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

RNA oxidation by hypochlorous acid (HOCl) is of major interest in vitro as well as in vivo settings. Conducting a thorough analysis of HOCl oxidation of ribonucleosides, oligoribonucleotides and native RNAs by LC-MS, we characterized dominant oxidation events and products in vitro. In addition to extensive cytidine chlorination, the predominant reactivity was guanosine oxidation leading to 8-oxoguanosine as an intermediate, which was further oxidized, thereby acting as a scavenger in protecting adjacent residues from oxidation, and eventually resulting in an abasic site, or a derivative with similar reactivity. We show that, while 8 oxoguanosine does not undergo -elimination under conventional aniline treatment, a combination with prior alkaline treatment does. On this basis, we developed a method that allows mapping of oxidative guanosine lesions in RNA sequences, not only detecting abasic sites but including direct detection of 8-oxoguanosine in a sequence context. An application to yeast ribosomal RNA oxidized as pure RNA, or in ribosome particles, or in living cells, revealed idiosyncratic features in each of these RNA preparations.