Modified Nucleosides as Tools for Molecular Biology

描述(由申请人提供)：本申请的目标是发展核酸酶学中的简并识别概念，作为理解或操纵生物系统的一般策略。正在考虑的假设是，对一组同位异构型和构象模糊的核苷酸碱基进行电子调谐可以产生有用的选择性探针，用于蛋白质-核酸相互作用。在验证这一假说的过程中，我们将探索改进DNA聚合酶的化学探针的机会，扩大基础生物化学研究的工具，以及加强生物技术。还将在对人类健康构成严重威胁的RNA依赖病毒的背景下探索应用退化识别原则的机会。组建了一组研究人员，将能够有效地结合化学、生化和生物物理方法来研究RNA和DNA聚合酶的分子、结构和动态性质。通过专注于新的生化工具的应用来研究重要的生物系统，我们的目标是促进我们对分子生物学的理解，以及对潜在治疗靶点的表征。 这项提议的目标是： 1.进一步发展唑甲酰胺碱基作为Pol I DNA聚合酶的机制探针。 2.扩大利用DNA聚合酶进行唑甲酰胺核苷酸三磷酸掺入和链延伸的技术基础。 3.以唑类碱基为随机诱变剂，研究新生嘌呤代谢酶5-氨基咪唑核糖核酸羧基酶的分子进化。 4.建立含有唑甲酰胺核苷酸碱基的RNA模板的化学酶方法，并分析这些模板被RNA依赖聚合酶识别的情况。

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.