Single-molecule chemical denaturation of riboswitches.

Paul A Dalgarno, Jorge Bordello, Rhodri Morris, Patrick St-Pierre, Audrey Dubé, Ifor D W Samuel, Daniel A Lafontaine, J Carlos Penedo

Nucleic acids research 2013 Apr

filter terms:

To date, single-molecule RNA science has been developed almost exclusively around the effect of metal ions as folding promoters and stabilizers of the RNA structure. Here, we introduce a novel strategy that combines single-molecule Förster resonance energy transfer (FRET) and chemical denaturation to observe and manipulate RNA dynamics. We demonstrate that the competing interplay between metal ions and denaturant agents provides a platform to extract information that otherwise will remain hidden with current methods. Using the adenine-sensing riboswitch aptamer as a model, we provide strong evidence for a rate-limiting folding step of the aptamer domain being modulated through ligand binding, a feature that is important for regulation of the controlled gene. In the absence of ligand, the rate-determining step is dominated by the formation of long-range key tertiary contacts between peripheral stem-loop elements. In contrast, when the adenine ligand interacts with partially folded messenger RNAs, the aptamer requires specifically bound Mg(2+) ions, as those observed in the crystal structure, to progress further towards the native form. Moreover, despite that the ligand-free and ligand-bound states are indistinguishable by FRET, their different stability against urea-induced denaturation allowed us to discriminate them, even when they coexist within a single FRET trajectory; a feature not accessible by existing methods.

Citation

Paul A Dalgarno, Jorge Bordello, Rhodri Morris, Patrick St-Pierre, Audrey Dubé, Ifor D W Samuel, Daniel A Lafontaine, J Carlos Penedo. Single-molecule chemical denaturation of riboswitches. Nucleic acids research. 2013 Apr;41(7):4253-65