Real-time detection of TDP1 activity using a fluorophore-quencher coupled DNA-biosensor

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaperJournal articleResearchpeer-review

  • Pia Wrensted Jensen
  • ,
  • Mattia Falconi, Department of Biology, University of Rome “Tor Vergata”, Via della Ricerca Scientifica, 00133, Interuniversity Consortium, National Institute Biostructure and Biosystem (INBB), Italy, Italy
  • Emil Laust Kristoffersen
  • Anita Tranberg Simonsen
  • Jéssica B. Cifuentes
  • ,
  • Lærke Bay Markussen, Department of Molecular Biology and Genetics, Aarhus University, Denmark, Denmark
  • Rikke Frøhlich Hougaard
  • Josephine Vagner
  • ,
  • Charlotte Harmsen, Denmark
  • Sissel Juul Jensen, Denmark
  • Yi-Ping Ho, Denmark
  • Marjorie A. Withers, Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA, United States
  • James R. Lupski, Department of Molecular and Human Genetics, Baylor College of Medicine & Department of Pediatrics, Baylor College of Medicine & Texas Children’s Hospital, Houston, TX, USA, United States
  • Jørn Erland Koch, Denmark
  • Alessandro Desideri, Department of Biology, University of Rome “Tor Vergata”, Via della Ricerca Scientifica, 00133, Interuniversity Consortium, National Institute Biostructure and Biosystem (INBB), Italy, Italy
  • Birgitta R. Knudsen
  • Magnus Stougaard
Real-time detection of enzyme activities may present the easiest and most reliable way of obtaining quantitative analyses in biological samples. We present a new DNA-biosensor capable of detecting the activity of the potential anticancer drug target tyrosyl-DNA phosphodiesterase 1 (TDP1) in a very simple, high throughput, and real-time format. The biosensor is specific for Tdp1 even in complex biological samples, such as human cell extracts, and may consequently find future use in fundamental studies as well as a cancer predictive tool allowing fast analyses of diagnostic cell samples such as biopsies. TDP1 removes covalent 3′DNA adducts in DNA single-strand break repair. This enzymatic activity forms the basis of the design of the TDP1-biosensor, which consists of a short hairpin-forming oligonucleotide having a 5′fluorophore and a 3′quencher brought in close proximity by the secondary structure of the biosensor. The specific action of TDP1 removes the quencher, thereby enabling optical detection of the fluorophore. Since the enzymatic action of TDP1 is the only “signal amplification” the increase in fluorescence may easily be followed in real-time and allows quantitative analyses of TDP1 activity in pure enzyme fractions as well as in crude cell extracts. In the present study we demonstrate the specificity of the biosensor, its ability to quantitatively detect up- or down-regulated TDP1 activity, and that it may be used for measuring and for analyzing the mechanism of TDP1 inhibition.
Original languageEnglish
JournalBiosensors and Bioelectronics
VolumeVolume 48
Pages (from-to)230–237
Number of pages8
ISSN0956-5663
DOIs
Publication statusPublished - 15 Oct 2013

    Research areas

  • Tyrosyl-DNA phosphodiesterase 1 (TDP1), Biosensor, Enzyme activity, Real-time measurement, Fluorophore–quencher

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