Oxidative base damage in RNA detected by reverse transcriptase

Oxidative base damage In DNA and metabolic defects In the recognition and removal of such damage play Important roles in mutagenesls and human disease. The extent to which cellular RNA is a substrate for oxidative damage and the possible biological conse-quences of RNA base oxidation, however, remain largely unexplored. Since oxidatively modified RNA may contribute to the high mutability of retrovlral genomic DNA, we have been interested in developing methods for the sequence specific detection of such damage. We show here that a primer extension assay using AMV reverse transcriptase (RT) can be used to reveal oxidatively damaged sites in RNA. This finding extends the currently known range of RNA modifica-tions detectable with AMV reverse transcriptase. Analogous assays using DNA polymerases to detect base damage in DNA substrates appear to be re-stricted to lesions at thymine. Oxidative base damage in the absence of any detectable chain breaks was produced by dye photosensitization of RNA. Six out of 20 dyes examined were capable of producing RT detectable lesions. RT stops were seen predominantly at purines, although many pyrimidine sites were also detected. Dye specific phot of oot prints revealed by RT analysis suggests differential dye binding to the RNA substrate. Some of the photoreactive dyes described here may have potential utility in RNA structural analysis, particularly in the identification of stem-loop regions in complex RNAs