis or purified through a CentriSEP spin-column to remove excess ATP-Linker-Fluorophore or ATP-Linker-Biotin that has not been utilized in the reaction. Samples are electrophoresed at 50W, 50 degrees Celsius, for a period of approximately 1-1.5 hours and then the gel is scanned with a BIORAD Molecular Imager FX Pro at the fluorophores specific emission wavelength filter setting. Subsequent gel analysis is performed with BIORAD Quantity One Quantitation software.
    • Enzyme Amount
  • To determine the optimal amount of T4 PNK needed for 5′ end-labeling with a fluorescent-ATP, two different concentrations (10U and 20U) of T4 PNK were tested with increasing concentrations of APT-L1-ROX as shown in FIGS. 13A-B. The results demonstrate that 20 Units of T4 PNK increases the efficiency of 5′ end-labeling at all concentrations of APT-L1-ROX tested. No improvements were observed when greater than 20U of T4 PNK were tested (data not shown).
    • Optimizing Enzyme Concentration
  • Referring now to FIG. 13A, a 5′ end-labeling of a 19-base oligonucleotide examining optimal T4 PNK activity (10U and 20U). Three concentrations of APT-L1-ROX (110, 220, and 550 micromolar) were tested. Reactions were incubated at 37 degrees Celsius for 18 hours, column purified, and electrophoresed on a 20% denaturing gel. Referring now to FIG. 13B, Graphical representation of quantitated bands from gel imaging.
    • Volume Exclusion Agents
  • High molecular weight polymers are reported to promote T4 PNK activity (i.e., transfer of the γ32P of a radio-labeled ATP) by stabilizing the enzyme via macromolecular crowding. Based on this earlier report, polyethylene glycol (PEG 8000) was added to end-labeling reactions to determine if a similar affect is observed when fluorescently-labeled ATP is used as the label source as shown in FIG. 14. Increases in the amount of 5′ ROX-labeled oligonucleotide are observed as a result of increased PEG in the reaction. Further increases in PEG past 12% did not improve the sefficiency of the fluorescent labeling reaction.
    • Macromolecular Crowding Improves 5′ End-labeling
  • Referring now to FIG. 14, an effect following addition of polyethylene glycol 8000 (PEG 8000) on the amount of labeled oligonucleotide. 5′ end-labeling reactions containing different concentrations of PEG 8000, (4, 6, 8, 12, and 18%) were examined with a 19-base oligonucleotide and APT-L1-ROX. The graph represents quantitated bands determined via gel imaging and analysis.
  • Chemical Properties that Affect the Labeling Reaction
  • In addition to examining reaction parameters to begin optimizing the 5′ end-labeling reaction, some of the chemical properties of the preliminary set of fluorescently labeled-ATPs were also investigated. The crystal structure of T4 PNK reveals that the active site resembles a shallow tunnel in which a gamma-phosphate of ATP can be positioned on one side of the tunnel for an in-line phosphoryl transfer to the 5′-OH of a nucleic acid substrate on the opposite side. Two features of the labeled-ATP which may influence the phosphoryl transfer by T4 PNK are the chemical characteristics of the fluorophore and the linker attachment. Their structural size and/or rigidity may be critical to enzymatic activity, depending on potential steric constraints within the active site tunnel.
  • Evidence which supports the differential effects that a DNA substrate has upon T4 PNK's end-labeling ability is the enzyme's decreased activity when confronted with recessed 5′ ends and nicks in double stranded DNA, and an apparent bias it exhibits towards preferentially phosphorylating a 5′ terminal guanosine base over other bases. Other considerations include the effect that hydrophobicity and polarity may have upon catalysis. This is particularly important due to the hydrophobic nature of many of the fluorescent dyes. T4 PNK's active site surface is almost entirely composed of charged or polar residues, however several hydrophobic residues are involved in formation of the walls. In order to ascertain whether the four linkers have differential effects on 5′ end-labeling, two sets of the fluorescently labeled-ATPs (ATP-Linker-ROX and ATP-Linker-Fluorescein) were examined shown in FIGS. 15A and B. Oligonucleotide labeling with the ROX-labeled ATPs show that T4 PNK's efficiency is higher when linker L1 joins the ATP and fluorophore, whereas linker L2 increases oligonucleotide labeling when fluorescein is used. Examining each of the fluorescent dyes (Fluorescein, ROX, TAMRA, Cy3, and Cy5) with linkers of different composition will enable the optimization of 5′ end-labeling oligonucleotides for these dyes and will allow a better understanding of the enzyme's phosphoryl transfer mechanism.
