Rare tautomeric and anionic nucleobases are believed to play 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 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 (10−3-10−5) 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.

Tautomeric and anionic Watson-Crick-like mismatches play important roles in replication and translation errors through mechanisms that are not fully understood. Using NMR relaxation dispersion, we resolved a sequence-dependent kinetic network connecting G•T/U wobbles with three distinct Watson-Crick mismatches consisting of two rapidly exchanging tautomeric species (Genol•T/U⇌G•Tenol/Uenol; population <0.4%) and one anionic species (G•T−/U−; population ≈0.001% at neutral pH). Inserting the sequence-dependent tautomerization/ionization step into a minimal kinetic mechanism for correct incorporation during replication following initial nucleotide binding leads to accurate predictions of dG•dT misincorporation probability across different polymerases, pH conditions, and for a chemically modified nucleotide, and provides mechanisms for sequence-dependent misincorporation. Our results indicate that the energetic penalty for tautomerization/ionization accounts for ≈10−2−10−3–fold discrimination against misincorporation, which proceeds primarily via tautomeric dGenol•dT and dG•dTenol with contributions from anionic dG•dT− dominating at pH ≥8.4 or for some mutagenic nucleotides.