title: Single-molecule FRET studies of RNA folding: a Diels-Alderase ribozyme with photolabile nucleotide modifications.
abstract: Enzymology at the single-molecule level by using fluorescence resonance energy transfer (smFRET) offers unprecedented insight into mechanistic aspects of catalytic reactions. Implementing spatiotemporal control of the reaction by using an external trigger is highly valuable in these challenging experiments. Here, we have incorporated a light-cleavable caging moiety into specific nucleotides of the Diels-Alderase (DAse) ribozyme. In this way, the folding energy landscape was significantly perturbed, and the catalytic activity was essentially suppressed. A careful smFRET efficiency histogram analysis at various Mg(2+) ion concentrations revealed an additional intermediate state that is not observed for the unmodified DAse ribozyme. We also observed that only a fraction of DAse molecules returns to the native state upon cleavage of the caged group by UV light. These constructs are attractive model RNA systems for further real-time single-molecule observation of the coupling between conformational changes and catalytic activity.
authors.affiliation: Institute of Applied Physics, Institute of Toxicology and Genetics and Center for Functional Nanostructures (CFN), Karlsruhe Institute of Technology (KIT) , Wolfgang-Gaede-Strasse 1, 76131 Karlsruhe, Germany.
authors.forename: Andrei Yu
authors.initials: AY
authors.lastname: Kobitski
authors.forename: Stefan
authors.initials: S
authors.lastname: Schäfer
authors.forename: Alexander
authors.initials: A
authors.lastname: Nierth
authors.forename: Marco
authors.initials: M
authors.lastname: Singer
authors.forename: Andres
authors.initials: A
authors.lastname: Jäschke
authors.forename: G Ulrich
authors.initials: GU
authors.lastname: Nienhaus
mesh_terms: D000838:Anions / Q000737:chemistry; D055162:Biocatalysis; D031541:Fluorescence Resonance Energy Transfer; D008274:Magnesium / Q000737:chemistry; D009154:Mutation; D009690:Nucleic Acid Conformation; D009711:Nucleotides / Q000737:chemistry*; D059370:RNA Folding / Q000528:radiation effects; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics; D014466:Ultraviolet Rays
chemical_list: D000838:Anions; C518747:Diels-Alder ribozyme; D009711:Nucleotides; D016337:RNA, Catalytic; D008274:Magnesium