AMINOGLYCOSIDE/RNA INTERACTIONS AND CORNEAL INFECTIONS

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RNA molecules are able to form precise three-dimensional structures which can be binding-sites for small organic molecules. An understanding of the rules that govern RNA/small molecule recognition processes rules would allow for an approach to our long-term goal of the de novo design of antagonists directed at particular RNA structures, in much the same way that inhibitors are designed for protein-based enzymes and receptors. Specific inhibitors designed to inhibit RNA molecules could be of enormous interest in ophthalmology and generally in medicine, in the design, for example, of small-molecules that can specifically interfere with the expression of mutant proteins that can lead to retinal degeneration, and in the design of small molecules that can antagonize RNA molecules from infectious disease producing organisms. This proposal describes approaches to gaining an understanding of the rules by which certain classes of naturally occurring RNA antagonists, the aminoglycosides, are recognized by specific RNA molecules. Random RNA molecules are selected by column methods to bind to defined aminoglycoside containing antibiotics with high affinity and specificity. New quantitative fluorescence methods are developed to determine the affinities and stoichiometries of antibiotic binding to the selected RNA aptamers. Novel chemical approaches are developed to reveal the regions of the aptamers which define the binding-sites for the specific antibiotic. High field NMR structural studies on the high affinity binding aptamers are planned. Those motifs in the specific RNA aptamers which recognize particular aminoglycosides will be determined and used as a guide for the future design of specific antagonists directed against naturally occurring RNA molecules. Two biologically occurring RNA molecules of particular interest in this context are the procaryotic 16S rRNA decoding region and the HIV-RRE transcriptional activator region. Quantitative structure-activity studies on aminoglycoside binding to the decoding and RRE regions leads to the design of novel aminoglycosidemimetic diversity libraries containing l,3(2)-hydroxylamine moieties. Specific antagonists directed against the decoding and RRE regions of RNA will be prepared and studies quantitatively. These antagonists are expected to be starting points for the design of drugs useful in the treatment of corneal infections. Overall, the studies described herein will serve as the basis for a general program on the design of specific RNA antagonists.