DNA-based sensor for real-time measurement of the enzymatic activity of human topoisomerase I

Lærke Bay Marcussen, Morten Leth Jepsen, Emil Laust Kristoffersen, Oskar Franch, Joanna Proszek, Yi Ping Ho, Magnus Stougaard*, Birgitta Ruth Knudsen

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

Sensors capable of quantitative real-time measurements may present the easiest and most accurate way to study enzyme activities. Here we present a novel DNA-based sensor for specific and quantitative real-time measurement of the enzymatic activity of the essential human enzyme, topoisomerase I. The basic design of the sensor relies on two DNA strands that hybridize to form a hairpin structure with a fluorophore-quencher pair. The quencher moiety is released from the sensor upon reaction with human topoisomerase I thus enabling real-time optical measurement of enzymatic activity. The sensor is specific for topoisomerase I even in raw cell extracts and presents a simple mean of following enzyme kinetics using standard laboratory equipment such as a qPCR machine or fluorimeter. Human topoisomerase I is a well-known target for the clinically used anti-cancer drugs of the camptothecin family. The cytotoxic effect of camptothecins correlates directly with the intracellular topoisomerase I activity. We therefore envision that the presented sensor may find use for the prediction of cellular drug response. Moreover, inhibition of topoisomerase I by camptothecin is readily detectable using the presented DNA sensor, suggesting a potential application of the sensor for first line screening for potential topoisomerase I targeting anti-cancer drugs.

Original languageEnglish
JournalSensors (Switzerland)
Volume13
Issue number4
Pages (from-to)4017-4028
ISSN1424-8220
DOIs
Publication statusPublished - 2013
Externally publishedYes

Keywords

  • Camptothecin
  • Cancer treatment
  • DNA sensor
  • Fluorophore-quencher pair
  • Real-time activity measurement
  • Topoisomerase I

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