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

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.

Temperature and Magnetic Field Driven Modifications in the I-V Features of Gold-DNA-Gold Structure

“…Meanwhile, the semiconducting properties of DNA-based devices in the presence of external electric and magnetic fields has received special attention for their prospective use as magnetic sensors [ 4 – 8 ]. The path-breaking discovery of the DNA structure by Watson and Crick stimulated rapid developments in the fields of biology, genetics, medicine [ 9 ] and nanoelectronics [ 10 , 11 ]. Previously, DNA-based biosensors were employed for gene analysis, detection of genetic disorders and tissue matching [ 12 – 14 ] using magnetic particle [ 15 ] and temperature sensing [ 16 ].…”

Optimized Detection of Plasmodium falciparum Topoisomerase I Enzyme Activity in a Complex Biological Sample by the Use of Molecular Beacons

“…DNA-based sensors and detection systems have in recent years been increasingly used in relation to detection of multiple DNA-modifying enzymes such as topoisomerases, nucleases and DNA repair enzymes [1,2,3,4,5,6,7,8,9]. In many cases, specially designed DNA oligonucleotides have been used to detect the enzymatic reaction by allowing for specific amplification of the DNA oligonucleotides, only after the correct enzymatic reaction has been performed [5,6,7,8,9,10].…”

“…In many cases, specially designed DNA oligonucleotides have been used to detect the enzymatic reaction by allowing for specific amplification of the DNA oligonucleotides, only after the correct enzymatic reaction has been performed [5,6,7,8,9,10]. These systems enable ultra-sensitive (down to the single-enzymatic-event-level) detection of enzymatic reactions.…”

Combining a Nanosensor and ELISA for Measurement of Tyrosyl-DNA Phosphodiesterase 1 (TDP1) Activity and Protein Amount in Cell and Tissue Extract

“…Moreover, for detection of DNA modifying enzymes a further advantage is that DNA-based nanosensors resemble the natural substrates of the enzymes. We have previously exploited these advantages for developing DNA-based nanosensors capable of detecting the speci¯c activities of the clinically relevant human enzymes topoisomerase I (TOPI) [3][4][5] and tyrosyl-DNA phosphodiesterase 1 (TDP1). 6 TOPI is a ubiquitous enzyme capable of regulating the topology of genomic DNA in nuclear processes such as replication, transcription and chromatin condensation.…”