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.
Labeling of long RNA molecules in a site-specific yet generally applicable manner is integral to many spectroscopic applications. Here we present a novel covalent labeling approach that is site-specific and scalable to long intricately folded RNAs. In this approach, a custom-designed DNA strand that hybridizes to the RNA guides a reactive group to target a preselected adenine residue. The functionalized nucleotide along with the concomitantly oxidized 3′-terminus can subsequently be conjugated to two different fluorophores via bio-orthogonal chemistry. We validate this modular labeling platform using a regulatory RNA of 275 nucleotides, the btuB riboswitch of Escherichia coli, demonstrate its general applicability by modifying a base within a duplex, and show its site-selectivity in targeting a pair of adjacent adenines. Native folding and function of the RNA is confirmed on the single-molecule level by using FRET as a sensor to visualize and characterize the conformational equilibrium of the riboswitch upon binding of its cofactor adenosylcobalamin. The presented labeling strategy overcomes size and site constraints that have hampered routine production of labeled RNA that are beyond 200 nt in length.
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