Guanine to Inosine Substitution Leads to Large Increases in the Population of a Transient G·C Hoogsteen Base Pair

[Image: see text] We recently showed that Watson–Crick base pairs in canonical duplex DNA exist in dynamic equilibrium with G(syn)·C(+) and A(syn)·T Hoogsteen base pairs that have minute populations of ∼1%. Here, using nuclear magnetic resonance R(1ρ) relaxation dispersion, we show that substitution of guanine with the naturally occurring base inosine results in an ∼17-fold increase in the population of transient Hoogsteen base pairs, which can be rationalized by the loss of a Watson–Crick hydrogen bond. These results provide further support for transient Hoogsteen base pairs and demonstrate that their population can increase significantly upon damage or chemical modification of the base.