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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM062248-13
Application #
8313994
Study Section
Macromolecular Structure and Function D Study Section (MSFD)
Program Officer
Preusch, Peter C
Project Start
2001-01-01
Project End
2014-07-31
Budget Start
2012-08-01
Budget End
2013-07-31
Support Year
13
Fiscal Year
2012
Total Cost
$312,098
Indirect Cost
$109,253
Name
Rutgers University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
001912864
City
New Brunswick
State
NJ
Country
United States
Zip Code
08901
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
Sengupta, Raghuvir N; Van Schie, Sabine N S; GiambaÅŸu, 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
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
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
GiambaÅŸu, George M; Gebala, Magdalena K; Panteva, Maria T et al. (2015) Competitive interaction of monovalent cations with DNA from 3D-RISM. Nucleic Acids Res 43:8405-15
Harris, Mike E; Piccirilli, Joseph A; York, Darrin M (2015) Enzyme transition states from theory and experiment. Biochim Biophys Acta 1854:1727-8
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
Chen, Haoyuan; Piccirilli, Joseph A; Harris, Michael E et al. (2015) Effect of Zn2+ binding and enzyme active site on the transition state for RNA 2'-O-transphosphorylation interpreted through kinetic isotope effects. Biochim Biophys Acta 1854:1795-800
Panteva, Maria T; Dissanayake, Thakshila; Chen, Haoyuan et al. (2015) Multiscale methods for computational RNA enzymology. Methods Enzymol 553:335-74
Dissanayake, Thakshila; Swails, Jason M; Harris, Michael E et al. (2015) Interpretation of pH-activity profiles for acid-base catalysis from molecular simulations. Biochemistry 54:1307-13

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