    • Examining Linker Effects on 5′ End-labeling
  • Referring now to FIGS. 15A&B, the differential effect of L1, L2, L3, and L4 on T4 PNK's utilization of fluorescently labeled ATP molecules in the 5′ end-labeling reaction. The reactions examining each of the four linkers were tested with a 19-base oligonucleotide and the set of (FIG. 15A) ATP-L-ROX (220 and 440 micromolar) molecules and (FIG. 15B) ATP-L-Fluorescein molecules (220 and 440 micromolar). Reactions were incubated at 37° C. for 18 hours, column purified, and electrophoresed on a 20% denaturing gel, not shown. The graph represents quantitated bands from gel imaging and analysis.
    • Specificity of Labeling at the 5′ End of the Oligonucleotide
  • To determine whether the 3′ end of the labeled oligonucleotide was available for enzymatic synthesis, DNA polymerase extension reactions were performed. Since polymerases are restricted to incorporation at the 3′ end, a 19-base oligonucleotide was 5′ end-labeled with APT-L1-ROX and then hybridized to a 20-base complementary oligonucleotide containing a single base overhang ‘A’ as shown in FIG. 16A. This overhanging ‘A’ was used by the polymerase as a template for the incorporation of an incoming base-labeled dUTP at the ROX-oligonucleotide's 3′ end as shown in FIG. 16B. Incorporation was determined by 19-base ROX-oligonucleotide conversion into a 20-base ROX-oligonucleotide and confirmed by its altered migration in the denaturing gel as shown in FIG. 16B. This experiment not only demonstrates the applicability of enzymatic fluorescent labeling of an oligonucleotide with the fluorescent-ATP reagents, but also reveals the potential utility for the creation of tailor-made probes with fluorescent dyes or fluorescent dyes and quenchers at either end, such as a TaqMan or Molecular Beacon probe.
    • Confirmation of ROX-Labeling at the Oligonucleotides 5′ End
  • Referring now to FIG. 16A, a Schematic of the 5′ end-labeled ROX-oligonucleotide duplexed to a complementary 20-base oligonucleotide and used as primer-template for incorporation of a base-labeled dUTP. Referring now to FIG. 16B, a gel electrophoresis of samples (Lane 1): . ROX-oligonucleotide with an incorporated base-labeled dUTP at its 3′ end (Lane 2): ROX-oligonucleotide without the base-labeled dUTP incorporated. Reaction products were detected using a ROX emission filter.
    • Primer Extension Reactions: Method Details
  • Oligonucleotides were annealed to form duplex by incubating primer and complementary template at equimolar amounts in a BioRad 96 microtube iCycler thermocycler with heated-lid starting at 96 degrees Celsius and slow-cooling to 20 degrees Celsius over a one hour period. Primer extension reactions contain the duplexed molecules, 1 μl HIV reverse transcriptase (1 mg/ml), 1 μl of 10× Promega Taq buffer (100 mM Tris-HCl (pH 9.0 at 25° C.), 500 mM KCl, 1.5 mM MgCl2, and 1% Triton X-100), and 200 μM dUTP-Alexa 488 (Invitrogen/Molecular Probes). The reaction was incubated in a 37° C. water bath for 2 hours.
