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 N⁶-methyladenosine (m⁶A), allowing secondary structure prediction for an alphabet of A, C, G, U, and m⁶A. 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 m⁶A, using experiments. Interestingly, N⁶-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 N⁶-methylation-activated protein recognition site from MALAT1 and human transcriptome-wide effects of N⁶-methylation on the probability of adenosine being buried in a helix.