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.
bioRxiv
; 2023 Aug 24.
Article
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| MEDLINE
| ID: mdl-37662220
ABSTRACT
Many biochemical processes use the Watson-Crick geometry to distinguish correct from incorrect base pairing. However, on rare occasions, mismatches such as Gâ¢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 RNADNA 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 dGâ¢rU and dTâ¢rG mismatches in two RNADNA hybrids transiently form tautomeric (G enol â¢T/U âGâ¢T enol /U enol ) and anionic (Gâ¢T - /U - ) Watson-Crick-like conformations. The tautomerization dynamics were like those measured in A-RNA and B-DNA duplexes. However, anionic dGâ¢rU - formed with a ten-fold higher propensity relative to dT - â¢rG and dGâ¢dT - and this could be attributed to the lower pK a (Δ pK a â¼0.4-0.9) of U versus T. Our findings suggest plausible roles for Watson-Crick-like Gâ¢T/U mismatches in transcriptional errors and CRISPR/Cas9 off-target gene editing, uncover a crucial difference between the chemical dynamics of Gâ¢U versus Gâ¢T, and indicate that anionic Watson-Crick-like Gâ¢U - could play a significant role evading Watson-Crick fidelity checkpoints in RNADNA hybrids and RNA duplexes.
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