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(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date _ . ... - 12 May 2011 (12.05.2011) W 2 11/05661 1 Al (51) International Patent Classification: (81) Designated States (unless otherwise indicated, for every C12Q 1/00 (2006.0 1) C12Q 1/48 (2006.0 1) kind of national protection available): AE, AG, AL, AM, C12Q 1/42 (2006.01) AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, (21) Number: International Application DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, PCT/US20 10/054171 HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, (22) International Filing Date: KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, 26 October 2010 (26.10.2010) ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PE, PG, PH, PL, PT, RO, RS, RU, SC, SD, (25) Filing Language: English SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, (26) Publication Language: English TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (30) Priority Data: (84) Designated States (unless otherwise indicated, for every 61/255,068 26 October 2009 (26.10.2009) US kind of regional protection available): ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, (71) Applicant (for all designated States except US): ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, MYREXIS, INC. [US/US]; 305 Chipeta Way, Salt Lake TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, City, Utah 84108 (US). EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, (72) Inventors; and SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, (75) Inventors/Applicants (for US only): OSTANIN, Kirill GW, ML, MR, NE, SN, TD, TG). [US/US]; 4601 Idlewild Road, Salt Lake City, Utah 84124 (US). GAO, Zhong-Hua [US/US]; 305 Chipeta Published: Way, Salt Lake City, Utah 84108 (US). MORHAM, — with international search report (Art. 21(3)) Scott, G. [US/US]; 305 Chipeta Way, Salt Lake City, Utah 84108 (US). — before the expiration of the time limit for amending the claims and to be republished in the event of receipt of (74) Agent: LEY III, Herbert, L.; c/o CPA Global, P. O. Box amendments (Rule 48.2(h)) 52050, Minneapolis, Minnesota 55402 (US). (54) Title: COUPLED REACTIONS FOR ANALYSIS OF NUCLEOTIDES AND THEIR HYDROLYSIS (57) Abstract: The present invention provides novel methods, reaction mixtures, and kits for the quantitative and qualitative anal ysis of nucleotides other than ATP, for the quantification of the hydrolysis of nucleotides other than ATP, and for the quantitative and qualitative analysis of the activity of enzymes that generate or consume nucleotides other than ATP. COUPLED REACTIONS FOR ANALYSIS OF NUCLEOTIDES AND THEIR HYDROLYSIS RELATED APPLICATIONS [000 1] This application claims the benefit of U.S. Provisional Patent Application Serial No. 61/255,068, filed October 26, 2009, the contents of which are incorporated by reference herein in their entirety. FIELD OF THE INVENTION [0002] The present invention generally relates to biochemical assays, and particularly to methods for the quantitative and qualitative analysis of nucleotides and their hydrolysis. BACKGROUND OF THE INVENTION [0003] Nucleotides play many roles in living cells. In addition to their somewhat mundane role as the building blocks of the nucleic acids, nucleotides play vital and central roles in cellular metabolism and bioenergetics. Nucleotides (particularly nucleoside triphosphates, or NTPs) serve as the "energy currency" of the cell. In their high-energy phosphorylated forms, nucleoside triphosphates provide a source chemical energy for numerous metabolic transactions and transformations. Nucleotides (and nucleosides) also serve as structural and functional components of a variety of enzyme cofactors, as well as metabolic intermediates in a wide variety of enzymatic reactions. Finally, nucleotides (and nucleosides) serve as small-molecule messengers that can activate or facilitate the response of cells to hormones and a variety of extracellular stimuli. [0004] As a consequence of the many important roles played by nucleotides in living systems, biochemists often desire to determine the types and amounts of nucleotides present both in vivo and in vitro, and measure their rates and extent of hydrolysis. In order to study various enzymatic reactions involving nucleotides, biochemists require simple and effective assays that allow for quantification of specific nucleotides, particularly just before, or immediately after, an enzymatic reaction occurs. Additionally, biochemists require assays that are specific for particular nucleotides that are involved in cellular signaling. [0005] To date, the only convenient and commercially available nucleotide hydrolysis assays are designed to measure adenosine triphosphate (i.e., ATP) hydrolysis. Assays for analyzing the hydrolysis of other nucleotide triphosphates are limited by high background caused by acidic reaction conditions and a lack of sensitivity (>10 µΜ detection capability). Indeed, the commonly used assay for nucleotide hydrolysis uses a malachite green based colorimetric assay. (See, e.g. : Kirchgesser & Dahlmann; A colorimetric assay for the determination of acid nucleoside triphosphatase activity; J. Clin. Chem. Clin. Biochem. 