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Simple and fast DNA based sensor system for screening of small-molecule compounds targeting eukaryotic topoisomerase 1

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  • Kamilla Vandsø Petersen
  • Asier Selas, University of the Basque Country
  • ,
  • Kirstine Mejlstrup Hymøller, Aarhus University
  • ,
  • Karol Mizielinski, VPCIR Biosciences ApS.
  • ,
  • Maria Thorsager
  • Magnus Stougaard
  • Concepcion Alonso, University of the Basque Country
  • ,
  • Francisco Palacios, University of the Basque Country
  • ,
  • Yolanda Pérez-Pertejo, University of Leon
  • ,
  • Rosa M. Reguera, University of Leon
  • ,
  • Rafael Balaña-Fouce, University of Leon
  • ,
  • Birgitta R. Knudsen
  • Cinzia Tesauro

Background: Eukaryotic topoisomerase 1 is a potential target of anti-parasitic and anti-cancer drugs. Parasites require topoisomerase 1 activity for survival and, consequently, compounds that inhibit topoisomerase 1 activity may be of interest. All effective topoisomerase 1 drugs with anti-cancer activity act by inhibiting the ligation reaction of the enzyme. Screening for topoisomer-ase 1 targeting drugs, therefore, should involve the possibility of dissecting which step of topoiso-merase 1 activity is affected. Methods: Here we present a novel DNA-based assay that allows for screening of the effect of small-molecule compounds targeting the binding/cleavage or the ligation steps of topoisomerase 1 catalysis. This novel assay is based on the detection of a rolling circle amplification product generated from a DNA circle resulting from topoisomerase 1 activity. Results: We show that the binding/cleavage and ligation reactions of topoisomerase 1 can be investigated separately in the presented assay termed REEAD (C|L) and demonstrate that the assay can be used to investigate, which of the individual steps of topoisomerase 1 catalysis are affected by small-mol-ecule compounds. The assay is gel-free and the results can be detected by a simple colorimetric readout method using silver-on-gold precipitation rendering large equipment unnecessary. Con-clusion: REEAD (C|L) allows for easy and quantitative investigations of topoisomerase 1 targeting compounds and can be performed in non-specialized laboratories.

Original languageEnglish
Article number1255
JournalPharmaceutics
Volume13
Issue8
Number of pages15
ISSN1999-4923
DOIs
Publication statusPublished - Aug 2021

    Research areas

  • Colorimetric readout, Drug screening, Enzyme activity, REEAD, Rolling circle amplification, Small-molecule compounds, Topoisomerase 1

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