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

核糖2‘-羟基(2’-OH)是RNA和DNA的关键化学区别。它们在结构、稳定性、功能和活性等方面的差异可以不同程度地归因于RNA中2‘-羟基的存在和DNA中2’-羟基的缺失。2‘-OH影响糖的构象平衡，从而影响成对的RNA链的几何形状，最终影响RNA相对于DNA的3D折叠空间。2‘-OH是核酸骨架中唯一的氢键供体，与DNA相比，它增加了RNA的水合作用和热力学稳定性。此外，2‘-OH在RNA剪接反应和核酶催化的磷酸二酯裂解中起亲核作用。由于X射线结晶学(XC)的技术限制和快速交换等溶液研究的挑战，结构生物学忽略了2‘-OH氢/取向。中子结晶学(NC)揭示了氢原子(2H、氘或D)在生物大分子中的位置。在该项目中，联合Cryo-NC/XC将用于几个RNA靶标，以评估：(I)2‘-OH在RNA可扩展折叠空间中的重要性，(Ii)结合蛋白可能如何影响2’-OH取向以塑造RNA主干，以及(Iii)5‘-3’和5‘-2’连接RNA中2‘-和3’-OH取向的差异，这可能解释后者在自然界中的消亡。严格的NC/XC组合方法将产生改进的水结构模型，并更好地理解核酸的结构、功能以及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。这项研究的结构改进和数据科学方面，包括PDB搜索和对褶皱模式的大规模分析，作为Vanderbilt的Aspirnaut计划的一部分，为高中生/本科生提供了理想的项目。中子结晶学(NC)揭示了氢原子(2H、氘或D)在生物大分子中的位置。相反，X射线结晶学(XC)即使在高分辨率下也不能精确定位H原子。CRYO NC还确定了第一层和更高层D2O的取向。然而，由于需要大晶体、过氚样品和有限的光束线，NC研究仍然存在高风险/-影响。最近，PI首次在橡树岭国家实验室(ORNL)使用最先进的仪器将低温NC应用于RNA结构。低温NC揭示了核糖羟基(2‘-OHS)在稳定非正则主干几何结构中的作用，2’-OH取向与糖折叠之间的结构相关性，以及2‘-OH作为氢键供体而不是受体的6：1偏好。这些见解导致了如下假设：(I)RNA相对于DNA的扩展折叠空间中2‘-OH部分的重要性(Ii)结合蛋白质如何影响2’-OH取向并翻转核糖折叠以塑造RNA骨架几何形状，以及(Iii)5‘-3’和5‘-2’连接RNA中2‘-OH和3’-OH取向的差异，这可能解释后者在自然界中的消亡。水在生命系统中无处不在，并参与调控蛋白质和核酸的折叠、几何、稳定性、动力学、功能和相互作用。严格的低温NC/XC方法将产生改进的水结构模型，对我们理解核酸结构、功能以及与结合蛋白的相互作用具有重要意义。该项目的工作最终将通过QM-MM和MD模拟实现对大分子和水合作用的精细理论处理。该奖项反映了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。

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.