Pathways to Tertiary Structure Interfaces in RNA: An Integrated NMR, Biochemical, and Computational Approach

RNA是DNA的化学近亲，最为人所熟知的是它的信使角色，即将信息从DNA基因组传递到合成由该基因组编码的蛋白质的细胞机器。然而，近几十年来，人们发现了RNA分子的各种非编码作用，包括催化化学反应、调节基因表达以及CRISPR等特殊功能。为了完成这些角色，RNA采取了各种各样的特定三维形状和形式。然而，人们对这些三维RNA结构的形成知之甚少，在寻找设计功能RNA分子和其他以细胞RNA为靶标的分子的预测框架方面留下了很大的空白。该项目将进行光谱、生物物理、生化和计算分析的综合计划，以绘制出RNA结构的能量学和折叠路径。这项工作将提供关于RNA三维结构相互作用形成的完整的原子水平的描述，并对非编码系统中的这种RNA-RNA相互作用进行科学的理解。这项提议将培训研究生和本科生中代表性不足的群体。尽管对DNA和RNA二级结构(螺旋形成)的潜在因素相对较好，但对复杂RNA三级结构形成的驱动力和机制的详细研究并没有跟上RNA结构生物学的最新进展。发夹状核酶的对接转变是一种罕见的仅三级相互作用的例子，适合分子间生物物理和光谱研究，具有丰富的现有结构和功能数据以及多种功能读数。该项目将结合核磁共振光谱和分子动力学计算来检查单个环的构象采样，并使用动态猝灭变体的功能分析来确定在每个环中确定的观察到的运动模式的相关性，并推导出对接能力状态的内部一致模型。将使用一种创新的核磁共振和活动初始化迭代马尔可夫分析(NAIMA)协议来整合所有数据，以计算确定整个对接过程的路径。在这项工作中，将完成为该领域一般使用的未折叠RNA核磁共振化学位移的参考数据库的推导。这个项目得到了生物科学局分子和细胞生物科学部分子生物物理学分部的支持。这个奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

RNA, a close chemical cousin of DNA, is most familiar in its messenger role of conveying information from the DNA genome to the cellular machinery that synthesizes proteins encoded by that genome. In recent decades, however, a great variety of noncoding roles for RNA molecules have been discovered, including in catalyzing chemical reactions, regulating gene expression, and in specialized functions such as CRISPR. To fulfill these roles, RNA takes up a wide variety of specific three-dimensional shapes and forms. Relatively little is known, however, about the formation of these three-dimensional RNA structures, leaving large gaps in the search for a predictive framework for the design of functional RNA molecules and of other molecules that target cellular RNAs. This project will carry out an integrated program of spectroscopic, biophysical, biochemical, and computational analyses in order to map out the energetics and folding pathways for RNA structures. The work will provide a full atomic-level description of the formation of an RNA three-dimensional structural interaction, and a scientific understanding of such RNA-RNA interactions in noncoding systems in general. This proposal will train underrepresented groups at the graduate and undergraduate levels. The PI’s efforts in mental-health awareness in the scientific workforce at regional and national conferences will be expanded.Although the factors underlying secondary structure (helix formation) in DNA and RNA are relatively well understood, the detailed study of the driving forces and mechanisms of formation of complex RNA tertiary structures has not kept pace with the great recent advances in RNA structural biology. The docking transition in the hairpin ribozyme is a rare example of a tertiary-only interaction amenable to intermolecular biophysical and spectroscopic investigation, with a wealth of existing structural and functional data and multiple functional readouts. This project will use a combination of NMR spectroscopy and molecular dynamics computations to examine conformational sampling of the individual loops and use functional assays of dynamically-quenched variants to determine the relevance of the observed motional modes determined in each loop and derive internally-consistent models of docking-competent states. An innovative protocol of NMR and Activity-initialized Iterative Markov Analysis (NAIMA) will be used to integrate all of the data in a computational determination of the pathway for the overall docking process. In the course of this work, the derivation of a reference database of unfolded RNA NMR chemical shifts for the general use of the field will be completed. This project is supported by the Molecular Biophysics Cluster of the Molecular and Cellular Biosciences Division in the Directorate for Biological Sciences.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.