Structure and Function of Non-Coding RNA

项目总结/摘要 我的团队开发并使用化学和生物化学工具来研究 非编码RNA（ncRNA）及其与蛋白质（RNP）和小分子（smRNA）的复合物。在 在本项目的背景下，我们将特别关注两个一般领域：1）工程片段抗原结合 （Fab）作为RNA/RNP/smRNA晶体学、冷冻电子显微镜（cryoEM）的伴侣，以及作为使 RNA生物学的工具，2）阐述RNA功能的机制，特别是催化RNA（核酶）。 陆地转录组包含大量编码和非编码的结构化RNA 转录本单独或与蛋白质协同作用，在几乎所有方面发挥至关重要的作用 细胞功能。了解这些RNA如何介导健康和疾病中的生物学功能需要 他们的三维结构的知识。我们将部署各种结构生物学，生物物理学， 生物化学、计算和化学方法，包括动力学同位素效应分析和 从人工扩增的遗传系统中提取核苷酸。从我们的重组Fab工作中得到的数据 将产生一个强大的平台来产生以高亲和力和特异性结合结构化RNA的Fab， 建立Fab作为结构和机制RNA生物学中强有力试剂并产生新的大分子 结构，这对我们理解潜在的生物学有深远的影响，并作为一个框架， 开发和测试功能假设。此外，我们对催化RNA的深入研究将首次定义 时间催化结构和过渡态结构之间的关系，一个缺失的环节，一直困扰着 努力设计具有新催化功能的大分子。除了概念上的进步， 这项工作有望为RNA社区提供强大的RNA/RNP结构Fab试剂， 功能研究，核苷酸类似物，以支持新的方法来定义RNA催化机制， 通过增加信息密度使大RNA更易处理的策略。

PROJECT SUMMARY/ABSTRACT My group develops and uses chemical and biochemical tools to investigate the structure and function of noncoding RNAs (ncRNA) and their complexes with proteins (RNPs) and small molecules (smRNAs). In the context of this project, we will specifically focus on two general areas: 1) engineering fragment antigen binding (Fabs) as chaperones for RNA/RNP/smRNA crystallography, cryoelectron microscopy (cryoEM) and as enabling tools for RNA biology, and 2) elaborating mechanisms of RNA function, especially catalytic RNAs (ribozymes). The terrestrial transcriptome contains vast numbers of structured RNAs within both coding and noncoding transcripts that act alone and in concert with proteins to carry out critically important roles in nearly every facet of cellular function. Understanding how these RNAs mediate biological function in health and disease requires knowledge of their three-dimensional architectures. We will deploy a variety of structural biology, biophysical, biochemical, computational and chemical approaches, including kinetic isotope effect analysis and the use of nucleotides from an artificially expanded genetic system. The data that emerge from our recombinant Fab work will engender a robust platform to generate Fabs that bind structured RNA with high affinity and specificity, establish Fabs as powerful reagents in structural and mechanistic RNA biology and yield new macromolecular structures, which have profound impact on our understanding of the underlying biology and serve as a framework to develop and test functional hypotheses. In addition, our in-depth work on catalytic RNA will define for the first time the relationship between catalytic structure and transition state structure, a missing link that has plagued efforts to engineer macromoleclules with novel catalytic functions. Beyond the conceptual advances that emerge, this work promises to deliver to the RNA community powerful Fab reagents for RNA/RNP structural and functional studies, nucleotide analogs to support novel approaches to define RNA catalytic mechanisms, and strategies to render large RNAs more tractable by increasing the density of information.