Abstract
Many biochemical processes use the Watson-Crick geometry to distinguish correct from incorrect base pairing. However, on rare occasions, mismatches such as G•T/U can transiently adopt Watson-Crick-like conformations through tautomerization or ionization of the bases, giving rise to replicative and translational errors. The propensities to form Watson-Crick-like mismatches in RNA:DNA hybrids remain unknown, making it unclear whether they can also contribute to errors during processes such as transcription and CRISPR/Cas editing. Here, using NMR R (1ρ) experiments, we show that dG•rU and dT•rG mismatches in two RNA:DNA hybrids transiently form tautomeric (G (enol) •T/U ⇄G•T (enol) /U (enol) ) and anionic (G•T (-) /U (-) ) Watson-Crick-like conformations. The tautomerization dynamics were like those measured in A-RNA and B-DNA duplexes. However, anionic dG•rU (-) formed with a ten-fold higher propensity relative to dT (-) •rG and dG•dT (-) and this could be attributed to the lower pK (a) (Δ pK (a) ∼0.4-0.9) of U versus T. Our findings suggest plausible roles for Watson-Crick-like G•T/U mismatches in transcriptional errors and CRISPR/Cas9 off-target gene editing, uncover a crucial difference between the chemical dynamics of G•U versus G•T, and indicate that anionic Watson-Crick-like G•U (-) could play a significant role evading Watson-Crick fidelity checkpoints in RNA:DNA hybrids and RNA duplexes.