Mapping of HOCl-oxidized RNA identifies abasic sites as major damage and oxidation product of oxo8G

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.