We have isolated RNA molecules from combinatorial libraries that catalyze C-C bond formation by Diels-Alder reaction. These catalysts show remarkable stereo-, regio- and chemoselectivities. We could demonstrate true catalysis of bimolecular reactions, enantioselective bond formation, and catalytically active L-nucleic acids. The mechanism and kinetics of the reaction are under investigation using spectroscopic, chromatographic, and electrophoretic methods in combination with mutation analysis and various substrate and product analogs, chemical and enzymatic probing, fast kinetic methods, and nucleotide analog interference mapping.
Fig.: Tertiary fold and three-dimensional topology in the crystal structure of the Diels-Alder ribozyme-product complex.
Furthermore, we are attempting to isolate catalysts for other chemical reactions. An exciting project is the identification of ribozymes that catalyze the cleavage of peptide bonds, a very difficult task to achieve due to the high stability of peptide bonds. Using a step-wise selection, we have enriched RNAs that covalently react with a well-known mechanistic protease inhibitor. This enriched library is now the starting point for further in vitro evolution.
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