A Test and Refinement of Folding Free Energy Nearest Neighbor Parameters for RNA Including N6-Methyladenosine

J Mol Biol. 2022 Sep 30;434(18):167632. doi: 10.1016/j.jmb.2022.167632. Epub 2022 May 16.


RNA folding free energy change parameters are widely used to predict RNA secondary structure and to design RNA sequences. These parameters include terms for the folding free energies of helices and loops. Although the full set of parameters has only been traditionally available for the four common bases and backbone, it is well known that covalent modifications of nucleotides are widespread in natural RNAs. Covalent modifications are also widely used in engineered sequences. We recently derived a full set of nearest neighbor terms for RNA that includes N6-methyladenosine (m6A). In this work, we test the model using 98 optical melting experiments, matching duplexes with or without N6-methylation of A. Most experiments place RRACH, the consensus site of N6-methylation, in a variety of contexts, including helices, bulge loops, internal loops, dangling ends, and terminal mismatches. For matched sets of experiments that include either A or m6A in the same context, we find that the parameters for m6A are as accurate as those for A. Across all experiments, the root mean squared deviation between estimated and experimental free energy changes is 0.67 kcal/mol. We used the new experimental data to refine the set of nearest neighbor parameter terms for m6A. These parameters enable prediction of RNA secondary structures including m6A, which can be used to model how N6-methylation of A affects RNA structure.

Keywords: RNA covalent modification; RNA folding stability; RNA methylation; RNA secondary structure prediction; optical melting.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Adenosine* / analogs & derivatives
  • Adenosine* / chemistry
  • Entropy
  • RNA Folding*
  • RNA* / chemistry


  • RNA
  • N-methyladenosine
  • Adenosine