Probing dNTP/DNA Polymerase Interactions

探测 dNTP/DNA 聚合酶相互作用

• 批准号: 7418610
• 负责人: LARRY W MCLAUGHLIN
• 金额: $ 22.84万
• 依托单位: BOSTON COLLEGE
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-05-01 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7418610
• 关键词: 2-hydroxypyridine; Antiviral Agents; Antiviral Therapy; Base Pairing; Biological Assay; Class; Complex; DNA; DNA-Directed DNA Polymerase; DNA-Directed RNA Polymerase; Deoxyribose; Discrimination; Effectiveness; Enzymes; Exonuclease; Gel; Goals; HIV; Human; Hydrogen; Hydrogen Bonding; Kinetics; Knowledge; Measures; Minor Groove; Modification; Monitor; Nitrogen; Nucleosides; Nucleotides; Polymerase; Preparation; Process; Prodrugs; Purines; Pyrimidine; Pyrimidines; RNA-Directed DNA Polymerase; Reaction; Shapes; Testing; Toxic effect; Viral; Virus; Virus Diseases; World Health; analog; base; design; functional group; inorganic phosphate; next generation; nuclease; nucleobase; nucleoside analog; phosphoramidite; phosphorothioate; purine; repaired; sugar; tripolyphosphate

DESCRIPTION (provided by applicant): This project will involve the preparation of analogue nucleosides and their triphosphate and phosphoramidite derivatives containing ribo, 2'-deoxyribose- or 2', 3'-dideoxyribose sugars. These analogues differ from the common nucleosides in that the minor groove functional groups, the pyrimdine O2-carbonyls (dT and dC) and the purine N3-nitrogens (dA and dG) are absent, but they maintain normal Watson-Crick hydrogen bonding. These minor groove functional groups are anticipated to be critically important for the activity of some of the DNA polymerases under study. The loss of these functional groups alters both the hydrogen-bonding contacts available to the probing polymerase and additionally alters the van der Waals shape of the nucleoside. In addition to Watson-Crick interstrand hydrogen bonding, van der Waals shape or minor groove contacts appear to be important to the dNTP recognition process. Analogue nucleosides will be prepared as triphosphates for examination of primer/template elongation and inhibition reactions, and from those assays of activity, we will extract kinetic parameters characterizing the reactions. We will initiate the study of RNA polymerases with the corresponding set of NTP derivatives to determine the importance of minor groove functional groups in RNA polymerase reactions. Nucleoside phosphoramidites will permit the preparation of primer/template complexes lacking minor groove functional group character in the "just synthesized" portion of the double-stranded complex. Such contacts may be critical to triggering the exonucleolytic repair activity. Whether the absence of minor groove functional groups permits primer elongation to continue, or results in nuclease activity will be monitored by gel analyses. Complementary structural studies will define the details of the designed analogue base pairs. Based upon the results from these studies, we will design and prepare additional (next-generation) derivatives that should function as better van der Waals shape mimics of the native nucleosides, but still lack minor groove functionality, as well as selected 3'-dNTPs for further studies of RNA polymerases. 3-Deaza-3-methyl purines will be prepared to introduce a significant steric block into the minor groove.

描述（由申请人提供）：该项目将涉及含有核糖、2'-脱氧核糖或2',3'-二脱氧核糖的类似核苷及其三磷酸和亚磷酰胺衍生物的制备。这些类似物与常见核苷的不同之处在于，不存在小沟官能团、嘧啶O2-羰基（dT和dC）和嘌呤N3-氮（dA和dG），但它们保持正常的沃森-克里克氢键。预计这些小沟官能团对于所研究的一些 DNA 聚合酶的活性至关重要。这些官能团的丢失不仅改变了探测聚合酶可用的氢键接触，还改变了核苷的范德华形状。除了 Watson-Crick 链间氢键外，范德华形状或小沟接触似乎对 dNTP 识别过程也很重要。类似核苷将被制备为三磷酸盐，用于检查引物/模板延伸和抑制反应，并且从这些活性测定中，我们将提取表征反应的动力学参数。我们将利用相应的 NTP 衍生物启动 RNA 聚合酶的研究，以确定小沟官能团在 RNA 聚合酶反应中的重要性。 核苷亚磷酰胺将允许制备在双链复合物的“刚合成的”部分中缺乏小沟官能团特征的引物/模板复合物。这种接触对于触发核酸外切修复活性可能至关重要。小沟官能团的缺失是否允许引物继续延伸，或者导致核酸酶活性将通过凝胶分析来监测。互补结构研究将定义设计的模拟碱基对的细节。 根据这些研究的结果，我们将设计和制备额外的（下一代）衍生物，这些衍生物应作为天然核苷的更好的范德华形状模拟物，但仍然缺乏小沟功能，以及选定的 3'-dNTP用于RNA聚合酶的进一步研究。将制备 3-脱氮杂-3-甲基嘌呤，以在小沟中引入显着的空间阻滞。