This proposal will continue studies on the solution structures of biologically active and potentially biomedically useful RNA enzymes (ribozymes). There has been a major breakthrough in this work during the previous grant period which was the development of methods for isotopically labeling RNAs and subsequent application of the powerful techniques of multi-dimensional heteronuclear magnetic resonance to the solution structure determination of RNAs. The ribozymes that will be studied include: i) a hammerhead ribozyme; ii) a hairpin ribozyme; and iii) a lead-dependent self-cleaving ribozyme termed the leadzyme. These ribozymes can all perform site-specific cleavage of a substrate RNA with multiple turnover. The RNAs will be 13C/15N labeled and then a variety of multi-dimensional heteronuclear magnetic resonance experiments will be used to assign the proton, carbon and nitrogen resonances in these molecules. Once resonance assignments have been made 2D, 3D, or 4D heteronuclear NOESY-type experiments will be performed to obtain proton- proton internuclear distances. Dihedral angle constraints will also be obtained from other multi-dimensional NMR experiments. This distance and dihedral angle information will be used as input for a distance geometry algorithm or constrained molecular dynamics calculations to generate three-dimensional structures consistent with the NMR data. The hammerhead and hairpin ribozymes have site-specific RNA endonuclease activity and have been shown to efficiently cleave specific sites in target RNAs in vitro. There have also been several in vivo applications of these ribozymes as RNA cleavage reagents against specific mRNAs, or as potential antiviral agents that target the genome of an RNA virus such as HIV-l. The structural information obtained from the NMR experiments proposed here will provide valuable data for the design of improved ribozymes and will be extremely helpful in interpretation of the wealth of biochemical, kinetic and mutagenesis data on these ribozyme systems.
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