Fundamental Studies of RNA Conformational Thermodynamics

摘要 非编码（nc）RNA是生物学中的关键角色，并且越来越多地被认为是治疗的靶点 传染病，癌症和遗传疾病，并作为生物工程和合成的分子工具， 生物学功能性和调节性RNA在多步生化循环中经历构象转变， 配体结合和信号传导。了解这些RNA结构如何形成以及它们如何形成非常重要 动态变化的细胞和化学线索，因为这些生物学的重要性， RNA，因为这种理解将为生物工程提供工具，并可能促进治疗 最根本的原因是，RNA是生命的基本分子，无论是现在还是过去。 RNA二级结构形成的热力学可以相当精确地预测 最近邻规则，并且在确定3D RNA和 RNA·蛋白质结构。然而，我们缺乏一个预测RNA三级构象的能量模型， 热力学，这是最终需要了解和操纵RNA的形式和功能， 生物过程。与RNA二级结构能量学的碱基对步骤的能量加和性不同， RNA三级结构能量学需要对构象系综进行统计力学建模 以及确定描述形成不同构象的概率的配分函数。 RNA的分子特性--分级折叠、重复结构基序和稀疏三级接触 界面使得RNA三级结构能量学比蛋白质更简单和更易处理。从 这些性质，一个重构模型已经开发出来，可以允许构象热力学 基于组分结构元素的构象集合进行预测：螺旋，连接，和 第三方联系伙伴。该建议的中心假设是，通过表征构象 任何RNA的构象热力学 可以确定。本提案的中心目标是测试和发展这一模式，并克服 确定数千个RNA元件的构象系综的巨大挑战。为了实现这一点， 将使用“RNA-MaP”--一种提供数百万次热力学测量的新技术， 成千上万的RNA螺旋、连接和三级接触的定量“热力学指纹” 元素并提供数据以获得每个元素的构象系综。该项目将（1）建立一个 RNA元件构象热力学图谱;（2）定义RNA元件构象系综名册 这些元素;然后（3）在重建模型中使用这些信息来设计和合理地 设计ncRNA的构象和能量特性。该项目还将提供一个免费的 计算工具RNAMake-ΔG，用于建模和设计动态RNA三级结构，并将提供一个 丰富的高精度热力学数据，以帮助指导社区范围内的模型开发。

Abstract Non-coding (nc)RNAs are key players in biology and are increasingly recognized as targets to treat infectious diseases, cancer, and genetic disorders, and as molecular tools for bioengineering and synthetic biology. Functional and regulatory RNAs undergo conformational transitions in multi-step biochemical cycles, ligand binding, and signaling. It is important to understand how these RNA structures form and how they dynamically change in response to cellular and chemical cues because of the biological importance of these RNAs, because this understanding will provide tools for bio-engineering and may facilitate therapeutic intervention, and, most fundamentally, because RNA is an essential molecule of life, both present and past. The thermodynamics of RNA secondary structure formation can be predicted with reasonable accuracy from nearest neighbor rules, and there have been remarkable advances in determining 3D RNA and RNA·protein structures. However, we lack a predictive energetic model for RNA tertiary conformational thermodynamics, which is ultimately required to understand and manipulate RNA form and function in biological processes. Unlike the energetic additivity of base pair steps for RNA secondary structure energetics, RNA tertiary structure energetics requires the statistical mechanical modeling of conformational ensembles and determination of partition functions that delineate the probabilities of forming different conformations. RNA's molecular properties—hierarchical folding, repeating structural motifs, and sparse tertiary contact interfaces—render tertiary structure energetics far simpler and more tractable for RNA than for proteins. From these properties, a Reconstitution Model has been developed that could allow conformational thermodynamics to be predicted based on conformational ensembles of component structural elements: helices, junctions, and tertiary contact partners. The central hypothesis of this proposal is that, by characterizing conformational thermodynamics for the array of component parts, the conformational thermodynamics of any arbitrary RNA can be determined. The central goals of this proposal are to test and develop this model and to overcome the vast challenge of determining conformational ensembles for thousands of RNA element. To accomplish this, `RNA-MaP' will be used—a novel technology that provides millions of thermodynamic measurements and quantitative `thermodynamic fingerprints' for tens of thousands of RNA helix, junction, and tertiary contact elements and provides data to obtain conformational ensembles for each element. This project will (1) build an atlas of conformational thermodynamics for RNA elements; (2) define a roster of conformational ensembles for these elements; and then (3) use this information within the Reconstitution Model to design and rationally engineer the conformational and energetic properties of ncRNAs. This project will also provide a freely available computational tool, RNAMake-ΔG, to model and engineer dynamic RNA tertiary structures, and will provide a wealth of high-precision thermodynamic data to help guide community-wide model development.