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）：拟议的研究将将技术从应用的分子进化基金会（FFAME）转移到火鸟生物分子科学LLC，从而使Firebird可以将其3'-oH基团被NH2组封闭的多使用核苷三磷酸盐商业化。这些三磷酸应通过末端转移酶，末端DNA聚合酶，HIV逆转录酶和TAQ DNA聚合酶的突变体接受。当在生长的寡核苷酸的3'末端掺入时，NH2组会阻止延伸（就像2'，3'-二维核苷酸一样）。但是，与二乙氧基核苷酸不同，这种阻塞是可逆的。简单，轻度试剂的应用快速但轻度切割3'-O-NH2键以再生天然3'-OH组，再次产生天然DNA。然而，与其他终结剂不同，3'-O-NH2组不仅可以阻止聚合酶扩展，而且提供了一种手柄，允许终止的DNA与某些官能团有选择性反应，从而使从复杂的生物混合物或SNP检测中恢复了扩展的，终止的DNA。为了实现这种商业化，我们将： （a）合成三磷酸胸苷的3'-O-NH2衍生物，2'-脱氧三磷酸，2'-脱氧腺苷三磷酸和2'-脱氧鸟苷三磷酸。 （b）使用支持TTP-O-NH2的掺入的突变型TAQ聚合酶演示其掺入。 （c）评估与标准（3'-OH）和2'-3'-二乙氧基核苷三磷酸盐竞争中融合的保真度。 （d）合成携带5位荧光组的胸苷3'-O-NH2模拟，并使用聚合酶演示其掺入。 重复融合和裂解的循环支持“使用环状可逆终止的测序”（SUCRT），这是一种可以高度多重的测序策略。根据与智能生物系统，Helicos和其他人的机密协议，目前正在评估这种终结者 - 聚合酶组合的仪器和测序体系结构。可逆终止的单个循环也具有价值。它们允许在体系结构中检测单核苷酸多态性（SNP）和多核苷酸多态性，这些体系结构的复杂性增加和风险增加。因此，这些终结者在各种研究环境中都有多个潜在的应用，以及可能参与人类诊断的体系结构。因此，将它们添加到Firebird待售的核酸类似物中不断增长的投资组合中，并强调了Firebird作为公司发展的下一阶段。 在过去的一个世纪中，我们将基因组信息与人体组织的分子表征联系起来，因此将分子与正常和患病生物学功能联系起来的能力是由新的化学作用驱动的，这些化学成分可实现新的分析架构，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底漆扩展由一个组可逆地终止，该组也可以用作捕获和分析扩展产品的临时手柄。