High Throughput Determination of RNA 3D Structures and Dynamics in Vivo

体内 RNA 3D 结构和动力学的高通量测定

• 批准号: 10276941
• 负责人: Zhipeng Lu
• 金额: $ 41.25万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-15 至 2026-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10276941
• 关键词: 3-Dimensional; Benchmarking; Binding; Biological; Biology; Cells; Chemicals; Clinic; Clinical; Code; Complex; Coupled; Crosslinker; Cryoelectron Microscopy; Crystallography; Development; Disease; Gene Expression; Genetic; Goals; Guide RNA; High-Throughput Nucleotide Sequencing; In Vitro; Instruction; Length; Life Cycle Stages; Ligation; Measures; Mediating; Medicine; Methods; Modeling; Molecular; Molecular Conformation; Nucleotides; Oligonucleotides; Physiological; Play; Proteins; RNA; RNA Sequences; RNA Viruses; RNA analysis; Research; Resolution; Role; Sampling; Signal Transduction; Structure; Technology; Therapeutic Intervention; base; computerized tools; crosslink; human disease; improved; in vivo; mathematical theory; programs; small molecule; stem; structural biology; targeted treatment; technology development; therapeutic development; three dimensional structure; three-dimensional modeling; transcriptome; viral RNA; virtual

In addition to coding proteins, RNA plays fundamental roles in virtually every aspect of biology. The extreme functional diversity of RNA stems from its ability to fold into complex structures and, like machines, dynamically take input, transmit signal and force, and execute genetic instructions. RNA structures regulate every step of gene expression in cells and control the life cycle of RNA viruses. As a result, physiological and abnormal activities underlie a variety of human diseases. In recent years, targeting RNA has transitioned from an interesting academic idea to a reality in the clinic, with the development of oligonucleotides and small molecules that bind specific RNA sequences and structures, ushering in a new era in RNA medicine. Despite decades of technology development, RNA structure analysis remains a major challenge, especially compared to proteins. Traditional physical methods such as crystallography, NMR and cryo-EM has only been applied to purified “well-behaving” samples in vitro, leaving the vast majority of cellular and viral RNAs beyond reach. Recent chemical probing methods provided experimental constraints that improved de novo modeling but has so far been limited to small and simple RNAs. This RNA structure analysis bottleneck has significantly limited functional studies and therapeutic development. In this MIRA application, I outline a research program to tackle the ultimate challenge in RNA structure biology: in vivo determination of structures and dynamics for any RNA in any biological sample at high resolution. This proposal is based on the simple mathematical theory that the 3D structure of any object is equivalent to the spatial distances among its components. Therefore, RNA 3D structure determination can be transformed into a problem of measuring spatial distances among the nucleotides. To achieve this goal, we will develop ic3D (in vivo crosslinking of 3D structures, or “I see 3D”), a technology that uses 3 new classes of “molecular rulers” - reversible chemical crosslinkers with defined lengths - to precisely measure inter-nucleotide distances at the atomic level. Coupled with proximity ligation, high throughput sequencing and Rosetta-based 3D modeling, ic3D enables in vivo global analysis of RNA structures and ensembles of conformations. We will perform rigorous benchmarking against a wide selection of simple and complex models that represent the full diversity of possible RNA structures in vivo. We will use ic3D to discover and model 3D structures across the transcriptome. The completion of this project will have broad impact in understanding the structural basis of RNA functions, mechanisms of RNA-mediated diseases, and revealing new structure targets for therapeutic interventions.

除了编码蛋白质之外，RNA 几乎在生物学的各个方面都发挥着重要作用。极端的 RNA 的功能多样性源于其折叠成复杂结构的能力，并且像机器一样， 动态地接受输入，传输信号和力，并执行遗传指令。 RNA结构调节 细胞中基因表达的每一步并控制RNA病毒的生命周期。结果，生理和 异常活动是多种人类疾病的根源。近年来，靶向RNA已经从 随着寡核苷酸和小分子药物的发展，一个有趣的学术想法在临床上成为现实 结合特定 RNA 序列和结构的分子，开创了 RNA 医学的新时代。尽管 经过数十年的技术发展，RNA 结构分析仍然是一个重大挑战，特别是与 蛋白质。传统的物理方法如晶体学、核磁共振和冷冻电镜仅应用于 在体外纯化“表现良好”的样本，使得绝大多数细胞和病毒 RNA 无法达到。 最近的化学探测方法提供了改进从头建模的实验约束，但 迄今为止仅限于小而简单的RNA。 RNA结构分析的瓶颈极大地限制了 功能研究和治疗开发。在这个 MIRA 申请中，我概述了一个研究计划 应对 RNA 结构生物学的终极挑战：体内结构和动力学测定 任何生物样品中的任何 RNA 都具有高分辨率。该建议基于简单的数学理论 任何物体的 3D 结构都相当于其组成部分之间的空间距离。因此，RNA 3D结构确定可以转化为测量物体之间空间距离的问题 核苷酸。为了实现这一目标，我们将开发 ic3D（3D 结构的体内交联，或“我看到 3D”），这是一种 使用 3 类新型“分子标尺”的技术 - 具有确定长度的可逆化学交联剂 - 在原子水平上精确测量核苷酸间的距离。加上邻近连接，高 通过通量测序和基于 Rosetta 的 3D 建模，ic3D 能够对 RNA 结构进行体内全局分析 和构象的集合。我们将针对各种简单的选择进行严格的基准测试 以及代表体内可能的 RNA 结构的全部多样性的复杂模型。我们将使用 ic3D 来 发现转录组中的 3D 结构并对其进行建模。该项目的完成将产生广泛 对理解 RNA 功能的结构基础、RNA 介导的疾病的机制以及 揭示治疗干预的新结构目标。