Probing dNTP/DNA Polymerase Interactions

描述（由申请人提供）：本项目将涉及制备类似物核苷及其含有核糖、2 '-脱氧核糖或2'，3 '-双脱氧核糖的三磷酸和亚磷酰胺衍生物。这些类似物与普通核苷的不同之处在于，小沟官能团，嘧啶O2-羰基（dT和dC）和嘌呤N3-氮（dA和dG）不存在，但它们保持正常的沃森-克里克氢键。这些小沟官能团预计是至关重要的一些DNA聚合酶的活性正在研究中。这些官能团的损失改变了探测聚合酶可用的氢键接触，并且另外改变了核苷的货车范德华形状。除了沃森-克里克链间氢键，货车德瓦尔斯形状或小沟接触似乎是重要的dNTP识别过程。将抗肿瘤核苷制备为三磷酸盐，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。我们将启动RNA聚合酶与相应的NTP衍生物的研究，以确定在RNA聚合酶反应小沟功能基团的重要性。 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是至关重要的。小沟官能团的缺失是否允许引物延伸继续，或导致核酸酶活性，将通过凝胶分析进行监测。互补结构研究将确定所设计的类似物碱基对的细节。 基于这些研究的结果，我们将设计和制备额外的（下一代）衍生物，其应该作为天然核苷的更好的货车德瓦尔斯形状模拟物，但仍然缺乏小沟功能，以及选择的3 '-dNTP用于RNA聚合酶的进一步研究。将制备3-脱氮-3-甲基嘌呤以将显著的空间嵌段引入小沟。

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.