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  Mg2+-dependent conformational changes and product release during DNA-catalyzed RNA ligation monitored by Bimane fluorescence.

Turriani, E., Höbartner, C., & Jovin, T. M. (2015). Mg2+-dependent conformational changes and product release during DNA-catalyzed RNA ligation monitored by Bimane fluorescence. Nucleic Acids Research, 43(1), 40-50. doi: 10.1093/nar/gku1268.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-0024-548B-1 Version Permalink: http://hdl.handle.net/11858/00-001M-0000-002A-21E3-8
Genre: Journal Article

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 Creators:
Turriani, E.1, Author              
Höbartner, C.2, Author              
Jovin, T. M.1, Author              
Affiliations:
1Emeritus Group Laboratory of Cellular Dynamics, MPI for Biophysical Chemistry, Max Planck Society, ou_578629              
2Research Group of Nucleic Acid Chemistry, MPI for biophysical chemistry, Max Planck Society, ou_578605              

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 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 Mg2+. 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 Mg2+ 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.

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Language(s): eng - English
 Dates: 2014-12-102015-01
 Publication Status: Published in print
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 Rev. Method: Peer
 Identifiers: DOI: 10.1093/nar/gku1268
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Title: Nucleic Acids Research
Source Genre: Journal
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Pages: - Volume / Issue: 43 (1) Sequence Number: - Start / End Page: 40 - 50 Identifier: -