N6-Hydroperoxymethyladenosine: a new intermediate of chemical oxidation of N6-methyladenosine mediated by bicarbonate-activated hydrogen peroxide

Abstract

N ⁶-Methyladenosine (m⁶A) represents a relatively abundant modification in eukaryotic RNA. Because m⁶A has similar properties to adenosine and a low reactivity, limited research has been focused on this nucleoside. In this study, we revealed an important intermediate in the oxidation of m⁶A through the bicarbonate-activated peroxide system. Over the course of oxidation, we found a new mechanism in which N⁶-hydroxymethyladenosine (hm⁶A), N⁶-formyladenosine (f⁶A) and N⁶-hydroperoxymethyladenosine (oxm⁶A) were intermediate products, and adenosine was the final product. In this study, oxm⁶A was isolated using HPLC and characterized by mass spectrometry, NMR and diphenyl-1-pyrenylphosphine (DPPP) fluorescence detection. This study provides a new modified nucleoside and demonstrates oxidative demethylation of m⁶A by reactive oxygen species at the nucleobase level and in RNA strands.