NMR measurements of transient low-populated tautomeric and anionic Watson-Crick-like GT/U in RNA:DNA hybrids: implications for the fidelity of transcription and CRISPR/Cas9 gene editing

Many biochemical processes use the Watson-Crick geometry to distinguish correct from incorrect base pairing. However, on rare occasions, mismatches such as G center dot 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 (1 rho) experiments, we show that dG center dot rU and dT center dot rG mismatches in two RNA:DNA hybrids transiently form tautomeric (G (enol) center dot T/U -><- G center dot T (enol) /U (enol) ) and anionic (G center dot T (-) /U (-) ) Watson-Crick-like conformations. The tautomerization dynamics were like those measured in A-RNA and B-DNA duplexes. However, anionic dG center dot rU (-) formed with a ten-fold higher propensity relative to dT (-) center dot rG and dG center dot dT (-) and this could be attributed to the lower pK (a) (Delta pK (a) similar to 0.4-0.9) of U versus T. Our findings suggest plausible roles for Watson-Crick-like G center dot T/U mismatches in transcriptional errors and CRISPR/Cas9 off-target gene editing, uncover a crucial difference between the chemical dynamics of G center dot U versus G center dot T, and indicate that anionic Watson-Crick-like G center dot U (-) could play a significant role evading Watson-Crick fidelity checkpoints in RNA:DNA hybrids and RNA duplexes.