5-Oxyacetic Acid Modification Destabilizes Double Helical Stem Structures and Favors Anionic Watson-Crick like cmo⁵...

Watson-Crick like G-U mismatches with tautomeric G^enol or U^enol bases can evade fidelity checkpoints and thereby contribute to translational errors. The 5-oxyacetic acid uridine (cmo⁵ U) modification is a base modification at the wobble position on tRNAs and is presumed to expand the decoding capability of tRNA at this position by forming Watson-Crick like cmo⁵ U^enol -G mismatches. A detailed investigation on the influence of the cmo⁵ U modification on structural and dynamic features of RNA was carried out by using solution NMR spectroscopy and UV melting curve analysis. The introduction of a stable isotope labeled variant of the cmo⁵ U modifier allowed the application of relaxation dispersion NMR to probe the potentially formed Watson-Crick like cmo⁵ U^enol -G base pair. Surprisingly, we find that at neutral pH, the modification promotes transient formation of anionic Watson-Crick like cmo⁵ U^- -G, and not enolic base pairs. Our results suggest that recoding is mediated by an anionic Watson-Crick like species, as well as bring an interesting aspect of naturally occurring RNA modifications into focus-the fine tuning of nucleobase properties leading to modulation of the RNA structural landscape by adoption of alternative base pairing patterns.