    • Preferential Elimination of Unlabeled Oligonucleotide
  • Experiments were conducted to examine if unlabeled oligonucleotide could be preferentially eliminated post-5′ end-labeling. The enzyme used to degrade the unlabeled oligonucleotide was lambda exonuclease, since the enzyme preferentially degrades phosphorylated DNA in the 5′ to 3′ direction if a 5′ phosphate is present. The inventors hypothesized that a fluorescent dye attached at the 5′ end of an oligonucleotide might provide protection against degradation by lambda exonuclease relative to one containing a 5′ phosphate added. The experiment where a ROX-labeled oligonucleotide and unlabeled oligonucleotide were treated with lambda exonuclease for different periods of time is shown in FIG. 17A. Intact ROX-oligonucleotide (Red) and unlabeled oligonucleotide (Green) are visible at timepoint 0. The amount of each remaining after treatment with lambda exonuclease is compared as shown in FIG. 17B. This experiment demonstrates that the presence of the fluorophore and linker masks the 5′ end of the labeled oligonucleotide, making it less susceptible to degradation by lambda exonuclease, and provides a method to enrich for the desired labeled product.
    • 5′ Fluorescently Labeled Oligonucleotide is Protected Against Lambda Exonuclease
  • Referring now to FIG. 17A&B, depict examining lambda exonuclease activity on ROX-labeled versus unlabeled oligonucleotide. Looking at FIG. 17A, a 5′ end-labeled ROX-oligonucleotide and unlabeled oligonucleotide that have been treated with lambda exonuclease for 0 to 10 minutes at 37° C. Reactions were electrophoresed and oligonucleotide integrity monitored by ROX emission for the ROX-oligonucleotide (RED) and SyBR Gold staining for unlabeled oligonucleotide (GREEN); gel images overlayed. The two green bands observed at timepoint 0 for the unlabeled oligonucleotide represent intact 19-base and 18-base oligonucleotide (a byproduct of incomplete oligonucleotide synthesis, −1). Looking at FIG. 17B, a graph represents quantitated bands, the intact band at timepoint 0 min. for ROX-1-oligonucleotide and unlabeled oligonucleotide (19-base) have each been assigned the relative activity of 1.0, with all other bands normalized to their resp. control values.
  • Lambda Exonuclease Treatment of a 19-base Oligonucleotide 5′ End-labeled with ATP-L1-ROX: Method Details
  • Standard 5′ end-labeling reactions were performed with ATP-L1-ROX with the exception that after overnight incubation natural ATP was added to a final concentration of 1 mM and reactions were allowed to incubate for an additional 2 hours at 37 degrees Celsius. The reactions were then purified on CentriSep columns and the eluent was vacuum dried. Treatment consisted of adding 5 μl of 10× lambda exonuclease buffer (1× buffer: 67 mM Glycine-KOH, 2.5 mM MgCl2, 50 μg/ml BSA, pH 9.4 at 25° C.), 5 μl (25U) of lambda exonuclease (NeW England Biolabs, MA), and then incubated for specified timepoints at 37 degrees Celsius. Timepoint aliquots were immediately removed to 5 μl stop solution (88% formamide, 1% bromophenol blue, 0.6M EDTA). Samples were electrophoresed on a 20% denaturing gel and the gel was scanned for ROX emission (red channel) and stained with SYBR Gold (Invitrogen/Molecular Probes) (green channel).
    • PDE2 Treatment for Removing Unlabeled Single-Stranded DNA or RNA.
  • The inventors also identified an alternative to the lambda exonuclease treatment for clearing unlabeled, single-stranded DNA and RNA from our T4 PNK labeling reactions; effectively raising the specific activity of the labeling reaction. The inventors have found that the phosphodiesterases 2 (PDE2) enzyme has several advantages over the Lambda exonuclease including: (1) no cold-phosphorylation of the unlabeled oligonucleotide is required, thus making pre-treatment of the reaction with natural ATP unnecessary, and (2) PDE2 will work on both DNA and RNA substrates.
