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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI030534-07
Application #
2003633
Study Section
Biochemistry Study Section (BIO)
Project Start
1990-12-01
Project End
1998-11-30
Budget Start
1996-12-01
Budget End
1997-11-30
Support Year
7
Fiscal Year
1997
Total Cost
Indirect Cost
Name
University of Vermont & St Agric College
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
066811191
City
Burlington
State
VT
Country
United States
Zip Code
05405
Zhang, Zhenxi; Burke, John M (2005) Inhibition of viral replication by ribozyme: mutational analysis of the site and mechanism of antiviral activity. J Virol 79:3728-36
Seyhan, Attila A; Vitiello, Danielle; Shields, Michele T et al. (2002) Ribozyme inhibition of alphavirus replication. J Biol Chem 277:25957-62
zu Putlitz, J; Yu, Q; Burke, J M et al. (1999) Combinatorial screening and intracellular antiviral activity of hairpin ribozymes directed against hepatitis B virus. J Virol 73:5381-7
Murray, J B; Seyhan, A A; Walter, N G et al. (1998) The hammerhead, hairpin and VS ribozymes are catalytically proficient in monovalent cations alone. Chem Biol 5:587-95
Yu, Q; Pecchia, D B; Kingsley, S L et al. (1998) Cleavage of highly structured viral RNA molecules by combinatorial libraries of hairpin ribozymes. The most effective ribozymes are not predicted by substrate selection rules. J Biol Chem 273:23524-33
Berzal-Herranz, A; Burke, J M (1997) Ligation of RNA molecules by the hairpin ribozyme. Methods Mol Biol 74:349-55
Yu, Q; Burke, J M (1997) Design of hairpin ribozymes for in vitro and cellular applications. Methods Mol Biol 74:161-9
Sargueil, B; Burke, J M (1997) In vitro selection of hairpin ribozymes. Methods Mol Biol 74:289-300
Sargueil, B; Pecchia, D B; Burke, J M (1995) An improved version of the hairpin ribozyme functions as a ribonucleoprotein complex. Biochemistry 34:7739-48
Butcher, S E; Heckman, J E; Burke, J M (1995) Reconstitution of hairpin ribozyme activity following separation of functional domains. J Biol Chem 270:29648-51