Development of thallium-205 NMR methods for the direct study of monovalent metal ions and ligands in nucleic acids

The requirement of monovalent cations for structure and function transcends all classes of biological macromolecules. In catalytic RNAs, a structural and/or catalytic requirement for monovalent ions has been identified in the ribosome, group I, and group II introns. Other R-NAs, such as the hammerhead, hairpin, and VS ribozymes, can perform catalysis the presence of only high concentrations of monovalent cations. Despite their importance, few techniques exist for the direct, solution study of these cations. Toward this goal, I have used 205Tl+, a K+ surrogate which is readily detectable by solution NMR, to study the binding of monovalent canons to a G-quadruplex, d(G4T4G4) 2. The NMR and crystal structures of the Tl+-form of d(G4T4G4)2, have been determined to assess the ability of Tl+ to mimic K+ in a nucleic acid setting. Direct detection 205Tl NMR studies have been used to characterize the binding of 205Tl + to d(G4T4G4)2 and provide evidence for a previously undetected mode of monovalent binding. I have also developed a 1H-205Il spin-echo difference experiment which was used to detect 1H-205Il scalar couplings and assign two of the experimentally observed 205Tl resonances to monovalent binding sites in d(G4T4G4) 2. These results comprise the first 1H-205Il scalar couplings observed in a biological system and the first 205 Tl heteronuclear experiment reported. Preliminary 205Tl NMR studies in RNA systems are also discussed.