The goal of this project is to develop the use of RNA enzymes as reagents for the site specific cleavage (a consequent inactivation) of HIV and other high molecular weight RNAs in vitro and in vivo. Recent success in several labs using the hammerhead ribozyme to inactivate a number of RNAs (including HIV sequences) in bacterial, plant and animal cells emphasize the value of this emerging technology. This project will focus on refining and expanding the biochemical description of the cleavage of small and large RNAs by the hammerhead ribozyme. A kinetic description of the cleavage of matched and mismatched targets will be developed with the goal of predicting both the rate and specificity of the reaction. A strategy is proposed to identify sites in large RNAs that are particularly susceptible to cleavage. The inhibition of hammerhead cleavage by antibiotics will be explored. Finally, recently developed methods for in vitro evolution will be used to select for improved versions of the hammerhead and for new ribozymes that cleave pre-defined RNA targets.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI030242-05
Application #
2065524
Study Section
AIDS and Related Research Study Section 4 (ARRD)
Project Start
1990-03-01
Project End
1998-03-31
Budget Start
1994-04-01
Budget End
1995-03-31
Support Year
5
Fiscal Year
1994
Total Cost
Indirect Cost
Name
University of Colorado at Boulder
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
City
Boulder
State
CO
Country
United States
Zip Code
80309
Seiwert, S D; Stines Nahreini, T; Aigner, S et al. (2000) RNA aptamers as pathway-specific MAP kinase inhibitors. Chem Biol 7:833-43
Stage-Zimmermann, T K; Uhlenbeck, O C (1998) Hammerhead ribozyme kinetics. RNA 4:875-89
Hertel, K J; Stage-Zimmermann, T K; Ammons, G et al. (1998) Thermodynamic dissection of the substrate-ribozyme interaction in the hammerhead ribozyme. Biochemistry 37:16983-8
Stage-Zimmermann, T K; Uhlenbeck, O C (1998) Circular substrates of the hammerhead ribozyme shift the internal equilibrium further toward cleavage. Biochemistry 37:9386-93
Baidya, N; Uhlenbeck, O C (1997) A kinetic and thermodynamic analysis of cleavage site mutations in the hammerhead ribozyme. Biochemistry 36:1108-14
Clouet-d'Orval, B; Uhlenbeck, O C (1997) Hammerhead ribozymes with a faster cleavage rate. Biochemistry 36:9087-92
Clouet-D'Orval, B; Uhlenbeck, O C (1996) Kinetic characterization of two I/II format hammerhead ribozymes. RNA 2:483-91
Hertel, K J; Herschlag, D; Uhlenbeck, O C (1996) Specificity of hammerhead ribozyme cleavage. EMBO J 15:3751-7
Stage, T K; Hertel, K J; Uhlenbeck, O C (1995) Inhibition of the hammerhead ribozyme by neomycin. RNA 1:95-101
Werner, M; Uhlenbeck, O C (1995) The effect of base mismatches in the substrate recognition helices of hammerhead ribozymes on binding and catalysis. Nucleic Acids Res 23:2092-6

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