    • 5′ End-Labeling an Oligonucleotide Using ATP-L1-Biotin
  • Due to the favorable results observed with the fluorescently labeled-ATPs, a biotin-labeled ATP was designed and synthesized for 5′ end-labeling an oligonucleotide. The molecule, APT-L1-biotin, is comprised of a biotin attached by linker L1 to the gamma-phosphate of ATP. As described for the fluorescently tagged-γ-ATP products, the attached biotin-linker moiety is transferred with the gamma-phosphate to the 5′ end of the oligonucleotide by T4 PNK. Detection of 5′ biotin-labeling of the oligonucleotide was observed by modifying an immunoblot analysis that is based on the strong interaction between biotin and streptavidin. Column purified (CentriSEP) 5′ biotinylated oligonucleotide was covalently attached to Whatman DE81 filter paper disc by spotting and allowed to air dry. Filter discs were processed with a streptavidin-alkaline-phosphatase conjugate and subsequently developed with NBT/BCIP reagent. Displayed are three DE81 filter discs that have undergone color development as shown in FIG. 18. Any residual APT-L1-biotin not utilized in the reaction was removed by CentriSEP column purification, as indicated by the lack of a spot on the negative control disc. The positive control (center filter) has been spotted with a dilution series of a chemically synthesized biotin-labeled oligonucleotide (Integrated DNA Technologies, Inc.) and is used as a comparison for labeling.
  • 5′ End-Labeling a 19-Base Oligonucleotide with APT-L1-Biotin
  • Referring now to FIG. 18, a 5′ end-labeling experiment of a 19-base oligonucleotide with APT-LI-biotin is shown. Three DE81 filters that have been color-processed (Left disc) Negative control: reaction without T4 PNK, (Center disc) Positive control: Chemically synthesized biotin-labeled oligonucleotide (Integrated DNA Technologies, Inc.) spotted are 1, 0.1, and 0.01 pmol indicated below each spot, (Right) APT-L1-biotin experiment: reaction containing T4 PNK.
    • Product Analysis of 5′ Biotin-Labeled Oligonucleotides: Method Details
  • Reaction products are CentriSEP column purified and the eluted sample is vacuum dried and resuspended in 5 μl of sterile milliQ water. The reaction is spotted onto Whatman DE81 filter paper in 1 μl aliquots with air drying between spottings. Filters are incubated in 10 mL of 1× phosphate buffered saline (PBS) solution for 10 minutes at room temperature with agitation. The filter is then incubated in 1× PBS+0.1% bovine serum albumin (BSA) as a block for 30 minutes at room temperature with agitation. Filters then underwent 3 ten minute washes with 1× PBS for 10 minutes at room temperature with agitation and were placed in a Promega streptavidin-alkaline phosphatase conjugate that was diluted 1/5000 in 1× PBS for an overnight incubation at 4 degrees Celsius with agitation. Three washes are conducted and the filters then underwent incubation in Promega Western Blue substrate (NBT/BCIP) at room temperature, covered with foil. The appearance of dark spots was monitored, and occurred within 1-15 minutes. Color reactions were terminated by washing filters in sterile milliQ water. Spots can be quantitated with a BioRad Gel Documentation System and analysis is performed with BIORAD Quantity One Quantitation software.
  • Calculating Labeling Efficiency. 5′ End-labeling an Oligonucleotide with ATP-L1-ROX: Method Details
  • A standard method utilized by Molecular Probes/Invitrogen (ULYSIS: Calculating the labeling efficiency of Nucleic Acid Labeling Kit, MP21650) to determine the labeling efficiency of nucleic acids was performed to examine the 5′ end-labeling of an oligonucleotide with VisiGen's ROX-labeled ATP. A reaction was performed as described previously (see Oligonucleotide 5′ end-labeling reaction: Method details) and a 2 microliter sample volume of the purified ROX-labeled oligonucleotide, 50 microMolar ATP-L1-ROX, and resuspension buffer were analyzed on a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Inc, USA) according to the manufacturer's recommendations. Absorbances were measured at 260 nm (λmax for nucleic acid) and 584 nm (λmax for ROX dye) to calculate the Base: Dye ratio. Also, the absorbance of the ATP-L1-ROX was measured at these wavelengths to correct for the dye contribution at the 260 nm reading, i.e., a correction factor (CF260). Equations are given in Research and Design Methods section, see Determining the relative use of the labeled-ATP product in oligonucleotide labeling reactions.
