title: Rolling circle amplification-based detection of human topoisomerase I activity on magnetic beads.
abstract: A high-sensitivity assay has been developed for the detection of human topoisomerase I with single molecule resolution. The method uses magnetic sepharose beads to concentrate rolling circle products, produced by the amplification of DNA molecules circularized by topoisomerase I and detectable with a confocal microscope as single and discrete dots, once reacted with fluorescent probes. Each dot, corresponding to a single cleavage-religation event mediated by the enzyme, can be counted due to its high signal/noise ratio, allowing detection of 0.3pM enzyme and representing a valid method to detect the enzyme activity in highly diluted samples.
authors: Department of Biology and Interuniversity Consortium, National Institute of Biostructure and Biosystems (INBB), University of Rome Tor Vergata, Rome 00133, Italy.
authors: Laura
authors: L
authors: Zuccaro
authors: Department of Biology and Interuniversity Consortium, National Institute of Biostructure and Biosystems (INBB), University of Rome Tor Vergata, Rome 00133, Italy.
authors: Cinzia
authors: C
authors: Tesauro
authors: Department of Chemical Sciences and Technologies, University of Rome Tor Vergata, Rome 00133, Italy.
authors: Barbara
authors: B
authors: Cerroni
authors: Department of Biology and Interuniversity Consortium, National Institute of Biostructure and Biosystems (INBB), University of Rome Tor Vergata, Rome 00133, Italy.
authors: Alessio
authors: A
authors: Ottaviani
authors: Department of Molecular Biology and Genetics and Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Aarhus 8000, Denmark.
authors: Birgitta Ruth
authors: BR
authors: Knudsen
authors: Max Planck Institute for Solid State Research, D-70569 Stuttgart, Germany.
authors: Kannan
authors: K
authors: Balasubramanian
authors: Department of Biology and Interuniversity Consortium, National Institute of Biostructure and Biosystems (INBB), University of Rome Tor Vergata, Rome 00133, Italy. Electronic address: desideri@uniroma2.it.
authors: Alessandro
authors: A
authors: Desideri
mesh_terms: D004264:DNA Topoisomerases, Type I / Q000032:analysis* / Q000378:metabolism; D004270:DNA, Circular / Q000737:chemistry / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D008280:Magnetics; D018613:Microscopy, Confocal; D021141:Nucleic Acid Amplification Techniques; D059629:Signal-To-Noise Ratio
chemical_list: D004270:DNA, Circular; D005456:Fluorescent Dyes; D004264:DNA Topoisomerases, Type I; C528909:TOP1 protein, human
keywords: Activity assay; Confocal microscope; Magnetic beads; Rolling circle amplification; Topoisomerase