More than two decades investigating nucleic acids and ribonucleic acids (RNA) by single molecule Förster Resonance Energy Transfer (smFRET) have passed. It turned out that sample heterogeneity in structure and function of RNA molecules as well as folding intermediates, kinetic subpopulations, and interconversion rates of conformational states of RNA biomolecules, all of which are usually hidden in ensemble type experiments, are often observed characteristics. Besides proteins, metal ions play a crucial role in RNA folding and dynamics, as well as RNA/RNA or RNA/DNA interactions. RNA molecules form discrete conformational intermediates before reaching the native three-dimensional fold, whereby metal ions guide the folding pathway by changing the energetic barriers between local and global minima in the energy landscape. Here we review recent advances in the characterization of the role of metal ions in folding and function of nucleic acid structures by means of smFRET. Subsequently, the workflow of smFRET data analysis is described and exemplified by the metal ion-depending folding and dynamics of the group IIB intron from S. cerevisiae and RNA-RNA binding kinetics of this ribozyme's 5'-splice site formation.

RNA G-quadruplexes, as their well-studied DNA analogs, require the presence of cations to fold and remain stable. This is the first comprehensive study on the interaction of RNA quadruplexes with metal ions. We investigated the formation and stability of two highly conserved and biologically relevant RNA quadruplex-forming sequences (24nt-TERRA and 18nt-NRAS) in the presence of several monovalent and divalent metal ions, namely Li, Na, K, Rb, Cs, NH, Mg, Ca, Sr, and Ba. Circular dichroism was used to probe the influence of these metal ions on the folded fraction of the parallel G-quadruplexes, and UV thermal melting experiments allowed to assess the relative stability of the structures in each cationic condition. Our results show that the RNA quadruplexes are more stable than their DNA counterparts under the same buffer conditions. We have observed that the addition of mainly Na, K, Rb, NH, as well as Sr and Ba in water, shifts the equilibrium to the folded quadruplex form, whereby the NRAS sequence responds stronger than TERRA. However, only K and Sr lead to a significant increase in the stability of the folded structures, which is consistent with their coordination to the O6 atoms from the G-quartet guanosines. Compared to the respective DNA motives, dNRAS and htelo, the RNA sequences are not stabilized by Na ions. Finally, the difference in response between NRAS and TERRA, as well as to the corresponding DNA sequences with respect to different metal ions, could potentially be exploited for selective targeting purposes.

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