Mapping of HOCl-oxidized RNA identifies abasic sites as major damage and oxidation product of oxo8G
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Abstract
RNA oxidation is an important yet understudied process, partly because methods to localize oxidized residues in RNA are lacking. We introduce OAbSeq, a deep-sequencing approach that maps oxidized sites with high sensitivity by exploiting aniline-induced strand scission at noncanonical nucleosides to generate unique ligation-competent fragments utilized for library preparation. Applied to yeast RNA, OAbSeq detects widespread signals predominating at purines, especially at guanosines. Exogenous oxidation increased signal intensity but preserved the guanosine-dominated pattern. Parallel quantification of 8-oxoguanosine (oxo8G) and abasic sites revealed that abasic sites are more abundant than oxo8G following oxidative treatment in vitro and under physiological conditions. These data support a model in which guanosine oxidation proceeds via transient oxo8G yielding abasic sites that can be mapped at nucleotide resolution by OAbSeq. Our findings also suggest abasic sites may be a more informative marker of RNA oxidative damage than oxo8G, facilitating studies of RNA oxidation dynamics in cells.