EAGER: JOINT CRYO NEUTRON/X-RAY CRYSTALLOGRAPHY OF RNA AND RNA-PROTEIN INTERACTIONS

核糖2 '-羟基（2'-OH）是RNA和DNA之间的关键化学差异。它们的不同特征，包括结构、稳定性、功能和活性，可以在不同程度上归因于RNA中2 '-OH的存在和DNA中2'-OH的缺失。2 '-OH影响糖构象平衡，从而影响成对RNA链的几何形状，并最终影响RNA与DNA相比大大扩展的3D折叠空间。2 '-OH是核酸骨架中唯一的氢键供体，导致RNA水合作用和相对于DNA的热力学稳定性增加。此外，2 '-OH在RNA剪接反应和核酶催化的磷酸二酯裂解中充当亲核试剂。由于X射线晶体学（XC）的技术限制和溶液研究的挑战，如快速交换，结构生物学忽略了2 '-OH氢/取向。中子晶体学（NC）揭示了氢原子（2 H，氘或D）在生物大分子中的位置。在该项目中，联合cryo-NC/XC将用于几种RNA靶标，以评估：（i）2 '-OH在RNA的广阔折叠空间中的重要性，（ii）结合蛋白如何影响2'-OH取向以塑造RNA骨架，以及（iii）分别在5 '-3'和5 '-2'连接的RNA中2 '-和3'-OH取向的差异，这或许可以解释后者在自然界中的消亡。严格的，结合NC/XC方法将产生改进的水结构模型和更好地了解核酸的结构，功能和与结合蛋白的相互作用。该项目的工作将最终实现对大分子和水合作用的精细理论处理，例如，通过分子动力学模拟。作为范德比尔特Aspirnaut计划的一部分，这项研究的结构优化和数据科学方面，包括PDB搜索和折叠模式的大规模分析，为高中生/本科生提供了理想的项目。中子晶体学（NC）揭示了氢原子（2 H，氘或D）在生物大分子中的位置。相反，X射线晶体学（XC）即使在高分辨率下也无法精确定位H原子。Cryo NC还确定了第一和更高壳层D2 O的取向。然而，由于需要大晶体、全氘样品和有限的光束线，核聚变研究仍然具有高风险/高影响。最近，PI在橡树岭国家实验室（ORNL）使用最先进的仪器首次将冷冻NC应用于RNA结构。Cryo NC揭示了核糖羟基（2 '-OH）在稳定非典型骨架几何结构中的作用，2'-OH取向和糖皱褶之间的结构相关性，以及2 '-OH作为H-键供体而不是受体的6：1偏好。这些见解导致以下方面的假设：（i）2 '-OH部分在RNA相对于DNA的扩展折叠空间中的重要性（ii）结合蛋白如何影响2'-OH方向并翻转核糖皱褶以塑造RNA骨架几何形状，以及（iii）分别在5 '-3'和5 '-2'连接的RNA中2 '-和3'-OH方向的差异，这也许可以解释后者在自然界中的消亡。水在生命系统中是无所不在的，并参与蛋白质和核酸的折叠，几何形状，稳定性，动力学，功能和相互作用。严格的cryo NC/XC方法将产生改进的水结构模型，对我们理解核酸结构，功能和与结合蛋白的相互作用具有重要意义。该项目的工作将最终使完善的理论处理的大分子和水化的QM-MM和MD模拟。这个奖项反映了NSF的法定使命，并已被认为是值得的支持，通过评估使用基金会的智力价值和更广泛的影响审查标准。

The ribose 2'-hydroxyl group (2'-OH) is the key chemical difference between RNA and DNA. Their divergent characteristics, including structure, stability, function and activity, can be attributed to various degrees to the presence of the 2'-OH in RNA and its absence in DNA. The 2'-OH affects the sugar conformational equilibrium and thereby the geometry of paired RNA strands, and ultimately the much-expanded 3D fold-space of RNA compared to DNA. The 2'-OH is the only hydrogen bond donor in the nucleic acid backbone and results in increased RNA hydration and thermodynamic stability relative to DNA. Moreover, the 2'-OH acts as nucleophile in RNA splicing reactions and ribozyme-catalyzed phosphodiester cleavage. Structural biology has ignored the 2'-OH hydrogen/orientation, owing to technical limitations of X-ray crystallography (XC) and challenges to solution studies such as rapid exchange. Neutron crystallography (NC) reveals the locations of hydrogen atoms (2H, deuterium or D) in biomacromolecules. In the project joint cryo-NC/XC will be used on several RNA targets to assess: (i) The importance of the 2'-OH in RNA’s expansive fold space, (ii) How binding proteins might influence 2'-OH orientation to sculpt the RNA backbone, and (iii) Differences in 2'- and 3'-OH orientations in 5'-3' and 5'-2' linked RNAs, respectively, that might explain the latter’s demise in nature. The rigorous, combined NC/XC approach will produce improved models of water structure and a better understanding of nucleic acid structure, function and interactions with binding proteins. The project work will ultimately enable refined theoretical treatments of macromolecules and hydration, e.g., by molecular dynamics simulations. Structural refinements and data science aspects of this research, including PDB searches and large-scale analysis of pucker modes provide ideal projects for high-school students/undergraduates as part of Vanderbilt’s Aspirnaut program.Neutron crystallography (NC) reveals the locations of hydrogen atoms (2H, deuterium or D) in biomacromolecules. Conversely, X-ray crystallography (XC) cannot pinpoint H atoms even at high resolution. Cryo NC also establishes the orientations of first- and higher-shell D2O. However, NC research has remained high-risk/-impact, owing to the need for large crystals, perdeuterated samples and limited access to beamlines. Recently the PI applied cryo NC for the first time to an RNA structure using state-of the-art instrumentation at Oak Ridge National Laboratory (ORNL). Cryo NC uncovered roles of ribose hydroxyls (2'-OHs) in stabilizing non-canonical backbone geometries, a structural correlation between 2'-OH orientation and sugar pucker, and a 6:1 preference of the 2'-OH to act as H-bond donor rather than acceptor. These insights lead to hypotheses in regard to: (i) The importance of the 2'-OH moiety in RNA’s expanded fold space relative to DNA (ii) How binding proteins might influence 2'-OH orientation and flip the ribose pucker to sculpt the RNA backbone geometry, and (iii) Differences in 2'- and 3'-OH orientations in 5'-3' and 5'-2' linked RNAs, respectively, that may explain the latter’s demise in nature. Water in living systems is omnipresent and is involved in governing folding, geometry, stability, dynamics, function and interactions of proteins and nucleic acids. The rigorous cryo NC/XC approach will produce improved models of water structure, with implications for our understanding of nucleic acid structure, function and interactions with binding proteins. The project work will ultimately enable refined theoretical treatments of macromolecules and hydration by QM-MM and MD simulations.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.