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DNA-Based Sensor for Real-Time Measurement of the Enzymatic Activity of Human Topoisomerase I

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  • Lærke Bay Marcussen, Danmark
  • Morten Leth Jepsen, Department of Molecular Biology and Genetics, Aarhus University, Danmark
  • Emil Laust Kristoffersen
  • Oskar Franch, Department of Molecular Biology and Genetics, Aarhus University, Danmark
  • Joanna Proszek, Department of Pathology, Aarhus University Hospital, Danmark
  • Yi-Ping Ho, Danmark
  • Magnus Stougaard
  • Birgitta Ruth Knudsen
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
Sider (fra-til)4017-4028
Antal sider12
StatusUdgivet - 25 mar. 2013

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