28:407-41 1, 1990.) In these assays, nucleotide hydrolysis releases inorganic phosphate (Pi). The Pi forms a yellowish complex with malachite green dye under highly acidic conditions. Because the assays are based upon changes in the optical density readout at a particular wavelength, the sensitivity of this assay type is relatively low (above 10 µΜ Pi). Additionally, this type of assay requires the use of sulfuric acid for color generation. Sulfuric acid itself can cause spontaneous acid hydrolysis of nucleotides, thereby interfering with the assays by increasing background. Further, such acidic assay conditions are unsuitable for high-throughput screening (HTS) machineries. [0006] In view of the above, there is a need for improved methods and assays that can be used for the quantitative and qualitative analysis of specific types of nucleotides, including nucleoside monophosphates, nucleoside diphosphates, nucleoside triphosphates (other than ATP), and cyclic nucleotides, and there is a need for improved methods and assays with which to measure the hydrolysis of nucleotides (other than ATP) by enzymes that utilize such nucleotides. BRIEF SUMMARY OF THE INVENTION [0007] The present invention provides a new approach and methods for developing assays for the qualitative and quantitative analysis of most biologically- relevant nucleotides other than ATP, as well as assays to monitor the hydrolysis of such nucleotides. Disclosed herein are working examples of specific assays for the qualitative and quantitative analysis of specific nucleotides and their hydrolysis that utilize this approach and these methods. Indeed, the inventive approach and methods described herein may be used to create a variety of assays that can be used to monitor a variety of enzymatic nucleotide hydrolysis reactions, and may be adapted to qualitatively and quantitatively assay most biologically-relevant ribonucleotides and deoxyribonucleotides, including, but not limited to, AMP, dAMP, GMP, dGMP, CMP, dCMP, UMP, TMP, IMP, XMP, ADP, dADP, GDP, dGDP, CDP, dCDP, UDP, TDP, CTP, GTP, TTP, UTP, dATP, dCTP, dGTP, dTTP cAMP, cGMP, and c-di-GMP. Thus the invention provides methods for the quantitative and qualitative analysis of a wide variety of nucleotides, other than ATP, and provides methods for quantifying their hydrolysis. [0008] The methods and assays of the present invention are applicable to a wide range of pursuits including, but not limited to, clinical diagnoses, drug development and academic research. As a result of their ready adaptability and broad utility, these assays are of value to biochemists, molecular biologists and clinicians, among others. [0009] The nexus of these methods and assays involves the coupling of an ATP- generating cyclic reaction referred to herein as a "Nucleotide Exchange Reaction" (or "NEPv") to any suitable reaction that serves as a means of detecting the ATP generated by the NER (i.e., the "ATP detection reaction" or "ADR"). The NER comprises a reversible reaction catalyzed by a single enzyme. That enzyme is a Nucleoside Monophosphate Kinase (or NMPK), which, in one direction (the "forward" reaction), acts to transfer a high-energy phosphate from a substrate ribonucleoside triphosphate (NTP) other than ATP, or in some cases, a deoxyribonucleoside triphosphate (dNTP), to a ribonucleoside monophosphate (NMP) or dexoyribonucleoside monophosphate (dNMP), to form two corresponding nucleoside diphosphates (NDP or dNDP). In the opposite direction (the "reverse" reaction), the NMPK catalyzes the transfer of a phosphate group from a ribonucleoside diphosphate (NDP), or in some cases a deoxyribonucleoside diphosphate (dNDP), to ADP to form ATP and a corresponding NMP or dNMP. With each "cycle" of the cyclic NER, a single ATP is produced, which then detected by the coupled ATP detection reaction or ADR. [0010] The NMPKs are a family of enzymes, the members of which have evolved to transfer phosphates between particular ribonucleotide triphosphates (NTPs) or, in some cases, a deoxyribonucleotidetriphosphates (dNTPs), and ADP. Hence, by the selective inclusion of a particular NMPK, and a particular
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  • Structure and Function Of

    Structure and Function Of

    8/5/2019 DNA and RNA The Code of Life The Human Genome Project Gene Expression Genetic code The alphabet of the genetic code contains Genes are DNA sequences that encode proteins (the gene product) only four letters (A,T,G,C). Gene expression refers to the process A number of experiments confirmed that the whereby the information contained in genes genetic code is written in 3-letter words, each begins to have effects in the cell. of which codes for particular amino acid. DNA encodes and transmits the genetic A nucleic acid word (3 nucleotide letters) is information passed down from parents to offspring. referred to as a codon. 1 8/5/2019 Nucleic acids Nucleotides Principle information molecule in the Nucleotides are the unit structure of cell. nucleic acids. Nucleotides composed of 3 All the genetic codes are carried out on components: the nucleic acids. Nitrogenous base (A, C, G, T or U) Pentose sugar Nucleic acid is a linear polymer of Phosphate nucleotides Nucleotides Nitrogenous bases There are four nitrogen bases making up four different nucleotides. There are 2 types: Purines(pyoo r-een): Adenine A Two ring structure Purines Adenine (A) and Guanine (G) Guanine G Pyrimidines(pahy-rim-i-deen,): N base Single ring structure Cytosine (C) and Thymine (T) or Uracil (U). Thymine T Pyrimidines Cytosine C 2 8/5/2019 A NUCLEOTIDE Nucleotide bases 1. Phosphate Group 2.3. 5Nitrogen-Carbon BaseSugar 1. Phosphate Group 2. 5-Carbon Sugar (Dexoyribose or Ribose) 3. (Dexoyribose or Ribose) 1. 3. Nitrogen Base 2. 3. Nucleotides, too O 1.