Rare tautomeric and anionic nucleobases are believed to have fundamental biological roles, but their prevalence and functional importance has remained elusive because they exist transiently, in low abundance, and involve subtle movements of protons that are difficult to visualize. Using NMR relaxation dispersion, we show here that wobble dG•dT and rG•rU mispairs in DNA and RNA duplexes exist in dynamic equilibrium with short-lived, low-populated Watson–Crick-like mispairs that are stabilized by rare enolic or anionic bases. These mispairs can evade Watson–Crick fidelity checkpoints and form with probabilities (10−3 to 10−5) that strongly imply a universal role in replication and translation errors. Our results indicate that rare tautomeric and anionic bases are widespread in nucleic acids, expanding their structural and functional complexity beyond that attainable with canonical bases.
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We thank S. Horowitz, H. Zhou, J. Lee, A. M. Mustoe, and E. N. Nikolova for assistance and critical comments. We are grateful for technical support and resources from the Duke Magnetic Resonance Spectroscopy Center and University of Michigan Flux HPC Cluster. This work was supported by an NIH grant (R01GM089846) and an Agilent Thought Leader Award given to H.M.A.
The authors declare no competing financial interests.
Extended data figures and tables
a, b, Shown are the hp-GT DNA (a) and Dickerson-GT (b) constructs with 13C/15N labelled dG•dT mispairs highlighted in red along with 2D imino [15N, 1H] HSQC, 2D aromatic [13C, 1H] HSQC and 2D C1′ [13C, 1H] HSQC spectra (pH 6.9, 25 °C).
Extended Data Figure 2 Rotating frame relaxation dispersion profiles of dG•dT mispairs in hp-GT and Dickerson-GT DNA constructs.
RD profiles showing chemical exchange (R2 + Rex) in the dG•dT mispair as a function of the spin lock offset (Ωeff 2π−1) and spin lock power (ωSL 2π−1, colour coded in insets). a, b, Shown are 15N (a) and 13C (b) RD profiles in hp-GT DNA. On-resonance profiles showing solid and dashed black lines indicate fits assuming no chemical exchange (solid) and simplified two-state exchange process (dash). The hp-GT dG15-N1 and dT5-N3 in brackets denote duplicate profiles (with an additional 800 Hz spinlock power for each) collected at pH 8.4 and 25 °C collected on a different spectrometer from the preceding profiles. c, 15N and 13C RD profiles for Dickerson-GT. Sample conditions are indicated on each profile. Error bars represent experimental uncertainty (one s.d., see Methods).
a, b, Global fitting of hp-GT DNA (a) and hp-GU-20 RNA (b) N1 and N3 RD profiles to two-state algebraic equation (equation (1), fit reduced χ2 shown in inset) and three-state algebraic equation (equation (2), fit reduced χ2 shown in inset). Numerical solutions to the Bloch–McConnell three-state equations assuming no minor exchange and input exchange parameters obtained based on the three-state algebraic fit are also shown to establish the validity of the three-state expression under these exchange scenarios (equation (2), see Methods). Sample conditions are indicated on each profile. Error bars represent experimental uncertainty (one s.d., see Methods).
a, RD-derived dG15-N1 and dT5-N3 chemical shifts (CSs) (referenced to GS WB) for ES1 (25 °C and pH 6.9) and ES2 (25 °C and pH 8.4) of hp-GT and ES1 of Dickerson-GT (25 °C and pH 6.9) are shown. Errors in all RD-derived fitted parameters (for example, Δω) reflect experimental uncertainty (one s.d.) from the weighted global fit (see Methods). b, RD-derived hp-GT dG•dT ES1 (blue) and ES2 (green) 15N CSs are shown as a function of temperature and pH for both dG15-N1 (square) and dT5-N3 (circle). c, Scheme used to calculate CSs using DFT (see Methods). Shown is a schematic representation of scenario used to for calculating CSs using DFT. Idealized B-form DNA helix is generated to give a central dG•dT mispair (red) that is flanked by canonical dG•dC pairs, analogous to the hp-GT construct. Residues are trimmed to 1-/9-methyl bases and i + 1/i − 1 pairs are frozen in place for subsequent geometry optimizations and NMR CS calculations. d, DFT-calculated CSs (referenced to an energy optimized WB geometry) are shown for various tautomeric and anionic configurations, where dGenol•dT/dG•dTenol represents population-weighted average over dGenol•dT (80%) and dG•dTenol (20%). e, RD-derived ES1 and ES2 CSs are plotted against DFT-calculated CSs of base opened dG•dT mispairs, taken from X-ray structures and pruned to 1-/9-methyl bases. f, DFT-calculated CSs (referenced to an energy optimized WB geometry) are plotted as a function of dG-N1 to dT-N3 inter-atomic distance for a WC-like dGenol•dT tautomeric pair. g, Computational studies31,32,70 predict that the tautomeric pathway proceeds via a planar dG+•dT– ion pair (charge delocalization is implied) that is highlighted by a network of five H-bonds. h, Predicted pair geometry of an anionic dG•dT– inverted wobble. Deprotonated dT-N3 is highlighted in red (charge delocalization is implied). i, Predicted pair geometry of a dGenol•dT Hoogsteen mispair.
