RUI: Atranyl-Nucleosides as Ribozyme Probes

Xavier University, Cincinnati OH

Investigators

Abstract

Current models for the catalytic mechanisms of protein enzymes are far more advanced than those for RNA enzymes (ribozymes). This project will contribute to understanding how the catalytic mechanisms of ribozymes compare with those of protein enzymes. The specific objectives of this project are to: (1) synthesize atranyl-nucleosides 1-3; (2) characterize and determine the aqueous stability of atranyl-nucleosides; (3) use atranyl-nucleosides as transition state analogues (TSAs) to study the hammerhead and lead-dependent (leadzyme) ribozymes via collaborations; and (4) train undergraduate students in synthetic organic techniques in preparation for graduate study and/or careers in science. TSAs are useful for enzyme studies; however, suitable TSAs for phosphoryl transfer reactions are lacking. Compounds 1-3 have an atrane moiety incorporated into a ribonucleoside. The atrane contains a trigonal bipyramidal silicon, germanium, tin, or titanium atom; and thus, 1-3 mimic the oxyphosphorane transition state of the ribozyme-catalyzed reaction. Compound 2 is a protected phosphoramidite that contains uridine and deoxyadenosine connected through the atrane moiety. Several syntheses of 2 starting from 5'-O-DMT-2'-amino-2'-deoxyuridine will be evaluated. Prof. William G. Scott of UC-Santa Cruz will incorporate 2 into the hammerhead substrate by solid-phase synthesis to obtain a hammerhead ribozyme-TSA crystal structure. Prof. Arthur Pardi of the University of Colorado-Boulder will similarly determine the structure in solution using NMR spectroscopy. Analogous approaches will be taken for leadzyme structural studies following the synthesis of phosphoramidite 3. These experiments will elucidate the magnitude of conformational rearrangement required to reach the transition state in each of these small RNA catalysts. This research will also help identify the role of specific RNA functional groups and metal cations in catalysis. This knowledge is paramount for understanding the pre-biotic "RNA world" and how our current life forms evolved from it.