title: Real-time detection of TDP1 activity using a fluorophore-quencher coupled DNA-biosensor.
abstract: Real-time detection of enzyme activities may present the easiest and most reliable way of obtaining quantitative analyses in biological samples. We present a new DNA-biosensor capable of detecting the activity of the potential anticancer drug target tyrosyl-DNA phosphodiesterase 1 (TDP1) in a very simple, high throughput, and real-time format. The biosensor is specific for Tdp1 even in complex biological samples, such as human cell extracts, and may consequently find future use in fundamental studies as well as a cancer predictive tool allowing fast analyses of diagnostic cell samples such as biopsies. TDP1 removes covalent 3'DNA adducts in DNA single-strand break repair. This enzymatic activity forms the basis of the design of the TDP1-biosensor, which consists of a short hairpin-forming oligonucleotide having a 5'fluorophore and a 3'quencher brought in close proximity by the secondary structure of the biosensor. The specific action of TDP1 removes the quencher, thereby enabling optical detection of the fluorophore. Since the enzymatic action of TDP1 is the only "signal amplification" the increase in fluorescence may easily be followed in real-time and allows quantitative analyses of TDP1 activity in pure enzyme fractions as well as in crude cell extracts. In the present study we demonstrate the specificity of the biosensor, its ability to quantitatively detect up- or down-regulated TDP1 activity, and that it may be used for measuring and for analyzing the mechanism of TDP1 inhibition.
authors.affiliation: Department of Pathology, Aarhus University Hospital, Denmark.
authors.forename: Pia W
authors.initials: PW
authors.lastname: Jensen
authors.forename: Mattia
authors.initials: M
authors.lastname: Falconi
authors.forename: Emil L
authors.initials: EL
authors.lastname: Kristoffersen
authors.forename: Anita T
authors.initials: AT
authors.lastname: Simonsen
authors.forename: Jèssica B
authors.initials: JB
authors.lastname: Cifuentes
authors.forename: Lærke B
authors.initials: LB
authors.lastname: Marcussen
authors.forename: Rikke
authors.initials: R
authors.lastname: Frøhlich
authors.forename: Josephine
authors.initials: J
authors.lastname: Vagner
authors.forename: Charlotte
authors.initials: C
authors.lastname: Harmsen
authors.forename: Sissel
authors.initials: S
authors.lastname: Juul
authors.forename: Yi-Ping
authors.initials: YP
authors.lastname: Ho
authors.forename: Marjorie A
authors.initials: MA
authors.lastname: Withers
authors.forename: James R
authors.initials: JR
authors.lastname: Lupski
authors.forename: Jørn
authors.initials: J
authors.lastname: Koch
authors.forename: Alessandro
authors.initials: A
authors.lastname: Desideri
authors.forename: Birgitta R
authors.initials: BR
authors.lastname: Knudsen
authors.forename: Magnus
authors.initials: M
authors.lastname: Stougaard
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D002460:Cell Line; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D057075:Enzyme Assays / Q000379:methods; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012680:Sensitivity and Specificity
chemical_list: D005456:Fluorescent Dyes; D009841:Oligonucleotides; D004247:DNA; D010727:Phosphoric Diester Hydrolases; C498508:TDP1 protein, human