a, 1D 13C spectra (without 13C-13C homonuclear decoupling) of the aromatic carbon region of protonated dGTP (black) and anionic dGTP (red) showing CS perturbations induced upon deprotonation of dGTP-N1. b, 13C spectra (without 13C-13C homonuclear decoupling) of the aromatic carbon region of protonated dTTP (black) and anionic dTTP (red) showing CS perturbations induced upon deprotonation of dTTP-N3. c, 2D [15N, 13C] HMQC spectra of dGTP showing CS of dGTP-N1 induced upon deprotonation. The spectra is rotated by 90°, to depict 15N CS along x-axis for visualization purposes. Red circles on inset structure highlight measured resonances (C6 and N1). d, 2D [15N, 13C] HMQC spectra of dTTP showing CS perturbation of dTTP-N3 induced upon deprotonation. The spectra is rotated by 90°, to depict 15N CS along x-axis for visualization purposes. Red circles on inset structure highlight measured resonances (C4 and N3). e, hp-GT DNA spectra of the dG/dT aromatic carbons upon increase in pH from 6.9 (black) to 10.7 (red). Minor upfield CSs are observed for dT5-C6 and dG9-C8, but not dG15-C8, indicating the dT5 in the dG•dT mispair is likely undergoing deprotonation and not the paired dG15. f, 8BrG15-hp-GT DNA construct bearing a 13C/15N site-labelled dT5 paired with a 8-bromo-2′-deoxyguanosine is shown (left) along with the 15N RD profile for the paired dT5-N3. Error bars represent experimental uncertainty (one s.d., see Methods).
a, Kinetic-thermodynamic diagram for exchange between GS and ES1 via a transition state for hp-GT DNA ES1 (left) and hp-GU-20 RNA ES1 (right), showing activation (G‡) and net free energy (G), enthalpy (H), and entropy (TS) changes (referenced to 0). b, Kinetic-thermodynamic parameters derived from RD data. Asterisk denotes parameters calculated using only a single temperature (see Methods), wherein enthalpic and entropic parameters cannot be derived. Here, dG15•dT5 ES2 values were calculated at 25.05 °C, rG16•rU5 ES2 values were calculated at 20.05 °C, and dG15•5BrdU5 ES1 and ES2 values were calculated at 10.05 °C. Error reflects experimental uncertainty (one s.d.) of the weighted global fits of the corresponding RD profiles. Error is propagated using the respective uncertainties in kex and pES.
a, m6G15-hp-GT DNA construct is shown (left) where dG15 is methylated at the O6 position to trap a near-WC “dGenol•dT”-like geometry (Fig. 3c). CS perturbations induced in the aromatic (centre) and sugar (right) resonances upon O6-methylation (blue) with the hp-GT DNA spectra (black) with the resonances for the dG•dT mispair from hp-GT DNA in red. m6dG•dT mispair and CSs are highlighted in red. b, Similarly, m6G4-Dickerson-GT DNA construct is shown (left) where dG4 is O6-methylated to trap a WC-like state, with similar colour scheme as a. c, 5BrU5-hp-GT DNA construct bearing a 13C/15N site-labelled dG15 paired with a 5-bromo-2′-deoxythymidine is shown (left) along with the 15N RD profile for the paired dG15-N1. Error bars represent experimental uncertainty (one s.d., see Methods).
