Shah, AarshPatel, HeerKanjarpane, ArjunSummers, MichaelMarchant, Jan2025-01-222025-01-222024-12-11https://doi.org/10.26434/chemrxiv-2024-qs8d7http://hdl.handle.net/11603/37361The application of NMR to large RNAs has been limited by the inability to perform heteronuclear correlation experiments essential for resolving overlapping 1H NMR signals, determining inter-proton distance restraints and inter-helical orientations for structure calculations, and evaluating conformational dynamics. Approaches exploiting 1H-13C correlations that are routinely applied to proteins and small RNAs of ~50 nucleotides or fewer are impractical for larger RNAs due to rapid dipolar relaxation of protons by their attached carbons. Here we report a 2H-enhanced, 1H-15N correlation approach that enables atom-specific NMR characterization of much larger RNAs. Purine H8 transverse relaxation rates are reduced ~20-fold with ribose perdeuteration, enabling efficient magnetization transfer via two-bond 1H-15N couplings. We focus on H8-N9 correlation spectra which benefit from favorable N9 chemical shift anisotropy. Chemical shift assignment is enabled by retention of protons at the C1? position, which allow measurement of H8-H1? NOEs and two-bond H1?-N9 correlation strategies with only a minor effect on H8 relaxation. The approach is demonstrated for the 232 nucleotide HIV-1 Rev response element, where chemical shift assignments, 15N-edited nuclear Overhauser effects, and 1H-15N residual dipolar couplings are readily obtained from sensitive, high-resolution spectra. Heteronuclear correlated NMR methods that have been essential for the study of proteins can now be extended to RNAs of at least 78 kDa.8 pagesen-USAttribution-NonCommercial-NoDerivatives 4.0 Internationalhttps://creativecommons.org/licenses/by-nc-nd/4.0/NMR SpectroscopyRNAIsotope LabelingStructure elucidationText