The goal of this project is to move quantitative, carefully controlled analysis of RNA enzymes from the test tube into the cell, through a systematic series of experiments designed to address both fundamental issues regarding biological catalysis by RNA, and the development of ribozymes as selective tools for targeted cleavage of cellular and viral RNAs. Following on collaborations in which we demonstrated that engineered hairpin ribozymes can selectively inhibit replication of HIV-1 and hepatitis B virus, we have focused on studies conducted entirely within the PI's lab in which we have demonstrated inhibition of Sindbis virus in BHK-21 cells stably expressing hairpin ribozymes.
Specific Aims of this proposal are-(1) Explore the activity of a new class of hairpin ribozymes that do not require G at the cleavage site; (2) Determine the inhibitory mechanism, site of action, and sequence selectivity of hairpin ribozyme-mediated inhibition of Sindbis virus replication; (3) Evaluate and optimize the enzymatic activity and sequence selectivity of engineered hairpin ribozymes in trans-cleavage reactions within mammalian cells; (4) Evaluate and optimize ribozyme inhibition of gene expression using Sindbis virus as a model system. By combining strength and experience in in vitro biochemistry with skills in virology and cell biology, we believe that we are poised to make strong and unique contributions to the fields of biological by RNA and to targeted RNA inactivation.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Experimental Virology Study Section (EVR)
Program Officer
Tseng, Christopher K
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University of Vermont & St Agric College
Schools of Medicine
United States
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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
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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
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Berzal-Herranz, A; Burke, J M (1997) Ligation of RNA molecules by the hairpin ribozyme. Methods Mol Biol 74:349-55
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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