Extended Data Figure 8 Rotating frame relaxation dispersion profiles for rG•rU mispairs in hp-GU-20, hp-GU-24 and xpt-G RNA constructs.
a, b, RNA constructs and the imino [15N, 1H] HSQC zoomed into the rG•rU wobble region of the spectra for hp-GU-20 and hp-GU-24. rG•rU mispair resonances are shown in red. c, The Bacillus subtilis guanine binding riboswitch (xpt-G RNA)71 construct and full imino [15N, 1H] HSQC of folded and guanine ligand-bound riboswitch. rG•rU mispair resonances are shown in red. d–f, 15N RD profiles for hp-GU-20 (d) hp-GU-24 (e) and xpt-G (f) riboswitch RNA. Error bars represent experimental uncertainty (one s.d., see Methods).
a, RD-derived rG16-N1 and rU5-N3 CSs (referenced to GS WB) are shown for ES1 (20 °C and pH 6.9) and ES2 of hp-GU-20 (20 °C and pH 7.9) and ES1 rG18-N1 and rU7-N3 CSs of hp-GU-24 (25 °C and pH 6.9). Errors in all RD-derived fitted parameters (for example, Δω) reflect experimental uncertainty (one s.d.) from the weighted global fit (see Methods). b, RD-derived CSs (referenced to GS WB) are shown for the ES of xpt-G riboswitch (rU17-N3 and rU69-N3) at 25 °C and pH 7.9. c, 2D [15N, 13C] HMQC spectra of rUTP showing CS of rUTP-N3 induced upon deprotonation. The spectra is rotated by 90°, to depict 15N CS along x-axis for visualization purposes. Red circles on inset structure highlight measured resonances (C4 and N3). d, 2D [15N, 13C] HMQC spectra of rGTP showing CS of rGTP-N1 induced upon deprotonation. The spectra is rotated by 90°, to depict 15N CS along x-axis for visualization purposes. Red circles on inset structure highlight measured resonances (C6 and N1). e, RD-derived hp-GU-20 rG•rU ES1 (blue) and ES2 (green) CSs are shown as a function of temperature and pH for both rG16-N1 (square) and rU5-N3 (circle). f, Scheme used to calculate CSs using DFT. Idealized A-form RNA helix is generated to give a central rG•rU mispair (red) that is flanked by canonical rG•rC and rA•rU pairs, analogous to the hp-GU-24 construct. Residues are trimmed to 1-/9-methyl bases and i + 1/i − 1 pairs are frozen in place for subsequent geometry optimizations and CS calculations (see Methods). g, DFT-predicted CSs (referenced to an energy optimized WB geometry) are shown for various tautomeric and anionic configurations, where rGenol•rU/rG•rUenol represents population weighted average CSs of rGenol•rU (60%) and rG•rUenol (40%). h, 15N rG-N1 and rU-N3 CS comparison between RD-derived ES1 CSs and population weighted DFT-predicted CSs (60:40 vs. 80:20). i, Computational studies32 predict that the tautomeric pathway for a rG•rU pair can proceed via a planar rG+•rU– ion pair (charge delocalization is implied) that is highlighted by a network of five H-bonds. j, Pair geometry of an anionic rG•rU– inverted wobble. Deprotonated rU-N3 is highlighted in red (charge delocalization is implied).
Extended Data Figure 10 dG•dT Misincorporation probabilities and correlation to WC-like excited states.
a, Explicit dGTP•dT and dG•dTTP kinetic misincorporation and base substitution probabilities (n = 53) and associated errors46,47,48,72,73,74,75,76,77 (see Supplementary Discussion 8) are plotted against hp-GT dG•dT ES1 (blue squares) and ES2 (green triangles). The pKa fit of ES2 probabilities to the Henderson–Hasselbalch equation (equation (4), see Methods) is shown as the green trend line. b, Red trend line shows the pKa fit to dGTP•dT misincorporation probabilities47 from pH 6.5–8.6 to the Henderson–Hasselbalch equation. The fit was weighted using reported experimental errors and gave a reduced χ2 of 3.56. c, d, Extrapolated dG•dT– ES2 probability (s.d. from the weighted global fit) is plotted against dGTP•dT (left) and dG•dTTP (right) misincorporation probabilities (errors as given)47 from pH 6.5–9.5.
This file contains Supplementary Discussions 1-10 and additional references. (PDF 370 kb)
This file contains relaxation dispersion related fit parameters and spinlock powers and offsets used for all constructs and nuclei reported in the manuscript. (XLSX 67 kb)
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Kimsey, I., Petzold, K., Sathyamoorthy, B. et al. Visualizing transient Watson–Crick-like mispairs in DNA and RNA duplexes. Nature 519, 315–320 (2015). https://doi.org/10.1038/nature14227
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