  • TABLE 2
    Calculating the Base:Dye Ratio for a 19 base DNA Oligonucleotide
    5′ end-labeled with ATP-L1-ROX
    Reagent A260 A584
    ROX-Oligonucleotide 0.6505 0.0220
    ATP-L1-ROX 0.0983 0.5388
    CF260 0.18 
    Reagent Extinction Coef. (ε)
    ROX Dye 82000
    ssDNA  8919
    Reagents in Ratio Ratio Value
    Base:Dye  82:1
    Oligonucleotide:Dye 4.3:1
    • 5′ End-Labeling RNA for Microarray Hybridization: Method Details
  • A 20 base RNA oligonucleotide was 5′ end-labeled with ATP-2-Cy3 for the purpose of examining labeling via signal intensity of the hybridized oligonucleotide to a DNA microarray. One microgram of the RNA oligonucleotide was used in a standard 5′ end-labeling reaction,(see Oligonucleotide 5′ end-labeling reaction: Method details) and then hybridized for 18 hours to the array. The array was composed of oligos consisting of the perfect match (PM), single base mismatch (MM1), two mismatched bases (MM2), a single base deletion (Del1), two deletions (Del2), or background spot which contains no oligonucleotide (BGD). The signal intensity profile and corresponding array cross-section illustrate that the Cy3-oligonucleotide is efficiently labeled and specifically hybridizes to the array. This demonstrates proof of principle for direct labeling of RNA and use of same in a microarray experiment, as shown in FIG. 19, below.
  • FIG. 19 depicts 5′ end-labeling an RNA oligonucleotide with ATP-L2-Cy3 and hybridization to a DNA microarray. Intensity diagram of a DNA microarray cross-section using a Cy3-5′ end-labeled-RNA oligonucleotide for hybridization. Intensity signal corresponding to individual microarray spot is shown for each. Microarray layout consists of BGD (Background), Perfect Match (PM), Mismatch of 1 base (MM1), Mismatch of 2 bases (MM2), Deletion of 1 base (Del1), and Deletion of 2 bases (Del2).
  • Develop Standard Method to Directly Label mRNA for Use in Microarray Studies
  • The labeling techniques used in current microarray technology to determine information about the levels of numerous transcripts in parallel are not reproducible, primarily due to labeling and hybridization biases. The present labeling technique will allow microarry researchers to directly label RNA and to characterize the labeling efficiency to determine if the labeled targets are adequate to detect their transcript of interest, prior to committing a costly microarray to a suboptimal experiment (due to insufficient labeling of the target).
  • One preferred application for the labeling technique of this invention is in direct labeling of RNA for use in microarray studies. The present technique transfers a labeled phosphate of an γ-phosphate labeled ATP to the 5′OH (the 5′ end) of a nucleic acid such as DNA, RNA, RNA/DNA mixed biomolecules, ribozymes, or other biomolecules having a 5′OH DNA or RNA terminus. To expose the 5′OH of a mRNA, the mRNA is fragmented prior to labeling. For other biomolecules, fragmentation may or may not be necessary depending on the type of DNA or RNA containing biomolecule. Further, for labeling consistency and associated reproducibility, the fragmentation process preferably produces fragments having an average length of about 200 bases, the targeted size for cDNA used in microarray hybridization experiments. Fragmentation conditions that produce the desired consistency in RNA length can be determined by first exposing total RNA and subsequently mRNA in an alkaline buffer having a pH 9 for increasing amounts of time, and examining the resulting size distribution of the fragmented RNA via denaturing polyacryamide gel electrophoresis. The fragmentation conditions is designed to produce similarly sized mRNA for use in the end-labeling reaction and minimize error.
  • The fragmented RNA are then end-labeled as described above. The end labeling efficiency is then determined for high, medium and low abundance transcripts. To determine an efficiency index for unknown RNA fragments, end labeling actin as an abundant RNA marker, glyceraldehydes-3-phosphate dehydrogenase(GAPDH) as a moderately abundant RNA marker and hypoxanthine-guanine phosphoribosyltransferase(HPRT) as a low abundance RNA marker.
