Direct evidence for (G)O6•••H2-N4(C)+ hydrogen bonding in transient G(syn)-C+ and G(syn)-m5C+ Hoogsteen base pairs in duplex DNA from cytosine amino nitrogen off-resonance R1ρ relaxation dispersion measurements

NMR relaxation dispersion studies have shown that Watson-Crick G-C and A-T base pairs in duplex DNA exist in dynamic equilibrium with their Hoogsteen counterparts. Hoogsteen base pairs form through concurrent rotation of the purine base about the glycosidic bond from an anti to a syn conformation and constriction of the C1'-C1' distance across the base pair by similar to 2 angstrom to allow Hoogsteen type hydrogen bonding. Owing to their unique structure, Hoogsteen base pairs can play important roles in DNA recognition, the accommodation, recognition, and repair of DNA damage, and in DNA replication. NMR relaxation dispersion experiments targeting imino nitrogen and protonated base and sugar carbons have provided insights into many structural features of transient Hoogsteen base pairs, including one of two predicted hydrogen bonds involving (G)N7 center dot center dot center dot H-N3(C)(+) and (A)N7 center dot center dot center dot H-N3(T). Here, through measurement of cytosine amino (N4)R-1 rho relaxation dispersion, we provide direct evidence for the second (G)O6 center dot center dot center dot H-2-N4 (C)(+) hydrogen bond in G(syn)-C+ transient Hoogsteen base pairs. The utility of cytosine N4 R-1 rho relaxation dispersion as a new sensitive probe of transient Hoogsteen base pairs, and cytosine dynamics in general, is further demonstrated by measuring G(syn)-C+ Hoogsteen exchange near neutral pH and in the context of the naturally occurring DNA modification 5-methyl cytosine (m(5)C), in DNA samples prepared using chemical synthesis and a N-15 labeled m(5)C phosphoramidite. (C) 2019 Elsevier Inc. All rights reserved.