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 分子参与与人类疾病有关的最基本的细胞过程。 RNA 包含对 RNA 功能做出关键贡献并具有吸引力的结构化模块药物靶标，尤其是无法治愈的疾病以及与“不可成药”蛋白质相关的知识。这些RNA的三维结构将有助于理解RNA功能的机制并可以大大加速药物发现工作然而，传统的 RNA 结构方法。测定、X射线晶体学和核磁共振波谱法，是费力且具有严格的技术性的与晶体学相比，单颗粒低温电子显微镜 (cryo-EM) 有许多优点。和 NMR，但仅适用于大分子或大分子组装体，不能用于大多数天然 RNA 由于其大小不足而被采用，该提案的重点是开发一种新型RNA。制备 RNA 冷冻电镜样品的方法，该方法规避了大小限制，省略了 RNA 纯化和 RNA 重折叠步骤，并允许轻松的冷冻电镜数据处理和结构解决方案。提出的概念验证研究结合了三个具体目标，具体目标 1 致力于开发。通过体外转录制备均一 RNA 种类的新生化方法的具体目标 2。将使用计算模型、生物化学和单粒子冷冻电镜实验来开发检测方法一种制备与中小型结构解决方案兼容的冷冻电镜样品的方法目标 3 将使用模型 RNA 分子和传统的单颗粒验证该方法。该提案集成了计算方法、生化分析和基于冷冻电镜的结构测定，开发一种通用且简单的方法来求解结构预计所提出的技术优于大多数 RNA 和 RNA 药物复合物。现有方法在劳动力、成本和适用性方面。