Rolling circle amplification-based detection of human topoisomerase I activity on magnetic beads

Laura Zuccaro; Cinzia Tesauro; Barbara Cerroni; Alessio Ottaviani; Birgitta Ruth Knudsen; Kannan Balasubramanian; Alessandro Desideri

doi:10.1016/j.ab.2014.02.003

Rolling circle amplification-based detection of human topoisomerase I activity on magnetic beads

Anal Biochem. 2014 Apr 15:451:42-4. doi: 10.1016/j.ab.2014.02.003. Epub 2014 Feb 11.

Authors

Laura Zuccaro¹, Cinzia Tesauro¹, Barbara Cerroni², Alessio Ottaviani¹, Birgitta Ruth Knudsen³, Kannan Balasubramanian⁴, Alessandro Desideri⁵

Affiliations

¹ Department of Biology and Interuniversity Consortium, National Institute of Biostructure and Biosystems (INBB), University of Rome Tor Vergata, Rome 00133, Italy.
² Department of Chemical Sciences and Technologies, University of Rome Tor Vergata, Rome 00133, Italy.
³ Department of Molecular Biology and Genetics and Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Aarhus 8000, Denmark.
⁴ Max Planck Institute for Solid State Research, D-70569 Stuttgart, Germany.
⁵ 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.

PMID: 24525043
DOI: 10.1016/j.ab.2014.02.003

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.

Keywords: Activity assay; Confocal microscope; Magnetic beads; Rolling circle amplification; Topoisomerase.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

DNA Topoisomerases, Type I / analysis*
DNA Topoisomerases, Type I / metabolism
DNA, Circular / chemistry
DNA, Circular / metabolism*
Fluorescent Dyes / chemistry
Humans
Magnetics*
Microscopy, Confocal*
Nucleic Acid Amplification Techniques*
Signal-To-Noise Ratio

Substances

DNA, Circular
Fluorescent Dyes
DNA Topoisomerases, Type I
TOP1 protein, human