A universal approach for determining three-dimensional RNA structures

确定三维 RNA 结构的通用方法

基本信息

批准号：
10724848
负责人：
Alexander Serganov
金额：
$ 29.66万
依托单位：
NEW YORK UNIVERSITY SCHOOL OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-09-01 至 2025-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10724848
关键词：
3-Dimensional Acceleration Address Binding Biochemical Biological Assay Biological Process Cell physiology Chemicals Classification Complex Computer Models Computing Methodologies Cryoelectron Microscopy Crystallography DNA DNA-Directed RNA Polymerase Data Collection Development Disease Drug Targeting Elements Future Genetic Transcription Heterogeneity Immobilization In Vitro Knowledge Length Lesion Location Methodology Methods Modeling Movement NMR Spectroscopy Oligonucleotides Pharmaceutical Preparations Pharmacologic Substance Positioning Attribute Preparation Proteins RNA RNA Folding RNA Stability RNA chemical synthesis RNA purification RNA-targeting therapy Reaction Resolution Role Sampling Site Small RNA Structure Technology Thermodynamics Transcript X-Ray Crystallography computerized data processing cost data structure design drug discovery effective therapy experimental study human disease hydrophilicity improved innovation insight interest macromolecular assembly nanoarchitecture new technology novel novel strategies particle promoter scaffold small molecule three dimensional structure tool viral RNA

项目摘要

Summary RNA molecules participate in the most fundamental cellular processes implicated in human disease. Many RNAs contain structured modules that make critical contributions to RNA functions and represent attractive drug targets, especially for diseases without a cure and associated with “undruggable” proteins. Knowledge of the three-dimensional structures of these RNAs would help to understand the mechanism of the RNA function and could greatly accelerate drug discovery efforts. However, traditional methods for RNA structure determination, X-ray crystallography and NMR spectroscopy, are laborious and have serious technical limitations. Single-particle cryogenic electron microscopy (cryo-EM) has many advantages over crystallography and NMR but is applicable only for large molecules or macromolecular assemblies and cannot be used for the majority of natural RNAs because of their insufficient size. This proposal is focused on developing a novel approach for preparing cryo-EM samples of RNA that circumvents the size restrictions, omits the RNA purification and RNA refolding steps, and allows facile cryo-EM data processing and structure solution. The proposed proof-of-concept study combines three specific aims. Specific Aim 1 is devoted to the development of the novel biochemical approach for preparing uniform RNA species by in vitro transcription. Specific Aim 2 will use computational modelling, biochemical assays, and single-particle cryo-EM experimentation to develop a methodology for preparing cryo-EM samples compatible with the structure solution of small- and medium- sized RNAs. Aim 3 will validate the methodology using model RNA molecules and conventional single particle cryo-EM structure solution pipeline. The proposal integrates computational methods, biochemical assays, and cryo-EM-based structure determination to develop a universal and simple approach for solving structures of the majority of RNA and RNA-drug complexes. The proposed technology is anticipated to be superior to the existing methods in labor, cost, and applicability.

概括 RNA分子参与了人类疾病实施的最基本的细胞过程。许多 RNA包含结构化模块，可为RNA功能做出重要贡献并代表吸引人药物靶标，尤其是对于没有治愈的疾病，并且与“不良”蛋白质有关。知识这些RNA的三维结构将有助于了解RNA功能的机制并且可以大大加速药物发现工作。但是，RNA结构的传统方法测定，X射线晶体学和NMR光谱是实验室，具有严重的技术限制。单粒子低温电子显微镜（Cryo-EM）比晶体学具有许多优势和NMR，但仅适用于大分子或大分子组件，不能用于大多数天然RNA的大小不足。该提议的重点是发展小说制备规避尺寸限制的RNA的冷冻EM样品的方法，省略了RNA 纯化和RNA重新折叠步骤，并允许轻松的冷冻EM数据处理和结构解决方案。拟议的概念证明研究结合了三个具体目标。具体目标1用于开发通过体外转录制备均匀RNA规格的新型生化方法。具体目标2 将使用计算建模，生化测定和单粒子冷冻EM实验一种方法，用于制备与中小型和中等的结构溶液兼容的冷冻EM样品大小的RNA。 AIM 3将使用模型RNA分子和常规单个粒子验证方法冷冻EM结构解决方案管道。该提案整合了计算方法，生化测定和基于冷冻EM的结构确定，以开发一种通用和简单的方法来解决结构大多数RNA和RNA-Prug复合物。预计拟议的技术将优于现有的人工，成本和适用性方法。