Thermodynamically Calibrated RNA Simulations to Decode Mechanisms of RNA Molecular Recognition

这MIRA建议详细介绍了一个研究计划，围绕发展的中心 以及应用改进的、精确的计算机模型来模拟RNA 原子分辨率的3D结构。这些模型与现有的RNA模型的不同之处在于， 它们被校准以再现关于以下物质的物理行为的溶液热力学数据： 核苷酸和核苷，这种方法很容易扩展到包括的影响， 非天然RNA和RNA-配体相互作用。这项技术尤其重要， 生物医学上重要的RNA不适用于传统的结构生物学技术， 这使得很难建立基本的结构-功能关系， 在设计潜在的治疗干预措施之前， 通常，感兴趣的RNA上唯一可用的结构信息是次要的 结构估计来自生物信息学或来自SHAPE化学探测实验。这 该提案建立在最近成功使用受稀疏限制的分子模拟的基础上。 SHAPE或NMR数据来模拟共转录折叠RNA的折叠途径，如 并描述了microRNA/mRNA复合物的灵活性如何影响它们如何结合 hAGO 2蛋白。在这些最新成果的基础上， 提出了三个主要部分。第一个是使用炼金术的自由能计算， 测量RNA碱基配对的能量，并根据实验重新校准它们。的 第二种是全自动、适应性RNA的二维复制交换方法 折叠结合可变强度二级结构约束-一种方法， 我们希望能扩展到包括三级基序的大（50-100 nt）RNA。 最后，我们提出了一种新的多维技术，同时折叠RNA适体 同时还结合小分子配体， 炼金术的自由能计算-这将是必要的捕捉“诱导适合”的 配体结合后的RNA适体。这些计算将用于预测配体结合 模式和工程最佳的RNA生物传感器通过靶向掺入化学 修饰的核酸。

This MIRA proposal details a research program that centers around the development and application of improved, thermodynamically accurate computer models for simulating RNA 3D structures at atomic resolution. These models differ from existing models for RNA in that they are calibrated to reproduce solution thermodynamic data on the physical behavior of nucleotides and nucleosides, an approach that is readily extended to include the effects of unnatural RNAs and RNA-ligand interactions. This technology is particularly important as many biomedically important RNAs are not amenable to traditional structural biology techniques, which makes it difficult to establish basic structure-function relationships that must be understood before potential therapeutic interventions could be designed. Often, the only available structural information on an RNA of interest are secondary structure estimates from bioinformatics or from SHAPE chemical probing experiments. This proposal builds on recent successes in using molecular simulations restrained by sparse SHAPE or NMR data to simulate the folding pathway of a co-transcriptionally folded RNA, as well as describe how the flexibility of microRNA/mRNA complexes affect how they bind the hAGO2 protein. Building on these recent results, a comprehensive research program is proposed in three major parts. The first is the use of alchemical free-energy calculations to measure the energetics of RNA base-pairing and recalibrate them against experiment. The second is a two-dimensional replica-exchange method for fully automated, adaptive RNA folding incorporating variable strength secondary structure constraints – a method that show promising results that we expect to scale to large (50-100 nt) RNAs including tertiary motifs. Lastly, we propose a novel multi-dimensional technique to simultaneously fold RNA aptamers while also binding small-molecule ligands using Hamiltonian replica-exchange combined with alchemical free energy calculations – which will be necessary to capture the “induced fit” of the RNA aptamer upon ligand binding. These calculations will be used to predict ligand binding modes and engineer optimal RNA biosensors through targeting incorporation of chemically modified nucleic acids.