    • Labeled-ATP Syntheses
  • The present invention also relates to a library of γ-phosphate labeled ATPs or other NTP. One preferred class of libraries comprise a plurality of γ-phosphate labeled ATPs where the linker is the same and the fluorophore is different. Another preferred class of libraries comprise a plurality of γ-phosphate labeled ATPs where the fluorophore is the same and the linker is different. Another preferred class of libraries comprise a plurality of γ-phosphate labeled ATPs where the fluorophore and linkers are different.
    • Labeled-ATP Synthesis and Characterization
  • Details of the labeled-ATP synthesis were described above. Once a labeled-ATP is synthesized, it undergoes quality control screening. First, Thin Layer Chromatography (TLC) is run on the product to ensure product integrity by comparing the intact labeled-ATP to phosphodiesterase (PDE) treated labeled-ATP. PDE treatment produces a cleavage event between the bond of the alpha and beta phosphates of the ATP, and results in the decomposition of the intact molecule. Product breakdown can be observed on TLC as spots which migrate differently in comparison to the intact molecule. Second, fluorometric analysis is run to verify the excitation and emission wavelengths of the intact molecule. The inventors have observed a discernible quenching effect between the ATP intermediate and the fluorescent dye that allows us to confirm the integrity of the labeled-ATP via a slight shift in its emission max wavelength and intensity provides us with an additional quality control.
    • Example 14
  • This example illustrates the preparation of ATP-L2-Cy5(TEA+). Although this example illustrates the preparation of ATP-L2-Cy5(TEA+), the preparation works equally well with L3 and L4.
    • Preparation of ATP(TEA+)
  • TEAB buffer (1M, 1L) was made from triethylamine (139 mL) and dry ice (˜200 g). Dowex resin (H+) (100 g) is treated with water (1L), ethanol (300 mL), water (2 L), TEAB (1 M, 1L), water (5L), to prepare the Dowex resin (TEA+) (˜95 g). ATP.Na2 (57 μmol) is transformed to ATP.TEA by passing through Dowex resin (TEA+) (2 g). The collected fractions are tested with a UV lamp and combined solution is lyophilized. The pellet is dissolved in water and its concentration is measured on UV spectrometer (174 mM*300 μL, 52 μmol).
  • Preparation of ATP-L2 (TEA+)
  • ATP.TEA (20 μmol) was rotavaped to dryness with TEA (20 μL) and then rotavaped with methanol (100 μL) three times. The pellet was dried on high vacuum overnight. Dry ice (3 kg)/acetone (500 mL) bath was used as the trap for the pump. DCC (75 μmol) is weighed out and dried on high vacuum overnight. L2 (200 μmol) is treated with TEA (20 μL) and methanol (100 μL) and rotavaped to dryness. It was dried on vacuum overnight. Pyridine (100 mL) was refluxed with CaH2 (8 g) and distilled onto 4A molecular sieves (5 g) under argon (50 mL distillate). Methanol (100 mL) was refluxed with Magnesium (5 g) and distilled onto 4A molecular sieves under argon (50 mL distillate). DMF (100 mL) was refluxed with CaH2 (8 g) and distilled (50 mL) onto 4A molecular sieves under argon (50 mL distillate).
  • The following procedure was carried out under argon. Dried DCC was dissolved in dry DMF/MeOH (200 μL/20 μL) and transferred to dry ATP TEA and stirred at room temperature for 3-4 hours. Pyridine (17 μL) was added and the solution was stirred for 5minutes before evaporated on high vacuum (protected by dry ice-acetone trap). This takes 1-2 hours. L2 in DMF (200-300 μL) was then added to the pellet and the resulting solution was stirred at room temperature overnight. After water (1 mL) was added, the reaction mixture was lyophilized. Water (1.5 mL) was added to the pellet and the solution was centrifuged 14000 rpm*3 minutes. The supernatant was then passed through a red 22 μm syringe filter. The sample was purified on HPLC (SAX column) with TEAB/H2O as elution buffer system (˜200 mL of 1M TEAB). In some cases two such purifications were needed for this amount of material. The collected product fraction was then lyophilized. The pellet was dissolved in HEPES buffer (5 mM, pH 8.5) and its concentration was measured on UV spectrometer (200 μL*48 mM, 9.6 μmol). Its purity was evaluated on silica TLC plate visualized by UV lamp and ninhydrin stain. Yield was 48%.
