The proposed research focuses on the structural and mechanistic characterization of metal-binding sites in ribozymes (RNAs with enzymatic activities), which have shown promise in the fight against AIDS and other retroviral diseases. The study of ribozymes is now limited by the structural information available about the active sites. It was recently demonstrated that metal ions are essential for both the structure and the function of ribozymes. This new class of metalloenzymes thus presents a timely challenge to bioinorganic chemists. The approach presented combines metal-ion-based spectroscopic techniques that have been used successfully in the characterization of metalloproteins and biochemical techniques that have been used in RNA laboratories, including a powerful method called directed selection and evolution. Initially, metal ions (such as Co2+) and nucleotide derivatives (such as phosphorothioates) with rich spectroscopic properties will be used to probe the metal-binding sites in hammerhead ribozymes. Other metal derivatives (such as Mn2+, Cd2+, Zn2+, Mg2+ and Ca2+) as well as other ribozymes (such as hairpin ribozymes and group I introns) will be studied in due course. The proposed research will advance our understanding of structural properties of the metal-binding sites such as their stoichiometry, ligands, geometry and electronic structure. The functional properties to be studied include how metal ions assist in binding the substrate, stabilizing the transition state, and releasing the product. Kinetic studies will also be carried out to elucidate the reaction mechanisms of ribozyme catalysis. A long-term goal of the research is to engineer ribozymes to maximize their potential as anti-viral drugs. Genetic engineering, mainly of metal- binding sites and their surrounding environments, combined with directed selection and evolution techniques, will be carried out to design more stable and efficient ribozymes. This project will open new frontiers in bioinorganic chemistry and biomedical engineering that are important both in fundamental research (role of metal ions in ribozymes) and in pharmaceutical applications (design of more effective drugs against AIDS and other retroviral diseases).
