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

除了编码蛋白质外，RNA在生物学的几乎每个方面都起着重要作用。极端 RNA的功能多样性源于其折叠成复杂结构的能力，就像机器一样， 动态地接受输入、传递信号和力，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。因此，生理和 异常活动是各种人类疾病的基础。近年来，靶向RNA已经从 一个有趣的学术想法，在临床上的现实，随着寡核苷酸和小的发展， 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 经过几十年的技术发展，RNA结构分析仍然是一个重大挑战，特别是与 proteins.传统的物理方法，如晶体学，NMR和cryo-EM，只应用于 在体外纯化的“行为良好”的样品，留下了绝大多数的细胞和病毒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， 目前仅限于小而简单的RNA。这一RNA结构分析瓶颈极大地限制了 功能研究和治疗开发。在这个MIRA应用程序中，我概述了一个研究计划， 解决RNA结构生物学的最终挑战：在体内确定结构和动力学， 任何生物样本中的任何RNA。这个建议是基于简单的数学理论 任何物体的3D结构都等同于其组成部分之间的空间距离。因此，RNA 3D结构确定可以被转换成测量三维结构之间的空间距离的问题。 个核苷酸为了实现这一目标，我们将开发ic 3D（3D结构的体内交联，或“我看到3D”）， 技术，使用3种新的“分子统治者”-可逆的化学交联剂与定义的长度 - 在原子水平上精确测量核苷酸间的距离。再加上邻近连接， 通过高通量测序和基于Rosetta的3D建模，ic 3D能够在体内对RNA结构进行全局分析 以及构象的集合。我们将对广泛的选择进行严格的基准测试， 和复杂的模型，代表了体内可能的RNA结构的全部多样性。我们将使用ic 3d 发现并模拟转录组中的3D结构。该项目的完成将具有广泛的 在理解RNA功能的结构基础，RNA介导的疾病的机制， 揭示了治疗干预的新结构靶点。

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.