Insight into the Conformational Ensembles Formed by U–U and T–T Mismatches in RNA and DNA Duplexes From a Structure-based Survey, NMR, and Molecular Dynamics Simulations

Abstract

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.