Arnold J R, Fisher J
School of Biology, University of Leeds, UK.
J Biomol Struct Dyn. 2000 Apr;17(5):843-56. doi: 10.1080/07391102.2000.10506573.
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 19F NMR signals for duplex and hairpin forms allow the duplex-hairpin equilibrium constant to be determined under a range of conditions, enabling thermodynamic characterisation and its salt dependence to be determined. Mg2+ also promotes duplex formation, but more strongly than Na+, such that at 25 degrees C, 10 mM MgCl2 has a comparable duplex-promoting effect to 300 mM NaCl. A similar 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.
我们已将5-氟尿苷掺入到一个基于MS2噬菌体翻译操纵子构建的19聚体RNA的多个位点中。19F NMR光谱显示了发夹二级结构中螺旋型和环型氟尿苷的不同化学位移。添加盐会产生一种物种，其中环型氟尿苷由于形成了另一种双分子双链体形式而获得了其螺旋对应物的化学位移。这得到了紫外热变性行为的支持，该行为变得高度依赖于RNA浓度。双链体和发夹形式的独特19F NMR信号使得能够在一系列条件下确定双链体-发夹平衡常数，从而能够确定热力学特征及其对盐的依赖性。Mg2+也促进双链体形成，但比Na+更强，以至于在25℃时，10 mM MgCl2具有与300 mM NaCl相当的促进双链体形成的效果。在Sr2+存在下也观察到类似的效果，但在Ca2+或Ba2+存在下未观察到。在Na+以及Mg2+、Ca2+、Sr2+和Ba2+离子存在下还观察到了额外的发夹物种。因此，整体的、总体平均的发夹构象是依赖于盐的。因此，静电因素参与了不同发夹构象体之间的平衡以及双链体-发夹平衡。此处给出的数据表明，19F NMR是研究RNA构象异质性的有力工具，这对于探究金属离子对RNA结构的影响尤为重要。双链体-发夹平衡的热力学特征在核酸结构理论模型的开发中也将具有重要价值。
