RNA-ligand interactions: simulation and experiment

描述（由申请人提供）：本项目代表了已建立的计算和实验实验室之间的合作，将生物物理方法结合起来，产生、评估和验证下一代生物分子模拟方法，以研究RNA分子。在过去的十年里，我们对RNA分子各种各样的功能角色的了解已经激增；RNA分子可以调节或催化，可以作为传感器，可以向上和向下调节基因表达，并且在所有门中都具有作为外源配体控制靶点的巨大潜力。RNA的结构是模块化的，包含多种结构元素，包括短双工区、发夹环、内部凸起、四向连接和受体位点。这些结构元素，以及它们的构象变化和动力学，是RNA功能的关键。然而，这些功能上重要的结构、动力学和能量特性还不能可靠地预测或完全通过实验理解。为了表征RNA结构、结构转变及其局部和全局动态，模拟和实验的协同作用是必要的。本项目的目标是利用理论和实验的生物物理方法，更全面地开发生物分子模拟工具，准确描述RNA分子，特别是它们如何与小分子、离子和蛋白质相互作用，并以实验数据为基准，对模型进行评估、验证和改进。Aim 1是围绕Varkud卫星核酶干环V RNA （SL5）构建的。这个小的RNA发夹有一个灵活的环，当它与离子结合时，会经历构象变化，并作为一个模型系统来测试静电、采样、结构精度，以及模拟离子对结构和动力学的微妙影响的模拟能力。基于最新AMBER核酸力场（ff99+parmbsc0）的新分子动力学模拟将评估力场参数，并应用新颖的偏态增强采样方法来更丰富地采样RNA环的构型空间。同时，NMR将进一步细化溶液结构，13C NMR弛豫实验将测量SL5在不同离子存在下的动力学。Aim 2将RNA模型系统扩展到核糖体的GTPase中心（GAC），以模拟离子结合、静电和抗生素结合。核磁共振用于结构和动力学测定，2-氨基嘌呤荧光报告了GAC的折叠，MD从58个核苷酸的GAC的晶体结构开始。这两个目标是协同的：从MD模拟中反向计算原始核磁共振数据，通过与实测核磁共振数据的直接比较来评估计算方法的准确性和精密度。目标3采用比较研究的结果来确定不足和实施改进。将通过哈密顿复制交换分子动力学促进力场修饰和构象采样，并最终应用于GAC RNA的三级折叠。

DESCRIPTION (provided by applicant): This project represents collaboration between established computational and experimental labs to combine biophysical methods to produce, assess, and validate the next generation of biomolecular simulation methods to study RNA molecules. In the past decade our knowledge of the widely varied functional roles of RNA molecules has exploded; RNA molecules can be regulatory or catalytic, can act as sensors, can both up- and down- regulate gene expression, and have great potential as targets for control by exogenous ligands in all phyla. The structures of RNA are modular and contain a mix of structural elements including short duplex regions, hairpin loops, internal bulges, four-way junctions, and receptor sites. These structural elements, along with their conformational changes and dynamics, are the keys to RNA function. However, these functionally important structural, dynamic, and energetic properties cannot yet be reliably predicted or fully experimentally understood. To characterize RNA structures, their structural transitions, and their local and global dynamics, a synergy of simulation and experiment is necessary. The goal of this project is to use theoretical and experimental biophysical methods to more fully develop biomolecular simulation tools to accurately describe RNA molecules, especially how they interact with small molecules, ions, and proteins, using experimental data as a benchmark to assess, validate and improve the models. Aim 1 is built around the Varkud satellite ribozyme Stem loop V RNA (SL5). This small RNA hairpin has a flexible loop that undergoes a conformational change when it binds to ions, and acts as a model system to test electrostatics, sampling, structural accuracy, and the ability of the simulations to model the subtle influences of ions on the structure and dynamics. New molecular dynamics simulations based on the latest AMBER nucleic acid force fields (ff99+parmbsc0) will assess the force field parameters and apply novel biased enhanced sampling methods to more richly sample the configurational space of the RNA loop. At the same time, NMR will further refine the solution structure, and 13C NMR relaxation experiments will measure dynamics of SL5 in the presence of different ions. Aim 2 expands the RNA model systems to the GTPase center (GAC) of the ribosome, to model ion binding, electrostatics, and antibiotic binding. NMR is used for structure and dynamics determination, 2-aminopurine fluorescence reports on folding of the GAC, and MD begins with crystal structures of the 58 nucleotide GAC. These two aims are synergistic: primary NMR data are back- calculated from the MD simulations to assess the accuracy and precision of the computational methods through direct comparison to the measured NMR data. Aim 3 takes the outcome of the comparative study to identify deficiencies and implement improvements. Force field modifications and conformational sampling will be facilitated through Hamiltonian replica exchange molecular dynamics and finally applied to the tertiary folding of the GAC RNA.