Probing dNTP/DNA Polymerase Interactions

描述(申请人提供)：该项目将涉及含有核糖核糖，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。这些类似物与常见的核苷的不同之处在于，没有次要的沟槽官能团、吡啶O2-羰基(dT和dc)和嘌呤N3-氮基(da和dg)，但它们保持了正常的Watson-Crick氢键。这些微小的沟槽官能团预计对一些正在研究的DNA聚合酶的活性至关重要。这些官能团的丧失改变了探测聚合酶可用的氢键接触，并另外改变了核苷的范德华形状。除了Watson-Crick链间氢键外，van der Waals形状或次要凹槽接触似乎对dNTP识别过程也很重要。类似的核苷将被制备为三磷酸盐，用于检测底物/模板延伸和抑制反应，并从这些活性测定中提取表征反应的动力学参数。我们将开始研究具有相应NTP衍生物的RNA聚合酶，以确定微小沟槽官能团在RNA聚合酶反应中的重要性。 核苷亚磷酰胺可用于制备在双链复合体的“刚合成”部分缺乏微小沟槽官能团特征的底物/模板复合体。这种接触可能是触发核外溶解修复活动的关键。是否缺少较小的沟槽官能团允许引物继续延伸，或导致核酸酶活性将通过凝胶分析进行监测。互补的结构研究将确定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，这些衍生物应该能够更好地模拟天然核苷的van der Waals形状，但仍然缺乏次要的凹槽功能，以及选择3‘-dNTPs用于进一步的RNA聚合酶研究。3-去氮-3-甲基嘌呤将被制备成在小凹槽中引入显著的空间嵌段。

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.