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

除了编码蛋白质外，RNA在生物学的几乎每一个方面都扮演着基本的角色。极端的 RNA的功能多样性源于它能够折叠成复杂的结构，就像机器一样， 动态接受输入，传递信号和力，执行遗传指令。RNA结构调控 基因在细胞中表达的每一步，并控制RNA病毒的生命周期。因此，生理上和 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变成了现实，随着寡核苷酸和小分子的发展 结合特定RNA序列和结构的分子，开创了RNA医学的新纪元。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 蛋白质。传统的物理方法，如结晶学、核磁共振和低温电子显微镜，仅应用于 在体外纯化的“行为良好”的样本，使绝大多数细胞和病毒RNA遥不可及。 最近的化学探测方法提供了改进从头模型的实验约束，但 到目前为止，仅限于小而简单的RNA。这个RNA结构分析的瓶颈已经大大限制了 功能研究和治疗发展。在这个Mira应用程序中，我概述了一个研究计划 解决RNA结构生物学的终极挑战：体内结构和动力学的测定 高分辨率的任何生物样本中的任何RNA。这一建议是基于简单的数学理论 任何物体的3D结构都等同于其组件之间的空间距离。因此，RNA 3D结构确定可以转化为测量 核苷酸。为了实现这一目标，我们将开发IC3D(体内3D结构的体内交联剂，或“我看到3D”)，一种 使用3类新的“分子尺子”的技术--限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。再加上近距离结扎，高 通过吞吐量测序和基于Rosetta的3D建模，IC3D能够在体内对RNA结构进行全局分析 以及各种构象的组合。我们将对广泛选择的简单 以及复杂的模型，代表了体内可能的RNA结构的全部多样性。我们将使用IC3D来 发现转录组中的3D结构并对其进行建模。该项目的建成将具有广阔的应用前景 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.