Divalent metal ions are a key component in the structure and function of nucleic acids in general and catalytic RNA molecules (ribozymes) in particular. Current biophysical methods, however, are inadequate to provide a full description of metal ion-RNA interactions. We therefore propose to develop pulsed EPR as a technology for the study of RNA metal ion sites in solution. Using monomeric systems, we will develop and optimize methodologies based on electron spin echo envelope modulation (ESEEM) and electron spin echo-electron nuclear double resonance (ESE-ENDOR) for analyzing ligation by various RNA functional groups to paramagnetic metal ions. A key element in this work is the design and construction of a novel Kalpha-band (31 GHz) ESE-ENDOR spectrometer using a solid-state microwave source and power amplifier. We will apply these methods to a number of outstanding problems in RNA metallobiochemistry, including the utility of """"""""ion cores"""""""" in promoting the folding of large RNAs, the structure of catalytic metal ion sites in the ground and transition states of various ribozymes, and the modulation of ion binding modes by base modification, sequence, and other factors. These studies have great potential to improve the fundamental understanding of RNA and ribonucleoprotein enzymes, as well as of biological catalysis in general. In addition, since the design of bioactive ribozyme derivatives for pharmaceutical applications depends on an adequate understanding of catalytic mechanism, this research may significantly advance ribozymes toward their potential as therapeutics for a number of devastating diseases, including AIDS, cancer, and hepatitis C.
