title: 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.
authors.affiliation: Department of Molecular Biology and Genetics, Aarhus University, Aarhus C 8000, Denmark. laerke_bay@hotmail.com
authors.forename: Lærke Bay
authors.initials: LB
authors.lastname: Marcussen
authors.forename: Morten Leth
authors.initials: ML
authors.lastname: Jepsen
authors.forename: Emil Laust
authors.initials: EL
authors.lastname: Kristoffersen
authors.forename: Oskar
authors.initials: O
authors.lastname: Franch
authors.forename: Joanna
authors.initials: J
authors.lastname: Proszek
authors.forename: Yi-Ping
authors.initials: YP
authors.lastname: Ho
authors.forename: Magnus
authors.initials: M
authors.lastname: Stougaard
authors.forename: Birgitta Ruth
authors.initials: BR
authors.lastname: Knudsen
mesh_terms: D001483:Base Sequence; D015374:Biosensing Techniques / Q000379:methods*; D002166:Camptothecin / Q000494:pharmacology; D003199:Computer Systems; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation / Q000187:drug effects; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000378:metabolism; D013050:Spectrometry, Fluorescence
chemical_list: D005456:Fluorescent Dyes; D004247:DNA; D004264:DNA Topoisomerases, Type I; C528909:TOP1 protein, human; D002166:Camptothecin