NMR measurements of transient low-populated tautomeric and anionic Watson-Crick-like G·T/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·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 R1ρ experiments, we show that dG·rU and dT·rG mismatches in two RNA:DNA hybrids transiently form tautomeric (Genol·T/U $ \mathbin{\lower.3ex\hbox{$\buildrel\textstyle\rightarrow\over {\smash{\leftarrow}\vphantom{_{\vbox to.5ex{\vss}}}}$}}$ G·Tenol/Uenol) 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 pKa (ΔpKa ∼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.