title: Mg2+-dependent conformational changes and product release during DNA-catalyzed RNA ligation monitored by Bimane fluorescence.
abstract: Among the deoxyribozymes catalyzing the ligation of two RNA substrates, 7S11 generates a branched RNA containing a 2',5'-linkage. We have attached the small fluorogenic probe Bimane to the triphosphate terminated RNA substrate and utilized emission intensity and anisotropy to follow structural rearrangements leading to a catalytically active complex upon addition of Mg(2+). Bimane coupled to synthetic oligonucleotides is quenched by nearby guanines via photoinduced electron transfer. The degree of quenching is sensitive to changes in the base pairing of the residues involved and in their distances to the probe. These phenomena permit the characterization of various sequential processes in the assembly and function of 7S11: binding of Mg(2+) to the triphosphate moiety, release of quenching of the probe by the 5'-terminal G residues of R-RNA as they engage in secondary base-pair interactions, local rearrangement into a distinct active conformation, and continuous release of the Bimane-labeled pyrophosphate during the course of reaction at 37°C. It was possible to assign equilibrium and rate constants and structural interpretations to the sequence of conformational transitions and catalysis, information useful for optimizing the design of next generation deoxyribozymes. The fluorescent signatures, thermodynamic equilibria and catalytic function of numerous mutated (base/substituted) molecules were examined.
authors.affiliation: Scuola Normale Superiore di Pisa, Piazza dei Cavalieri 7, I-56126 Pisa, Italy Laboratory for Cellular Dynamics, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany.
authors.forename: Elisa
authors.initials: E
authors.lastname: Turriani
authors.affiliation: Max Planck Research Group Nucleic Acid Chemistry, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany and Institute for Organic and Biomolecular Chemistry, Georg August University Göttingen, Tammannstrasse 2, 37077 Göttingen, Germany.
authors.forename: Claudia
authors.initials: C
authors.lastname: Höbartner
authors.affiliation: Laboratory for Cellular Dynamics, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany tjovin@gwdg.de.
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Jovin
mesh_terms: D001643:Bridged Bicyclo Compounds / Q000737:chemistry*; D021881:DNA, Catalytic / Q000737:chemistry* / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry*; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry*; D009154:Mutation; D012313:RNA / Q000737:chemistry* / Q000378:metabolism
chemical_list: D001643:Bridged Bicyclo Compounds; D021881:DNA, Catalytic; D005456:Fluorescent Dyes; D012313:RNA; D008274:Magnesium; C028381:monobromobimane