title: Multiplexed detection of site specific recombinase and DNA topoisomerase activities at the single molecule level.
abstract: We previously demonstrated the conversion of a single human topoisomerase I mediated DNA cleavage-ligation event happening within nanometer dimensions to a micrometer-sized DNA molecule, readily detectable using standard fluorescence microscopy. This conversion was achieved by topoisomerase I mediated closure of a nicked DNA circle followed by rolling circle amplification leading to an anchored product that was visualized at the single molecule level by hybridization to fluorescently labeled probes (Stougaard et al. ACS Nano 2009, 3, 223-33). An important inherent property of the presented setup is, at least in theory, the easy adaptability to multiplexed enzyme detection simply by using differently labeled probes for the detection of rolling circle products of different circularized substrates. In the present study we demonstrate the specific detection of three different enzyme activities, human topoisomerase I, and Flp and Cre recombinase in nuclear extracts from human cells one at a time or multiplexed using the rolling circle amplification based single-molecule detection system. Besides serving as a proof-of-principle for the feasibility of the presented assay for multiplexed enzyme detection in crude human cell extracts, the simultaneous detection of Flp and Cre activities in a single sample may find immediate practical use since these enzymes are often used in combination to control mammalian gene expression.
authors: Department of Molecular Biology and Interdisciplinary Nanoscience Center, Aarhus University, 8000 Aarhus C, Denmark.
authors: Felicie Faucon
authors: FF
authors: Andersen
authors: Magnus
authors: M
authors: Stougaard
authors: Hanne Laerke
authors: HL
authors: Jørgensen
authors: Simon
authors: S
authors: Bendsen
authors: Sissel
authors: S
authors: Juul
authors: Kristoffer
authors: K
authors: Hald
authors: Anni Hangaard
authors: AH
authors: Andersen
authors: Jørn
authors: J
authors: Koch
authors: Birgitta Ruth
authors: BR
authors: Knudsen
mesh_terms: D002460:Cell Line; D004264:DNA Topoisomerases, Type I / Q000737:chemistry* / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D007668:Kidney / Q000201:enzymology* / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000379:methods*; D015336:Molecular Probe Techniques; D045522:Recombinases / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D045522:Recombinases; D004264:DNA Topoisomerases, Type I