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Sensors 2013, 13(4), 4017-4028; https://doi.org/10.3390/s130404017

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

1
Department of Molecular Biology and Genetics, Aarhus University, Aarhus C 8000, Denmark
2
Department of Pathology, Aarhus University Hospital, Aarhus C 8000, Denmark
3
Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Aarhus C 8000, Denmark
These authors contributed equally to this work.
*
Authors to whom correspondence should be addressed.
Received: 1 February 2013 / Revised: 16 February 2013 / Accepted: 19 March 2013 / Published: 25 March 2013
(This article belongs to the Special Issue Enzymatic Biosensors)
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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. View Full-Text
Keywords: real-time activity measurement; DNA sensor; fluorophore-quencher pair; topoisomerase I; cancer treatment; camptothecin real-time activity measurement; DNA sensor; fluorophore-quencher pair; topoisomerase I; cancer treatment; camptothecin
This is an open access article distributed under the Creative Commons Attribution License (CC BY 3.0).

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Marcussen, L.B.; Jepsen, M.L.; Kristoffersen, E.L.; Franch, O.; Proszek, J.; Ho, Y.-P.; Stougaard, M.; Knudsen, B.R. DNA-Based Sensor for Real-Time Measurement of the Enzymatic Activity of Human Topoisomerase I. Sensors 2013, 13, 4017-4028.

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