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.
Keywords: real-time activity measurement; DNA sensor; fluorophore-quencher pair; topoisomerase I; cancer treatment; camptothecin
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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.
Marcussen LB, Jepsen ML, Kristoffersen EL, Franch O, Proszek J, Ho Y-P, Stougaard M, Knudsen BR. DNA-Based Sensor for Real-Time Measurement of the Enzymatic Activity of Human Topoisomerase I. Sensors. 2013; 13(4):4017-4028.
Marcussen, Lærke B.; Jepsen, Morten L.; Kristoffersen, Emil L.; Franch, Oskar; Proszek, Joanna; Ho, Yi-Ping; Stougaard, Magnus; Knudsen, Birgitta R. 2013. "DNA-Based Sensor for Real-Time Measurement of the Enzymatic Activity of Human Topoisomerase I." Sensors 13, no. 4: 4017-4028.