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

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} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} $ \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.


Figure S1 .Figure S2 .
Figure S1.NMR spectra of the two RNA:DNA hybrids.(A) 1D 1 H spectra and (B) 2D [ 15 N, 1 H] HSQC spectra of the imino region of dT•rG (left) and dG•rU (right) measured at pH 7.4.Shown in the 2D HSQC spectra are the imino resonances of G-N1/H1 and T/U-N3/H3 targeted for relaxation dispersion measurements.The chemical shift of both G-H1 and T/U-H3 is up-field shifted relative to other resonances, indicative of the G•T/U wobble geometry.(C) 1D 1 H spectra and (D) 2D [ 15 N, 1 H] HSQC spectra of the imino region of dT•rG (left) and dG•rU (right) measured at pH 7.8.

Figure S3 .Figure S4 .Figure S5 .
Figure S3.Extrapolating exchange rates using pH dependent data.Exchange rates were extracted from fits to off resonance R1ρ profiles measured in a prior study.(3)The natural logarithm of the exchange rates is plotted against the pH for hpTG-CGC (left) or hpTG-GGC (right).Forward and backward rates for ES1 (blue) and ES2 (green) are shown as filled and open symbols, respectively.Minor exchange rates between ES1 and ES2 are shown as open triangles.Solid lines are linear fits to the pH dependent data points.

Figure S6 .
Figure S6.Anionic Watson-Crick-like G•U -contribution to misincorporation errors at different pH.Relative flux quantifying the fractional percentage (fA) of misincorporations proceeding through the anionic Watson-Crick-like G•U -conformation as a function of varying k2 computed using the kinetic model presented in Figure 4A.Results are shown when using the chemical dynamics measured by NMR for dG•dT (DNA) and rG•rU (RNA) with a CGC trinucleotide sequence context(3) and dG•rU and dT•rG in the two RNA:DNA hybrids examined in this work.The flux through the two pathways is computed using extrapolated exchange rates (dotted lines, see Materials and Methods) at pH 6.9 (A) or NMR-derived exchange rates (full lines) at pH 7.8 for the two hybrids (B, left), or at pH 8.4 for B-DNA and A-RNA (B, right).The calculations assume that the Watson-Crick-like G•T/U is an obligatory

Table S2 .
Exchange parameters obtained from fitting 15 N R1ρ data in dG•rU and dT•rG at pH 7.4, 25 °C.GS corresponds to the wobble ground state of G•T/U, ES1 corresponds to the tautomeric Watson-Crick-like species which consists of G enol •T/U and G•T enol /U enol conformations in rapid equilibrium, and ES2 corresponds to the anionic G•T -/U - conformation.Red c 2 is the reduced c 2 obtained on fitting the data.The exchange parameters that best fit the data based on statistical tests and chemical shift criteria as outlined in Methods are highlighted in bold.

Table S3 .
Exchange parameters obtained from fitting 15 N R1ρ data in dG•rU and dT•rG at pH 7.8, 25 °C.GS corresponds to the wobble ground state of G•T/U, ES1 corresponds to the tautomeric Watson-Crick-like species which consists of G enol •T/U and G•T enol /U enol conformations in rapid equilibrium, and ES2 corresponds to the anionic G•T -/U - conformation.Red c 2 is the reduced c 2 obtained on fitting the data.The exchange parameters that best fit the data based on statistical tests and chemical shift criteria as outlined in Methods are highlighted in bold.