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

EAGER:RNA 和 RNA-蛋白质相互作用的联合冷冻中子/X 射线晶体学

基本信息

  • 批准号:
    2224897
  • 负责人:
  • 金额:
    $ 30万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
    Standard Grant
  • 财政年份:
    2022
  • 资助国家:
    美国
  • 起止时间:
    2022-05-15 至 2024-10-31
  • 项目状态:
    已结题

项目摘要

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.
核糖2'-羟基(2'-OH)是RNA和DNA之间的关键化学区别。它们的不同特征,包括结构、稳定性、功能和活性,可在不同程度上归因于RNA中存在2'-OH而DNA中不存在2'-OH。2'-OH会影响糖的构象平衡,从而影响成对RNA链的几何形状,最终影响RNA与DNA相比更大的三维折叠空间。2'-OH是核酸主链中唯一的氢键供体,与DNA相比,它能提高RNA的水合性和热力学稳定性。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试剂的作用。由于x射线晶体学(XC)的技术限制和溶液研究(如快速交换)的挑战,结构生物学忽略了2'-OH氢/取向。中子晶体学(NC)揭示了生物大分子中氢原子(2H,氘或D)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) 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死亡。严谨的NC/XC结合方法将产生改进的水结构模型,更好地理解核酸的结构、功能和与结合蛋白的相互作用。该项目的工作最终将使大分子和水合作用的精细化理论处理成为可能,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pucker模式的大规模分析,为高中学生/本科生提供了理想的项目,作为范德比尔特大学Aspirnaut项目的一部分。中子晶体学(NC)揭示了生物大分子中氢原子(2H,氘或D)的位置。相反,x射线晶体学(XC)即使在高分辨率下也不能精确定位氢原子。Cryo NC还建立了第一壳层和高壳层D2O的取向。然而,由于需要大晶体,渗透样品和有限的光束线,NC研究仍然是高风险/影响。最近,PI首次使用橡树岭国家实验室(ORNL)最先进的仪器将冷冻NC应用于RNA结构。Cryo NC揭示了核糖羟基(2′-OH)在稳定非规范骨架几何结构中的作用,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。这些见解导致了关于以下方面的假设:(i)相对于DNA, 2'- oh片段在RNA扩展折叠空间中的重要性;(ii)结合蛋白如何影响2'- oh取向并翻转核糖褶皱以塑造RNA骨干几何形状;(iii) 5‘-3’和5‘-2’连接RNA中2'-和3'- oh取向的差异,分别可以解释后者在自然界中的消亡。水在生命系统中无所不在,参与控制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、功能和与结合蛋白的相互作用具有重要意义。该项目工作最终将通过QM-MM和MD模拟实现大分子和水合作用的精细理论处理。该奖项反映了美国国家科学基金会的法定使命,并通过使用基金会的知识价值和更广泛的影响审查标准进行评估,被认为值得支持。

项目成果

期刊论文数量(0)
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Martin Egli其他文献

Expanding the binding space of argonaute-2: incorporation of either emE/em or emZ/em isomers of 6′-vinylphosphonate at the 5′ end of the antisense strand improves RNAi activity
扩展 argonaute-2 的结合空间:在反义链 5' 端掺入 6'-乙烯基膦酸酯的 emE/em 或 emZ/em 异构体可提高 RNAi 活性
  • DOI:
    10.1039/d5cc00348b
  • 发表时间:
    2025-04-02
  • 期刊:
  • 影响因子:
    4.200
  • 作者:
    Dhrubajyoti Datta;Jayanta Kundu;Patrick Miller;Mehreen S. Khan;Juan Salinas;June Qin;Sarah LeBlanc;Tuyen Nguyen;Haiyan Peng;Christopher S. Theile;Martin Egli;Muthiah Manoharan
  • 通讯作者:
    Muthiah Manoharan
Synthesis and Biophysical Studies of High-Affinity Morpholino Oligomers Containing G-Clamp Analogs.
含有 G-Clamp 类似物的高亲和力吗啉代低聚物的合成和生物物理研究。
  • DOI:
  • 发表时间:
    2023
  • 期刊:
  • 影响因子:
    3.6
  • 作者:
    Arnab Das;Atanu Ghosh;Jayanta Kundu;Martin Egli;M. Manoharan;Surajit Sinha
  • 通讯作者:
    Surajit Sinha
A. Sigel, H. Sigel, R. K. O. Sigel (Eds): The alkali metal ions: their role for life. Volume 16 of metal ions in life sciences
  • DOI:
    10.1007/s11243-016-0067-y
  • 发表时间:
    2016-06-03
  • 期刊:
  • 影响因子:
    1.700
  • 作者:
    Martin Egli
  • 通讯作者:
    Martin Egli
Structure and Stability of Ago2 MID‐Nucleotide Complexes: All‐in‐One (Drop) His6‐SUMO Tag Removal, Nucleotide Binding, and Crystal Growth
Ago2 MID-核苷酸复合物的结构和稳定性:一体化(滴)His6-SUMO 标签去除、核苷酸结合和晶体生长
  • DOI:
  • 发表时间:
    2024
  • 期刊:
  • 影响因子:
    0
  • 作者:
    Li Lei;J. Harp;John C. Chaput;Kelly Wassarman;M. Schlegel;Muthiah Manoharan;Martin Egli
  • 通讯作者:
    Martin Egli
Improved emIn Vivo/em Metabolic Stability and Silencing Efficacy of siRNAs with Phosphorothioate Linkage-Free, GalNAc-Conjugated Sense Strands Containing Morpholino-LNA Modifications
具有无硫代磷酸酯连接、含吗啉代-LNA 修饰的 GalNAc 偶联正义链的 siRNA 的体内代谢稳定性和沉默效率的提高
  • DOI:
    10.1021/acs.orglett.4c02903
  • 发表时间:
    2024-11-29
  • 期刊:
  • 影响因子:
    5.000
  • 作者:
    Dhrubajyoti Datta;Pawan Kumar;Jayanta Kundu;June Qin;Jason A. Gilbert;Sally Schofield;Daniel P. Donnelly;Ju Liu;Rohan Degaonkar;Martin Egli;Muthiah Manoharan
  • 通讯作者:
    Muthiah Manoharan

Martin Egli的其他文献

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