Modified Nucleosides as Tools for Molecular Biology

描述（由申请人提供）：本申请的目的是发展核酸酶学中简并识别的概念，作为理解或操纵生物系统的一般策略。所考虑的假设是，电子调谐的一组电子等排和构象模糊的核碱基可以导致在蛋白质-核酸相互作用的选择性的有用的探针。在测试这一假设的过程中，我们将探索改进DNA聚合酶的化学探针的机会，并扩展基础生物化学研究的工具，以及提高生物技术。应用简并识别原理的机会也将在对人类健康构成严重威胁的RNA依赖性病毒的背景下进行探索。一个研究小组的组装，将使化学，生物化学和生物物理方法的有效结合，以解决RNA和DNA聚合酶的分子，结构和动态特性。通过专注于新的生物化学工具的应用，以调查重要的生物系统，我们的目标是推进我们的分子生物学的理解，并表征潜在的治疗靶点。 该提案的目标是： 1.进一步开发唑酰胺核苷碱基作为Pol Ⅰ DNA聚合酶的机理探针。 2.目的：为利用DNA聚合酶进行氮唑羧酰胺核苷三磷酸掺入和链延伸奠定技术基础。 3.利用唑类碱基作为随机诱变剂，研究嘌呤从头代谢酶5-氨基咪唑核苷酸羧化酶（AIRC）的分子进化。 4.开发用于产生含有唑羧酰胺核碱基的RNA模板的化学酶方法，并分析这些模板在RNA依赖性聚合酶的识别中的作用。

DESCRIPTION (provided by applicant): The goals of this application are to develop the concepts of degenerate recognition in nucleic acid enzymology as a general strategy for understanding or manipulating biological systems. The hypothesis under consideration is that the electronic tuning of a set of isosteric and conformationally ambiguous nucleobases can result in useful probes of selectivity in protein-nucleic acid interactions. In the course of testing this hypothesis, we will explore opportunities to refine chemical probes of DNA polymerases and expand the tools for basic biochemical studies as well as enhance biotechnology. The opportunities for applying the principle of degenerate recognition will be also explored in the context of RNA dependent viruses that pose serious threats to human health. A team of investigators are assembled that will enable an effective combination of chemical, biochemical, and biophysical methods to address the molecular, structural and dynamic properties of the RNA and DNA polymerases. By focusing on the application of novel biochemical tools to investigate important biological systems, we aim to advance both our understanding of molecular biology, and the characterization of potential therapeutic targets. The goals of this proposal are: 1. To further develop azole carboxamide nucleobases as mechanistic probes of Pol I DNA polymerases. 2. To expand the technology base for azole carboxamide nucleotide triphosphate incorporation and strand extension using DNA polymerases. 3. To implement azole nucleobases as random mutagens to study the molecular evolution of 5- aminoimidazole ribonucleotide carboxylase (AIRC), an enzyme in de novo purine metabolism. 4. To develop chemoenzymatic methodologies for creating RNA templates containing azole carboxamide nucleobases and to analyze these templates in their recognition by RNA dependent polymerases.