What types of chemical reactions can RNA enzymes perform and how do the three dimensional structures of these RNAs specify their function? All naturally-occurring ribozymes catalyze a limited set of reactions involving the RNA phosphate backbone. By developing and applying in vitro selection methods, we will isolate novel classes of RNA enzymes that perform a wide range of new catalytic activities. Characterization of these and previously isolated RNAs will be performed to determine the chemical and structural basis for ribozyme catalysis. Starting from a pool of random sequence RNA molecules, we have identified well-defined RNA motifs that recognize the carboxylation co-factor biotin and the fluorophore sulforhodamine with high affinity. Using the biotin- binding RNA as a starting point for mutagenesis, we have successfully evolved ribozymes that utilize a biotin-based substrate to perform a self- alkylation reaction. Preliminary characterization shows that trivial models to account for the high activity of one of these enzymes are invalid, indicating that more involved catalytic mechanisms are exploited to achieve catalysis. FIRST, we will adapt the selection method used to obtain self-alkylating ribozymes to target other types of ligation chemistry, isolating RNAs that catalyze peptide and glycoside formation. SECOND, we will develop a selection strategy for isolating RNA enzymes by immunoaffinity chromatography. This method should make it possible to obtain ribozymes with many different types of catalytic activities. THIRD, we will use a direct fluorescence detection method to isolate multiple turnover ribozymes that catalyze hydrolysis reactions with fluorigenic substrates. FOURTH, we will characterize the biotin- and sulforhodamine-binding motifs, the self-biotinylating ribozymes, and any additional isolated ribozymes using a combination of genetic, biochemical, and biophysical methods. These studies will aim towards building complete structural models for each RNA that explain how nucleotides interact with one another to direct folding to a unique tertiary structure and how nucleotides and ligands interact to yield high affinity binding. FIFTH, we will conduct a series of enzymological experiments to probe the specific mechanisms used by the self-biotinylating ribozymes to achieve catalysis. IN SUMMARY, these experiments should allow us to isolate many different classes of ribozymes that catalyze a wide range of chemical reactions. These enzymes will then serve as simple model systems for answering basis questions about the nature of ribozyme catalysis.
