Secondary structure prediction for RNA sequences including N6-methyladenosine

There is increasing interest in the roles of covalently modified nucleotides in RNA. There has been, however, an inability to account for modifications in secondary structure prediction because of a lack of software and thermodynamic parameters. We report the solution for these issues for N 6 -methyladenosine (m 6 A), allowing secondary structure prediction for an alphabet of A, C, G, U, and m 6 A. The RNAstructure software now works with user-defined nucleotide alphabets of any size. We also report a set of nearest neighbor parameters for helices and loops containing m 6 A, using experiments. Interestingly, N 6 -methylation decreases folding stability for adenosines in the middle of a helix, has little effect on folding stability for adenosines at the ends of helices, and increases folding stability for unpaired adenosines stacked on a helix. We demonstrate predictions for an N 6 -methylation-activated protein recognition site from MALAT1 and human transcriptome-wide effects of N 6 -methylation on the probability of adenosine being buried in a helix. RNA folding free energy nearest neighbor parameters were determined for sequences with the nucleotide m 6 A. The RNAstructure software package can accommodate modified nucleotides, enabling secondary structure prediction of sequences with m 6 A.