¹⁹F NMR Strategy for Probing Site-specific RNA Dynamics in Large RNA-protein Complexes: Application to the Hfq RNA...

Nuclear magnetic resonance (NMR) spectroscopy is uniquely suited to probing motions on the microsecond to millisecond timescales that underlie RNA recognition and restructuring of ribonucleoprotein (RNP) complexes. However, the large size and conformational dynamics of most RNPs results in poor sensitivity, spectral crowding, and resolution loss in traditional NMR approaches. Here, we present a ¹⁹F NMR strategy for resolving residue-level RNA conformational dynamics within a ∼76 kDa Hfq-RNA complex at micromolar concentrations. Site-specific incorporation of dual-labeled [6-²H, 5-¹⁹F]-uridine (6D, 5FU) into a 27-nt model RNA yields excellent spectral quality when in complex with the Hfq chaperone. ¹⁹F resonance broadening and ¹⁹F Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments reveal specific changes in RNA dynamics on different surfaces of Hfq. Our NMR strategy provides a framework for studying how RNA-binding proteins like Hfq selectively alter the conformational dynamics of their RNA targets.