Multipurpose reversible terminators

多用途可逆终结器

• 批准号: 7482019
• 负责人: Daniel Hutter
• 金额: $ 10万
• 依托单位: FIREBIRD BIOMOLECULAR SCIENCES, LLC
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-17 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7482019
• 关键词: 2' -deoxyadenosine triphosphate; 2' -deoxycytidine 5' -triphosphate; Adenine; Agreement; Aldehydes; Architecture; Biological; Biological Process; Businesses; Chemistry; Cleaved cell; Complex; Condition; Confidentiality; Cytosine; DNA; DNA Primers; DNA-Directed DNA Polymerase; Detection; Development; Diagnostic; Dideoxynucleosides; Enzymes; Figs - dietary; Foundations; Funding; Genetic Polymorphism; Genomics; Guanine; HIV; Human; Hydrogen; Indium; Medicine; Molecular; Molecular Evolution; Natural regeneration; Nucleic Acids; Nucleotides; Object Attachment; Oligonucleotides; Oximes; Phase; Polymerase; Polymorphism Analysis; Positioning Attribute; Primer Extension; Property; RNA-Directed DNA Polymerase; Reaction; Reagent; Recovery; Research; Risk; Sales; Science; Scientist; Single Nucleotide Polymorphism; Standards of Weights and Measures; System; Tail; Taq Polymerase; Technology; Technology Transfer; Thymidine; Transferase; Transition Elements; Water; Work; analog; commercial application; commercialization; deoxyguanosine triphosphate; dideoxynucleotide; functional group; human tissue; hydroxyl group; instrument; mutant; novel; nucleic acid analog; nucleoside triphosphate; size; thymidine 5' -triphosphate; tripolyphosphate

DESCRIPTION (provided by applicant): The proposed research will transfer technology from the Foundation for Applied Molecular Evolution (FfAME) to Firebird Biomolecular Sciences LLC, allowing Firebird to commercialize multi-use nucleoside triphosphates whose 3'-OH groups are blocked by a NH2 group. These triphosphates should be accepted by terminal transferase, Terminator DNA polymerase, HIV reverse transcriptase, and mutants of Taq DNA polymerase. When incorporated at the 3'-end of a growing oligonucleotide, the NH2 group blocks extension (just as a 2',3'-dideoxynucleotide does). Unlike a dideoxynucleotide, however, this blockage is reversible; application of a simple, mild reagent rapidly but mildly cleaves the 3'-O-NH2 bond to regenerate the natural 3'-OH group, yielding natural DNA again. Unlike other such terminators, however, the 3'-O-NH2 group not only blocks polymerase extension but also offers a handle that allows the terminated DNA to selectively react with certain functional groups, allowing for the recovery of the extended, terminated DNA from complex biological mixtures, or SNP detection, among others. To enable this commercialization, we shall: (a) Synthesize the 3'-O-NH2 derivatives of thymidine triphosphate, 2'-deoxycytidine triphosphate, 2'-deoxyadenosine triphosphate, and 2'-deoxyguanosine triphosphate. (b) Demonstrate their incorporation using the mutant Taq polymerase that supported the incorporation of TTP-O-NH2. (c) Evaluate the fidelity of incorporation in competition with standard (3'-OH) and 2',3'- dideoxynucleoside triphosphates. (d) Synthesize a thymidine 3'-O-NH2 analog carrying a 5-position fluorescent group and demonstrate its incorporation using polymerases. Repeating the cycle of incorporation and cleavage supports "sequencing using cyclic reversible termination" (SuCRT), a sequencing strategy that can be highly multiplexed. Under confidentiality agreements with Intelligent Bio-Systems, Helicos, and others, this terminator-polymerase combination is presently being evaluated for their instruments and their sequencing architectures. Single cycles of reversible termination also have value. They permit the detection of single nucleotide polymorphisms (SNPs) and multinucleotide polymorphisms in architectures that have increasing complexity and increasing risk. Therefore, these terminators have multiple potential applications in a variety of research settings, and in architectures that might see involvement in human diagnostics. Therefore, they will be added to the growing portfolio of nucleic acid analogs offered by Firebird for sale, and underline the next phase in the development of Firebird as a corporation. Over the past century, our ability to connect genomic information to the molecular characterization of human tissues, and therefore connect molecules to normal and diseased biological function, has been driven by new chemistries that enable new analytical architectures because they deliver new "specs". The proposed research will facilitate the commercialization of one of those new chemistries, which allows DNA primer extension to be reversibly terminated by a group that also can serve as a temporary handle to capture and analyze the extended product.

描述（由申请人提供）：拟议的研究将技术从应用分子进化基金会（FfAME）转移到Firebird Biomolecular Sciences LLC，使Firebird能够商业化其3 '-OH基团被NH 2基团阻断的多用途核苷三磷酸。这些三磷酸应该被末端转移酶、终止子DNA聚合酶、HIV逆转录酶和Taq DNA聚合酶的突变体所接受。当在生长的寡核苷酸的3 '-末端掺入时，NH 2基团阻断延伸（就像2'，3 '-双脱氧核苷酸一样）。然而，与双脱氧核苷酸不同的是，这种阻断是可逆的;应用一种简单、温和的试剂快速但温和地切割3 '-O-NH 2键以再生天然的3'-OH基团，再次产生天然DNA。然而，与其他这样的终止子不同，3 '-O-NH 2基团不仅阻断聚合酶延伸，而且还提供允许终止的DNA选择性地与某些官能团反应的手柄，从而允许从复杂的生物混合物中回收延伸的终止的DNA，或SNP检测等。为了实现这一商业化，我们将： (a)合成胸苷三磷酸、2 '-脱氧胞苷三磷酸、2'-脱氧腺苷三磷酸和2 '-脱氧鸟苷三磷酸的3'-O-NH 2衍生物。(b)使用支持TTP-O-NH 2掺入的突变体Taq聚合酶证明其掺入。(c)评价与标准（3 '-OH）和2'，3 '-二脱氧核苷三磷酸竞争掺入的保真度。(d)合成携带5位荧光基团的胸苷3 '-O-NH 2类似物，并使用聚合酶证明其掺入。 重复掺入和切割的循环支持“使用循环可逆终止的测序”（SuCRT），这是一种可以高度多重化的测序策略。根据与Intelligent Bio-Systems、Helicos和其他公司的保密协议，目前正在评估这种终止子-聚合酶组合的仪器和测序结构。可逆终止的单循环也有价值。它们允许在具有增加的复杂性和增加的风险的结构中检测单核苷酸多态性（SNP）和多核苷酸多态性。因此，这些终止子在各种研究环境中以及可能参与人类诊断的架构中具有多种潜在应用。因此，它们将被添加到Firebird提供的越来越多的核酸类似物产品组合中进行销售，并强调Firebird作为一家公司发展的下一个阶段。 在过去的世纪中，我们将基因组信息与人体组织的分子表征联系起来，从而将分子与正常和患病的生物功能联系起来的能力，一直受到新化学的推动，这些化学能够实现新的分析架构，因为它们提供了新的“规格”。拟议的研究将促进其中一种新化学物质的商业化，该化学物质允许DNA引物延伸被一个基团可逆地终止，该基团也可以作为捕获和分析延伸产物的临时手柄。