RNA enzymes, or ribozymes, are fascinating molecules that are of great potential utility as sequence-specific antiviral therapeutic agents. This project directly addresses the two central scientific questions regarding ribozymes-- First, how does the molecular structure of ribozymes result in catalytic activity? Second, can engineered ribozymes be used effectively within the mammalian cell? Our experiments utilize the catalytically versatile 50 nt hairpin ribozyme. Progress to date includes the development and exploitation of a novel and powerful in vitro selection system, and the use of more traditional methods, to determine structural and biochemical requirements for the ribozyme, especially its substrate recognition mechanism. The Specific Aims of this project are to: (1) Identify all essential structural elements required for substrate binding, RNA folding, and catalytic function of the hairpin ribozyme. (2) Map the tertiary structure of the ribozyme and ribozyme-substrate complex. (3) Characterize and optimize the enzymatic activity, selectivity and stability of ribozymes in cytoplasmic extracts. (4) Rigorously demonstrate and optimize intracellular catalysis by engineered hairpin ribozymes. (5) Explore two novel strategies for directing ribozymes to specific cellular locations. The project will result in development of the technical capacity to rationally design therapeutic ribozymes for the treatment of essentially any current or future viral disease. The results of this project will be of immediate importance to those working in the areas of biomolecular catalysis, RNA structure, molecular recognition, developmental therapeutics, infectious disease, and gene therapy.