  • ATP-L2-Cy5 (TEA+)
  • ATP-2 (0.8 μmol, 17 μL) in NaHCO3 buffer (1M, pH 9, 40 μL) and Cy5-NHS (1 μmol) in DMF (42 μL) were mixed and the resulting mixture is set on the shaker to react for 7 hours. Water (1.5 mL) was added and the mixture is lyophilized. A water solution (200 μL) of the pellet was passed through a 1*28 cm Sephadex G-25 column. The right fractions are collected and the combined solution was lyophilyzed. The pellet is dissolved in TEAA (100 mM, 1.5 mL) and purified on HPLC (C18) with TEAA (200 mL, 100 mM)/CH3CN. The product fraction is collected and lyophilized. The pellet was dissolved in HEPES buffer (5 mM, pH8.5). Its concentration was determined on UV spectrometer at 646 nm (80 μL*2.8 mM, 0.22 μmol). Yield was 28%. If necessary, a phosphate assay was carried out as an independent concentration determination. Enzymatic assay was done and analyzed on TLC (silica or PEI cellulose). Mass Spectrometry was determined if necessary.
    • Example 15
  • This example illustrates the preparation of ATP-L1-Cy5 (TEA+). This reaction does not work efficiently for the linker L2, L3, and L4. Although ethylene diamine was used as the linker, the preparation is well suited for other alkenyldiamines.
  • ATP-L1(TEA+) (100 mL) as Elution System
  • ATPNa2 (12.7 μmol) was reacted with L1 (110 μmol) in the presence of EDC (110 μmol) at room temperature for 3 hours and pH was maintained at ˜5.7 over the time. The reaction was monitored by silica TLC. When completed, the solution was adjusted to pH˜7.5 and rotavaped. The pellet was dissolved in water (1.5 mL) and the sample was purified on HPLC (C18) with TEAA (100 mM, 200 mL)/CH3CN (100 mL) as elution system. If needed, the sample was split into two halves for two purifications. The product fraction was lyophilized and the pellet was dissolved in HEPES buffer (5 mM, pH 8.5). Concentration was determined on UV spectrometer (200 μL*24 mM, 4.8 μmol). Yield was 37%. Its purity was evaluated on silica TLC.
  • ATP-L1-Cy5(TEA+)
  • APT-L1 (1 μmol) in NaHCO3 buffer (1 M, pH9, 50 μL) and Cy5-NHS (1.3 μmol) in DMF (100 μL) were mixed and the resulting mixture was set on the shaker and reacted for 6 hr. Water (1.5 mL) was added and the mixture is lyophilized. A water solution (200 μL) of the pellet was passed through a 1*28 cm Sephadex G-25 column. The right fractions were collected and the combined solution was lyophilyzed. The pellet was dissolved in TEAA (100 mM, 1.2 mL) and purified on HPLC (C18) with TEAA (100 mM, 200 mL)/CH3CN (100 mL) as elution system. The product fraction was collected and lyophilized. The pellet was dissolved in HEPES buffer (5 mM, pH8.5). Its concentration was determined on UV spec at 646 nm (1.1 mM*470 μL). Yield is 52%. Enzymatic assay was done and analyzed on TLC. MS was determined if necessary.
  • The above examples also work for GTP and other NTPs.
  • All references cited herein are incorporated by reference. While this invention has been described fully and completely, it should be understood that, within the scope of the appended claims, the invention may be practiced otherwise than as specifically described. Although the invention has been disclosed with reference to its preferred embodiments, from reading this description those of skill in the art may appreciate changes and modification that may be made which do not depart from the scope and spirit of the invention as described above and claimed hereafter.

