A universal approach for determining three-dimensional RNA structures

确定三维 RNA 结构的通用方法

• 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
• 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

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.

总结 RNA分子参与人类疾病中涉及的最基本的细胞过程。许多 RNA包含结构化模块，这些模块对RNA功能做出关键贡献，并代表了吸引人的功能。 药物靶点，特别是对于无法治愈的疾病和与“不可药”蛋白质相关的疾病。知识 这些RNA的三维结构将有助于理解RNA功能的机制 并且可以大大加速药物发现的努力。然而，传统的RNA结构分析方法 测定、X射线晶体学和NMR光谱学是费力的并且具有严重的技术问题。 局限性。单粒子低温电子显微镜（cryo-EM）与晶体学相比有许多优点 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，因为它们的大小不足。这项提案的重点是开发一种新的 一种制备RNA的冷冻EM样品的方法，该方法避开了大小限制，省略了RNA 纯化和RNA重折叠步骤，并允许简易的冷冻-EM数据处理和结构解决方案。的 拟议的概念验证研究结合了三个具体目标。具体目标1致力于发展 通过体外转录制备均一RNA种类的新生物化学方法。具体目标2 将使用计算建模，生化分析和单粒子冷冻EM实验来开发 一种用于制备与中小微生物结构溶液相容的低温EM样品的方法， 大小的RNA目标3将使用模型RNA分子和常规单颗粒验证方法学 cryo-EM结构溶液管道。该提案整合了计算方法，生物化学测定， 基于cryo-EM的结构确定，以开发一种通用且简单的方法来解决 大部分RNA和RNA-药物复合物。预计所提出的技术将上级 现有的方法在劳动力、成本和适用性方面。