Multipurpose reversible terminators

描述（由申请人提供）：此第二阶段STTR申请将允许应用分子进化基金会（FfAME）完成其向火鸟生物分子科学有限责任公司的技术转让，允许火鸟商业化三磷酸核苷，其中3'-OH基团被3'-ONH2基团阻断。具有这种修饰的三磷酸被DNA聚合酶和其他酶所接受。在一个生长中的寡核苷酸的3‘端被结合后，3’ -ONH2基团阻断了延伸（就像2',3'-二脱氧核苷酸一样）。然而，这种阻塞是可逆的；缓冲的亚硝酸钠迅速切割O-NH2键，生成天然的3'-OH基团，在温和的条件下产生引物，可以再次被DNA聚合酶延长。此外，3'-ONH2基团提供了一个手柄，允许终止的DNA选择性地与某些功能基团反应，允许从复杂的生物混合物中恢复延长的终止的DNA。这在许多分析DNA的架构中都很有用，包括承诺个性化诊断和治疗的“下一代”测序，检测单核苷酸多态性的工具，为医学治疗进行“有效性研究”的策略，以及其他复杂性和风险不断增加的架构。因此，这里开发的产品在各种研究环境、遗传疾病发现和人类诊断中具有多种应用。在Firebird的内部资金支持下，该二期项目将建立一条生产管道，以制备12种携带可逆终止物的不同三磷酸盐，以及4种酰胺磷，以支持使用这些终止物的试剂盒。在第一阶段开发的合成化学将支持这一管道。此外，在I期用于开发处理非天然核苷三磷酸的聚合酶的工具将被应用于开发更好的聚合酶，用于利用我们的三磷酸的检测。最后，利用这些可逆终止物的DNA分析试剂盒将被生产出来，在许多情况下，与FfAME、Firebird和其他公司专有的其他试剂创新。一些客户已经在开发这些化合物。其中包括外国客户，他们购买这些试剂，通过在国际上销售产品，为美国创造了就业机会。扩大其核酸类似物的投资组合是火鸟公司发展的下一个阶段的基础。

DESCRIPTION (provided by applicant): This Phase 2 STTR application will allow the Foundation for Applied Molecular Evolution (FfAME) to complete its transfer of technology to Firebird Biomolecular Sciences LLC, allowing Firebird to commercialize nucleoside triphosphates where the 3'-OH groups are blocked by a 3'-ONH2 group. Triphosphates having this modification are accepted by DNA polymerases and other enzymes. After being incorporated at the 3'-end of a growing oligonucleotide, the 3'-ONH2 group blocks extension (just as a 2',3'-dideoxynucleotide does). However, this blockage is reversible; buffered sodium nitrite rapidly cleaves the O-NH2 bond to generate the natural 3'-OH group, yielding under mild conditions a primer that can again be extended by DNA polymerase. Further, the 3'-ONH2 group 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. This is useful in many architectures that analyze DNA, including "next generation" sequencing that promises to personalize diagnostics and therapeutics, tools to detect single nucleotide polymorphisms, strategies to do "effectiveness research" for medical treatments, and other architectures that have increasing complexity and risk. Therefore, the products to be developed here have multiple applications in a variety of research settings, genetic disease discovery, and human diagnostics. This Phase 2 project, together with Firebird's internal funding, will build a production pipeline to prepare 12 different triphosphates carrying the reversible terminator, as well as four phosphoramidites to support kits that use these. The synthetic chemistry developed in the Phase 1 period will support this pipeline. Further, tools used in the Phase I period to develop polymerases to handle unnatural nucleoside triphosphates will be applied to create still better polymerases for assays that exploit our triphosphates. Finally, kits for DNA analysis will be produced that exploit these reversible terminators, in many cases with other reagent innovations that are proprietary to FfAME, Firebird, and others. Several customers are already exploiting these compounds. These include foreign customers, whose purchase of these reagents is creating American jobs through the sale of products internationally. Growing its portfolio of nucleic acid analogs underlies the next phase in the development of Firebird as a corporation. PUBLIC HEALTH RELEVANCE: Firebird Biomolecular Sciences LLC commercializes innovative reagents, polymerases that exploit them, and assays and architectures based on them, for the detection, analysis and sequencing of nucleic acids. Firebird also develops software to exploit genomic sequence data, and what it means in biology and medicine. This Phase 2 STTR project will create over two dozen jobs through the development of a series of reversible terminators suitable for next generation DNA sequencing, SNP detection, and DNA capture.