Claims (20)

1-43. (canceled)
44. The method of claim 80, wherein the nucleotides are the same or different.
45. The method of claim 44, wherein the gamma labeled nucleotide or nucleotide analog comprises a nucleotides-selected from the group consisting of adenine triphosphate (ATP), cytosine triphosphate (CTP), guanine triphosphate (GTP), inosine triphosphate (ITP), thymine triphosphate (TTP), uridine triphosphate (UTP), pseudouridine triphosphate (YTP), xanthine triphosphate (XTP), Orotidine triphosphate (OTP), 5-bromouridine triphosphate (BTP), thiouridine triphosphate (STP), 5,6-dihydrouridine triphosphate (DTP), dATP, dGTP, dTTP, dUTP, dCTP and analogs thereof.
46-48. (canceled)
49. The method of claim 80, wherein the gamma label of the gamma labeled nucleotide or nucleotide analog comprises a linker and a fluorescent dye.
50. The method of claim 49, wherein each linker has the same or different properties, where the properties are selected from the group consisting of chain length, bulk or size, rigidity, polarity and combinations thereof.
51. The method of claim 50, wherein the linker has the general formula -E-R-E-, where E is a main group element selected from the group consisting of C, N, O, P, S and combinations thereof and R is a carbon-containing group having between 1 and about 20 main group atoms selected from the group consisting of B, C, N, O, P, S and combinations thereof, with the valency being completed by H.
52. The method of claim 80, wherein the detecting comprises detecting a detectable property of the gamma label of the gamma labeled nucleotide or nucleotide analog.
53. The method of claim 80, wherein the detectable property is selected from the group consisting of light emission, emission frequency, emission duration, emission intensity, quenching, electron spin, radio-activity, nuclear spin, color, absorbance, near IR absorbance, UV absorbance, and far UV absorbance.
54. The method of claim 52, wherein the analytical technique comprises an analytical chemical or physical instrument for detecting and/or monitoring the property.
55. The method of claim 54, wherein the instrument is selected from the group consisting of a camera, an electron spin resonance spectrometry instrument, a nuclear magnetic resonance (NMR) spectrometry instrument, a UV and visible light spectrometry instrument, a far IR, IR or near IR spectrometry instrument, and an X-ray spectrometromety instrument.
56-74. (canceled)
75. The method of claim 80, wherein the phosphatase comprises an alkaline phosphatase.
76. The method of claim 75, wherein the intestinal phosphatase is selected from the group consisting calf intestinal alkaline phosphatase, shrimp alkaline phosphatase and mixtures or combinations thereof.
77. The method of claim 80, wherein the solution is contained in a container.
78. The method of claim 55, wherein the solution is viewable within a viewing field of a camera, the detectable property is fluorescence and the fluorescence is detectable in a single pixel or pixel-bin of the viewing field of the camera.
79. The method of claim 80, wherein the-label of the gamma labeled nucleotide or nucleotide analog comprises a fluorescent dye.
80. A method comprising:
purifying one or more gamma labeled nucleotides or nucleotide analogs from a solution comprising both unlabeled and gamma labeled nucleotides or nucleotide analogs by contacting the solution with a phosphatase to produce one or more purified gamma labeled nucleotides or nucleotide analogs;
using the one or more purified gamma labeled nucleotides or nucleotide analogs in a polymerase reaction;
detecting each of one or more incorporations of a gamma labeled nucleotide or nucleotide analog into a nascent nucleic acid strand by the polymerase; and
determining the base identity of the at least one incorporated nucleotide or nucleotide analog.
81. The method of claim 80, wherein one or more gamma labeled nucleotides or nucleotide analogs comprise a deoxynucleotide or analog thereof.
82. The method of claim 80, wherein one or more gamma labeled nucleotides or nucleotide analogs comprise a fluorescent or fluorogenic label attached to or associated with the γ-(gamma) phosphate of the nucleotide or nucleotide analog.
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US11/648,138 Abandoned US20070172863A1 (en) 2000-07-07 2006-12-29 Compositions and methods for sequence determination
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