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

TY - JOUR

T1 - DNA-Based Sensor for Real-Time Measurement of the Enzymatic Activity of Human Topoisomerase I

AU - Marcussen, Lærke Bay

AU - Jepsen, Morten Leth

AU - Kristoffersen, Emil Laust

AU - Franch, Oskar

AU - Proszek, Joanna

AU - Ho, Yi-Ping

AU - Stougaard, Magnus

AU - Knudsen, Birgitta Ruth

PY - 2013/3/25

Y1 - 2013/3/25

N2 - 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.

AB - 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.

KW - real-time activity measurement

KW - DNA sensor

KW - fluorophore-quencher pair

KW - topoisomerase I

KW - cancer treatment

KW - camptothecin

U2 - 10.3390/s130404017

DO - 10.3390/s130404017

M3 - Journal article

C2 - 23529147

SN - 1424-8220

VL - 13

SP - 4017

EP - 4028

JO - Sensors

JF - Sensors

IS - 4

ER -