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Abstract

Using fluorescence microscopy as a tool to study biological systems has been proven to be useful in both experiments in cells and in vitro. However, current methods for preparing fluorescent RNAs for use in vitro fluorescence assays are challenging and inefficient. Recently, we have shown that deoxyribozymes (DNAzymes) can be used to site-specifically label RNA molecules with fluorescent GMP moieties. We believe that this method will prove generally useful for incorporation of fluorescent labels into different sites within an RNA molecule for use in biochemical studies. We propose that the annealing of two RNA molecules (the substrate RNA and a “helper” RNA) to the DNAzyme results in formation of a ternary complex that brings key catalytic sites within close proximity in order to facilitate GMP transfer. Initial experiments suggested that this ternary complex is not stable during native PAGE analysis. I am currently studying a unimolecular RNA/DNA hybrid in which the substrate RNA, the helper RNA, and the DNA are all part of a single oligonucleotide. Our goal is to use this simplified construct to characterize the folding and reaction pathways for DNAzyme catalysis and RNA labeling. These experiments will lead to development of next generation deoxyribozymes with improved function. This work was supported by a Beckman Young Investigators award to AAH and a Hilldale Undergraduate Research Fellowship to MA.