The goal of this project is to understand the biochemical properties of the """"""""hammerhead"""""""" and """"""""hairpin"""""""" RNA enzymes (ribozymes). These two small RNA catalysts have the potential to cause site-specific cleavage (and consequent inactivation) of HIV and other high molecular weight RNAs in vitro and in vivo. The major focus will be on the design of optimally active cleavage reagents in vitro. Important variables include the possibility of interfering structure in the target RNA, the conformational homogeneity of the ribozyme and target RNAs, the length and sequence of the RNA helices which join the RNA enzyme to the target and the possibility of certain RNA structures which stimulate hybridization rates. The approach will involve the use of pre-steady state and steady state kinetics to analyze the cleavage reaction. The rate limiting step of each reaction will be determined since it is anticipated that it may change depending on reaction conditions. If possible,, the Kappa/m will be related to the known free energy parameters of helix formation, making it possible to predict the Kappa/m for any ribozyme-substrate pair.
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