Insight into the Conformational Ensembles Formed by U-U and T-T Mismatches in RNA and DNA Duplexes From a...

Nucleic acid base pairs interconvert between alternative conformations on a free energy landscape, and these dynamics play critical roles in recognition, folding, and catalysis. U-U and T-T mismatches can adopt two nearly isoenergetic wobble conformations, distinguished by their relative shearing displacements. Experimental NMR evidence suggests that these conformations dynamically interconvert in RNA motifs containing tandem U-U mismatches. However, whether such motions occur ubiquitously across U-U and T-T mismatches remains unknown, as high-resolution nucleic acid structures typically report only a single conformation. Here, we used NMR spectroscopy, a structure-based survey of the Protein Data Bank, and molecular dynamics (MD) simulations to investigate wobble dynamics in U-U and T-T mismatches when flanked by canonical Watson-Crick base pairs in RNA and DNA duplexes. The structure-based survey revealed that U-U mismatches have propensities to adopt alternative wobble conformations even when controlling for sequence and identified potential intermediates along the wobble transition. Off-resonance R_1ρ relaxation dispersion experiments detected no micro- to millisecond dynamics for U-U mismatches in duplex RNA and T-T mismatches in duplex DNA. However, alternative conformer refinement of the electron density in X-ray structures, inter-proton NOEs, carbonyl carbon chemical shifts, an RDC-derived conformational ensemble, and MD simulations indicated that U-U and T-T mismatches exist in a dynamic equilibrium between two wobble conformations, with the minor state exceeding 30% and the transitions occurring on the nanosecond timescale. Our findings suggest that U-U and T-T ubiquitously undergo sub-microsecond wobble motions, contributing to the energetic landscape and dynamic plasticity of nucleic acids, with important implications for processes that generate and act on these mismatches.