Probing rare and short-lived conformational states in nucleic acids using off-resonance carbonyl and guanidino carbon...

Chemical exchange-based NMR techniques provide powerful approaches for characterizing lowly-populated and short-lived conformational states in nucleic acids. Among possible probes, the nucleobase carbonyl and guanidino carbons stand out due to the sensitivity of their chemical shifts to hydrogen bonding and keto-enol tautomerization. However, chemical exchange measurements targeting these carbon nuclei have not yet been reported in studies of nucleic acids. Here, we present an experiment for measuring off-resonance R_1ρ relaxation dispersion for guanine-C2, guanine-C6, thymine/uracil-C2, and thymine/uracil-C4 carbons in uniformly ¹³C/¹⁵N labeled nucleic acids. We demonstrate the utility of the experiment by characterizing chemical exchange in a G•T mismatch in duplex DNA between a dominant wobble conformation and two lowly-populated, short-lived, and rapidly interconverting Watson-Crick-like tautomeric states (G^enol•T ⇌ G•T^enol) implicated in DNA replicative errors. The population and exchange rate deduced from the guanine-C6, guanine-C2, and thymine-C4 off-resonance R_1ρ relaxation dispersion profiles were in excellent agreement with counterparts obtained from R_1ρ measurements on proton-bound carbon and nitrogen nuclei. The carbon chemical shifts of the minor state were downfield shifted relative to the wobble ground state, consistent with (G)C6 = O···HO-C4(T^enol) and (G^enol)C6-OH···O=C4(T) hydrogen bonding in the Watson-Crick-like tautomeric state. As a second application, we did not detect any exchange contribution to uracil-C2 and uracil-C4 R_1ρ profiles measured for a U·U mismatch in RNA, consistent with isomerization between two alternative wobble conformations occurring on the sub-microsecond timescale. These results establish carbonyl and guanidino carbons as valuable probes for chemical exchange measurements of micro-to-millisecond motions in nucleic acids.