Probing dNTP/DNA Polymerase Interactions

描述（由申请人提供）：该项目将涉及制备类似核苷及其含有核糖，2'-脱氧核糖或2',3'-二脱氧核糖糖的三磷酸和磷酸酰胺衍生物。这些类似物与普通核苷的不同之处在于，它们不存在较小的凹槽官能团，pypymd02 -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。这些小凹槽官能团被认为对一些正在研究的DNA聚合酶的活性至关重要。这些官能团的缺失既改变了探测聚合酶可用的氢键接触，也改变了核苷的范德华形状。除了沃森-克里克链间氢键外，范德华形状或小槽接触似乎对dNTP识别过程很重要。类似的核苷将被制备成三磷酸盐，用于检测引物/模板的延伸和抑制反应，并从这些活性分析中提取表征反应的动力学参数。我们将启动具有相应NTP衍生物的RNA聚合酶的研究，以确定次要凹槽官能团在RNA聚合酶反应中的重要性。

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.