Structural Equilibria in RNA as Revealed by ¹⁹NMR

Abstract

We have incorporated 5-fluorouridine into several sites within a 19-mer RNA modelled on the translational operator of the MS2 bacteriophage. The ¹⁹F NMR spectra demonstrate the different chemical shifts of helical and loop fluorouridines of the hairpin secondary structure. Addition of salt gives rise to a species in which the loop fluorouridine gains the chemical shift of its helical counterparts, due to the formation of the alternative bi-molecular duplex form. This is supported by UV thermal melting behaviour which becomes highly dependent on the RNA concentration. Distinct ¹⁹FNMR signals for duplex and hairpin forms allow the duplex- hairpin equilibrium constant to be determined under a range of conditions, enabling thermo- dynamic characterisation and its salt dependence to be determined. Mg²⁺ also promotes duplex fomation, but more strongly than Na²⁺, such that at 25°C, 10 mM MgCl₂ has a comparable duplex-promoting effect to 300 mM NaCl. A simliar effect is observed with Sr²⁺, but not Ca²⁺ or Ba²⁺. Additional hairpin species are observed in the presence of Na⁺ as well as Mg²⁺, Ca²⁺, Sr²⁺ and Ba²⁺ ions. The overall, ensemble average, hairpin conformation is therefore salt-dependent. Electrostatic considerations are thus involved in the balance between different hairpin conformers as well as the duplex-hairpin equilibrium. The data presented here demonstrate that ¹⁹F NMR is a powerful tool for the study of conformational heterogeneity in RNA, which is particularly important for probing the effects of metal ions on RNA structure. The thermodynamic characterisation of duplex-hairpin equilibria will also be valuable in the development of theoretical models of nucleic acid structure.