Probing conformational transitions towards mutagenic Watson–Crick-like G·T mismatches using off-resonance sugar carbon R(1ρ) relaxation dispersion

NMR off-resonance R(1ρ) relaxation dispersion measurements on base carbon and nitrogen nuclei have revealed that wobble G·T/U mismatches in DNA and RNA duplexes exist in dynamic equilibrium with short-lived, low-abundance, and mutagenic Watson–Crick-like conformations. As Watson–Crick-like G·T mismatches have base pairing geometries similar to Watson–Crick base pairs, we hypothesized that they would mimic Watson–Crick base pairs with respect to the sugar-backbone conformation as well. Using off-resonance R(1ρ) measurements targeting the sugar C3′ and C4′ nuclei, a structure survey, and molecular dynamics simulations, we show that wobble G·T mismatches adopt sugar-backbone conformations that deviate from the canonical Watson–Crick conformation and that transitions toward tautomeric and anionic Watson–Crick-like G·T mismatches restore the canonical Watson–Crick sugar-backbone. These measurements also reveal kinetic isotope effects for tautomerization in D(2)O versus H(2)O, which provide experimental evidence in support of a transition state involving proton transfer. The results provide additional evidence in support of mutagenic Watson–Crick-like G·T mismatches, help rule out alternative inverted wobble conformations in the case of anionic G·T(−), and also establish sugar carbons as new non-exchangeable probes of this exchange process. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s10858-020-00337-7) contains supplementary material, which is available to authorized users.