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

TL;DR

This study uses NMR to show that rare G·T/U mismatches in RNA:DNA hybrids can form stable Watson–Crick-like structures, which may cause errors in transcription and CRISPR/Cas9 editing.

Contribution

The study reveals that anionic G·T/U mismatches form more readily in RNA:DNA hybrids than previously known, with implications for genetic fidelity.

Findings

G·T/U mismatches in RNA:DNA hybrids transiently form tautomeric and anionic Watson–Crick-like conformations.

Anionic dG·rU− forms with ten-fold higher propensity than dT−·rG and dG·dT− due to lower pKa of uracil.

These findings suggest a role for G·T/U mismatches in transcription errors and CRISPR/Cas9 off-target effects.

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 R1ρ experiments, we show that dG·rU and dT·rG mismatches in two RNA:DNA hybrids transiently form tautomeric (Genol·T/U \documentclass[12pt]{minimal}
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$ \mathbin{\lower.3ex\hbox{$\buildrel\textstyle\rightarrow\over {\smash{\leftarrow}\vphantom{_{\vbox to.5ex{\vss}}}}$}}$\end{document} 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.

Graphical Abstract

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