The short-term goal of this work is to develop a method of temporary functionalization of an RNA scaffold with a transition metal-Schiff base complex such that a library of RNA structures can be screened, as their metal chelates, for catalytic oxidation chemistry. After selection of catalytic RNAs from the pool, the metal ion and Schiff base ligand will be removed by facile hydrolysis after EDTA treatment, allowing normal PCR amplification of the selected RNAs. Two systems will be studied: (1) the recognition and oxidation of 8-oxoguanine, a common oxidative lesion in DNA, permitting selective tagging of this lesion with an aldehyde functional group such that it can be biotinylated or fluoresceinylated, and (2) the recognition and tagging of a 5-MeC lesion in a CpG site, a hotspot for methylation. In both of these systems, the DNA substrates to be tagged are derived from mutagenic hotspots in the p53 gene. The proposed research will result in two significant, long-term advances in nucleic acid chemistry: (a) the method, and (b) the product. Originally, catalytic RNAs developed by in vitro selection were limited to the four natural bases; more recently, additional functional groups were introduced by replacing one of the four nucleotides with a synthetic nucleotide analog bearing a pendant, functionalized sidechain. However, this approach is problematic because the side chain is present at all sites containing that nucleoside and because the side chains often interfere with enzymes used in amplification. This new method will introduce covalent, but temporary, transition metal chelates using only the four natural nucleotides. The product will be ribozymes capable of recognizing and tagging DNA lesions in the genome.
