Structure and Thermodynamics of N6-Methyladenosine in RNA: A Spring-Loaded Base Modification

N-6-Methyladenosine (m(6)A) modification is hypothesized to control processes such as RNA degradation, localization, and splicing. However, the molecular mechanisms by which this occurs are unclear. Here, we measured structures of an RNA duplex containing m(6)A in the GGACU consensus, along with an unmodified RNA control, by 2D NMR. The data show that m(6)AU pairing in the double-stranded context is accompanied by the methylamino group rotating from its energetically preferred syn geometry on the WatsonCrick face to the higher-energy anti conformation, positioning the methyl group in the major groove. Thermodynamic measurements of m6A in duplexes reveal that it is destabilizing by 0.5-1.7 kcal/mol. In contrast, we show that m6A in unpaired positions base stacks considerably more strongly than the unmodified base, adding substantial stabilization in single-stranded locations. Transcriptome-wide nuclease mapping of methylated RNA secondary structure from human cells reveals a structural transition at methylated adenosines, with a tendency to single-stranded structure adjacent to the modified base.