A universal approach for determining three-dimensional RNA structures

摘要 RNA分子参与了与人类疾病有关的最基本的细胞过程。许多 RNA包含对RNA功能做出关键贡献的结构化模块，并代表有吸引力的 药物靶点，尤其是针对无法治愈的和与“无法下药的”蛋白质有关的疾病。了解以下内容 这些RNA的三维结构将有助于理解RNA的作用机制 并可能极大地加速药物发现工作。然而，传统的RNA结构研究方法 X-射线结晶学和核磁共振波谱测定，费时费力，技术含量高。 限制。单粒子低温电子显微镜(Cryo-EM)具有许多优于结晶学的优点 和核磁共振，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。这项提议的重点是开发一部小说 一种绕过大小限制、省略RNA的冷冻-EM RNA样品制备方法 纯化和RNA复性步骤，并允许方便的冷冻-EM数据处理和结构解决方案。这个 提出的概念验证研究结合了三个具体目标。具体目标1致力于发展 体外转录制备均一核糖核酸的生物化学新方法。具体目标2 将使用计算模型、生化分析和单粒子低温电磁实验来开发 一种与中小型金属结构溶液相容的低温EM样品的制备方法 大小的RNA。AIM 3将使用模型RNA分子和传统的单粒子来验证该方法 低温电磁结构溶液管道。该方案综合了计算方法、生化分析和 基于CRYO-EM的结构确定发展了一种通用而简单的求解结构的方法 大多数是RNA和RNA-药物复合体。这项拟议的技术预计将优于 在人工、成本和适用性方面的现有方法。

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.