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+ o...