title: Single-molecule chemical denaturation of riboswitches.
abstract: 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.
authors: SUPA, School of Physics and Astronomy, University of St Andrews, St Andrews, Fife, KY16 9SS, UK.
authors: Paul A
authors: PA
authors: Dalgarno
authors: Jorge
authors: J
authors: Bordello
authors: Rhodri
authors: R
authors: Morris
authors: Patrick
authors: P
authors: St-Pierre
authors: Audrey
authors: A
authors: Dubé
authors: Ifor D W
authors: ID
authors: Samuel
authors: Daniel A
authors: DA
authors: Lafontaine
authors: J Carlos
authors: JC
authors: Penedo
mesh_terms: D031541:Fluorescence Resonance Energy Transfer; D008024:Ligands; D008274:Magnesium / Q000737:chemistry; D009691:Nucleic Acid Denaturation; D058928:Riboswitch; D014508:Urea / Q000737:chemistry
chemical_list: D008024:Ligands; D058928:Riboswitch; D014508:Urea; D008274:Magnesium