The objective of this proposal is to bring to bear state of the art theoretical methods to the study of the mechanisms of ribozyme catalysis and the factors that regulate reactivity. An overarching theme in the proposal is to facilitate active collaborations with a network of experimental groups in order to progress toward a consensus view of mechanism that may, ultimately, contribute to a deeper understanding of more complex cellular catalytic RNA systems such as the ribosome and spliceosome. The proposal focuses on application to a series of archetype ribozymes that catalyze site-specific phosphodiester bond cleavage (and ligation) but that have different active site architectures and catalytic requirements. As a baseline, the same reaction will be studied in a non-enzymatic dinucleotide model in solution, and catalyzed by the protein enzyme analog, RNase A. The tandem study of alternate mechanistic strategies and the factors that govern reactivity will provide penetrating insight into the rational design of new biomedical technology. However, these applications demand new methodological advances. Of key importance are accurate and efficient quantum mechanical/molecular mechanical (QM/MM) methods for catalysis, reliable molecular simulation force fields for RNA and metal ions, and efficient methods for sampling free energy surfaces and conformational transitions. Toward this end, we propose to: 1) improve the QM, MM and QM/MM models for ribozyme catalysis, 2) develop a new QM/MM method for prediction of pKa shifts in ribozymes;3) develop a novel free energy expansion approach to extend our QM/MM methods to the ab initio level;4) develop efficient path methods to study chemical mechanisms and conformational transitions in ribozymes. In this way we hope to greatly extend the predictive capability and range of application of state of the art theoretical methods to RNA catalysis.
The goal of this proposal is to use quantum mechanical and molecular simulations methods to study the mechanisms whereby molecules of RNA catalyze important chemical reactions. The insight gained by these studies will enhance our understanding of the fundamental role RNA plays in cells and facilitate the design of new RNA-based biomedical technology.
|Chen, Haoyuan; Giese, Timothy J; Golden, Barbara L et al. (2017) Divalent Metal Ion Activation of a Guanine General Base in the Hammerhead Ribozyme: Insights from Molecular Simulations. Biochemistry 56:2985-2994|
|Lee, Tai-Sung; Radak, Brian K; Harris, Michael E et al. (2016) A Two-Metal-Ion-Mediated Conformational Switching Pathway for HDV Ribozyme Activation. ACS Catal 6:1853-1869|
|Sengupta, Raghuvir N; Van Schie, Sabine N S; Giamba?u, George et al. (2016) An active site rearrangement within the Tetrahymena group I ribozyme releases nonproductive interactions and allows formation of catalytic interactions. RNA 22:32-48|
|Zhang, Shuming; Gu, Hong; Chen, Haoyuan et al. (2016) Isotope effect analyses provide evidence for an altered transition state for RNA 2'-O-transphosphorylation catalyzed by Zn(2+). Chem Commun (Camb) 52:4462-5|
|Gaines, Colin S; York, Darrin M (2016) Ribozyme Catalysis with a Twist: Active State of the Twister Ribozyme in Solution Predicted from Molecular Simulation. J Am Chem Soc 138:3058-65|
|Panteva, Maria T; Giamba?u, George M; York, Darrin M (2015) Comparison of structural, thermodynamic, kinetic and mass transport properties of Mg(2+) ion models commonly used in biomolecular simulations. J Comput Chem 36:970-82|
|Gebala, Magdalena; Giamba?u, George M; Lipfert, Jan et al. (2015) Cation-Anion Interactions within the Nucleic Acid Ion Atmosphere Revealed by Ion Counting. J Am Chem Soc 137:14705-15|
|Giese, Timothy J; Panteva, Maria T; Chen, Haoyuan et al. (2015) Multipolar Ewald Methods, 2: Applications Using a Quantum Mechanical Force Field. J Chem Theory Comput 11:451-461|
|Harris, Michael E; Piccirilli, Joseph A; York, Darrin M (2015) Integration of kinetic isotope effect analyses to elucidate ribonuclease mechanism. Biochim Biophys Acta 1854:1801-8|
|Giamba?u, George M; York, Darrin M; Case, David A (2015) Structural fidelity and NMR relaxation analysis in a prototype RNA hairpin. RNA 21:963-74|
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