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）：拟议的研究将把技术从应用分子进化基金会 (FfAME) 转让给 Firebird Biomolecular Sciences LLC，使 Firebird 能够将其 3'-OH 基团被 NH2 基团封闭的多用途三磷酸核苷商业化。这些三磷酸应该被末端转移酶、Terminator DNA 聚合酶、HIV 逆转录酶和 Taq DNA 聚合酶突变体所接受。当掺入正在生长的寡核苷酸的 3' 端时，NH2 基团会阻止延伸（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。使用简单、温和的试剂可快速但温和地裂解 3'-O-NH2 键，从而重新生成天然 3'-OH 基团，从而再次产生天然 DNA。然而，与其他此类终止子不同的是，3'-O-NH2 基团不仅阻止聚合酶延伸，而且还提供了一个手柄，允许终止的 DNA 选择性地与某些官能团反应，从而允许从复合物中恢复延伸的终止 DNA生物混合物或 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-NH2 衍生物。 (b) 使用支持 TTP-O-NH2 掺入的突变型 Taq 聚合酶证明它们的掺入。 (c) 评估与标准 (3'-OH) 和 2',3'- 双脱氧核苷三磷酸竞争掺入的保真度。 (d) 合成带有 5 位荧光基团的胸苷 3'-O-NH2 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等公司签订的保密协议，目前正在评估这种终止子-聚合酶组合的仪器和测序架构。单周期可逆终止也具有价值。它们允许检测复杂性和风险不断增加的结构中的单核苷酸多态性 (SNP) 和多核苷酸多态性。因此，这些终结器在各种研究环境以及可能涉及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 提供的不断增长的核酸类似物产品组合中进行销售，并强调 Firebird 作为一家公司的下一阶段的发展。 在过去的一个世纪里，我们将基因组信息与人体组织的分子特征联系起来，从而将分子与正常和患病的生物功能联系起来的能力，一直受到新化学物质的推动，这些新化学物质提供了新的“规格”，从而实现了新的分析架构。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 引物延伸被一组可逆地终止，该组也可以作为捕获和分析延伸产物的临时句柄。