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

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

Marta Szabat  1 , Martina Prochota  1 , Ryszard Kierzek  1 , Elzbieta Kierzek  2 , David H Mathews  3
Affiliations

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

Marta Szabat et al. J Mol Biol. .

Abstract

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.

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Conflict of interest statement

Declaration of Competing Interest The authors declare that they have no known competing financial interests of personal relationships that could have appeared to influence the work reported in this paper.

Figures

Figure 1.
Figure 1.
Overview of Approach. Our goal is to test and refine nearest neighbor parameters for the prediction of folding stability (ΔG°37) for sequences that contain m6A. We designed sequences that include the RRACH motif that is the consensus for N6-methylation. We then melted pairs of sequences with or without methylation and also estimated the folding free energy change with nearest neighbor parameters. Throughout this work, we use 6 in sequences to represent m6A. The difference between the experimental and predicted ΔG°37, termed ΔΔG°37, is the measure of success. Perfect estimates have ΔΔG°37 = 0 kcal/mol. Where trends were observed in ΔΔG°37, we used the experiments to improve the nearest neighbor parameterization.
Figure 2.
Figure 2.
Distribution of ΔΔG°37 for 40 matched duplexes with m6A (orange) and with A (blue). ΔΔG°37 = ΔG°37 experimental - ΔG°37 estimated and therefore negative values indicate the estimate is understabilizing compared to the experiment.
Figure 3.
Figure 3.
Predicted ΔG°37 as a function of experimentally-determined ΔG°37 for duplexes placing A (blue) or m6A (orange) in a helical context. Perfect predictions would be on the diagonal, which is shown for reference.
Figure 4.
Figure 4.
Predicted ΔG°37 as a function of experimentally-determined ΔG°37 for duplexes placing A (blue) or m6A (orange) in or adjacent to a dangling end. Perfect predictions would be on the diagonal, which is shown for reference.
Figure 5.
Figure 5.
Predicted ΔG°37 as a function of experimentally-determined ΔG°37 for duplexes placing A (blue) or m6A (orange) in or adjacent to an internal or bulge loop. Perfect predictions would be on the diagonal, which is shown for reference.
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