Thermodynamically Calibrated RNA Simulations to Decode Mechanisms of RNA Molecular Recognition

这份Mira提案详细说明了一个以开发为中心的研究计划 以及用于模拟RNA的改进的、热力学上准确的计算机模型的应用 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 对它们进行了校准，以重现溶液热力学数据中的物理行为 核苷酸和核苷，这一方法很容易扩展到包括 非自然的RNA和RNA与配体的相互作用。这项技术特别重要，因为有许多 生物医学上重要的RNA不适用于传统的结构生物学技术， 这使得难以建立基本的结构-功能关系 在设计潜在的治疗干预措施之前，必须了解这一点。 通常，关于感兴趣的RNA的唯一可用的结构信息是次要的 来自生物信息学或形状化学探测实验的结构估计。这 该提案建立在最近在使用稀疏约束的分子模拟方面取得成功的基础上 形状或核磁共振数据，以模拟共转录折叠的RNA的折叠路径，如 并描述了microRNA/mRNA复合体的灵活性如何影响它们如何结合 HAGO2蛋白。在这些最新成果的基础上，一项全面的研究计划是 分三大部分提出。第一个是使用炼金术自由能计算来 测量RNA碱基配对的能量学，并根据实验重新校准它们。这个 第二种是用于全自动、自适应RNA的二维副本交换方法 结合可变强度二级结构约束的折叠--一种显示 很有希望的结果，我们预计将扩大到大(50-100个核苷酸)的RNA，包括第三基序。 最后，我们提出了一种新的多维技术来同时折叠RNA适配子 同时也使用哈密顿复制交换结合结合小分子配体 炼金术自由能计算-这将是必要的，以捕捉“诱导匹配” 配基结合时的RNA适体。这些计算将被用来预测配体结合 通过化学靶向掺入构建最佳的RNA生物传感器 修饰过的核酸。

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.