Metal interactions and activities of truncated and extended hammerhead ribozyme constructs

The hammerhead ribozyme (HHRz) catalyzes a site-specific phosphodiester bond cleavage reaction that is enhanced by the presence of metal cations. Metal cations are thought to aid in the folding and possibly the catalytic mechanism of this ribozyme. The goal of this research is to characterize the activities and metal interactions of minimal and extended HHRz constructs using kinetic and spectroscopic studies. Metal binding to the cleavage site of the HHRz was probed using 31P NMR to monitor Cd2+ titrations of HHRzs with a phosphorothioate modification at the cleavage site. Either a 2'-F or a 2'-NH2 substitution at the nucleophile position was used to block cleavage. With a 2'-F, no metal binding to the cleavage site phosphate was observed. However, with a 2'-NH2 substitution, a large change in 31P chemical shift of the phosphorothioate peak suggests Cd2+ binding. A 2'-NH2 is a potential metal ligand, but a 2'-F is not. This suggests that a metal ion binds to the cleavage site phosphate when the 2' nucleophile position also provides a ligand. Minimal HHRzs with only one stem loop structure show little activity in presence of physiologically relevant concentrations of divalent cations. A kinetic and thermodynamic characterization of an extended HHRz derived from Schistosoma mansoni with loops in stems I and II was performed. High catalytic activity was observed with low concentrations of divalent cations, and loss of potential loop-loop interactions resulted in a large decrease in activity. An electrostatic surface plot of a HHRz crystal structure revealed an area of high negative electrostatic potential in the cleft between stems I and II with contributions from nucleotides U7, A6, and C17 of the HHRz that could serve to trap metal ions. To probe this putative metal site, kinetic studies of HHRz constructs with phosphorothioate substitutions 5' to U7 or C17 or with an A6 2'- OMe substitution were performed. Results of these studies suggest that a metal interaction at this site would include direct coordination to A6 2'-OH, but indirect interactions with the phosphates.