title: A homogeneous time-resolved fluorescence detection of telomerase activity.
abstract: The homogeneous time-resolved fluorescence (HTRF) technology is an assay developed to study the interaction between biomolecules. This detection system is based on a fluorescence resonance energy transfer (FRET) between a Tris-bipyridine europium cryptate used as a long-lived fluorescent donor and a chemically modified allophycocyanine as acceptor. This technology is characterized by both a spectral selectivity and a temporal selectivity (due to the time-resolved mode), ensuring a highly specific signal. Here a europium-cryptate-labeled deoxyuridine triphosphate analogue (K-11-dUTP) was used to monitor the extension reaction on a biotinylated oligonucleotide used as substrate for telomerase in a telomeric repeat amplification protocol (TRAP). After the addition of an allophycocyanine-streptavidin conjugate, the extension products give rise to a FRET between the incorporated cryptate moieties and the allophycocyanine acceptor that then displays a specific long-lived emission. The TRAP-HTRF format was validated as a screening tool by using a 2,6-diaminoanthraquinone analogue, a known inhibitor of telomerase activity. The IC(50) measured was consistent with the reported values, showing the convenience of the HTRF technology for the study of telomerase activity and inhibitors.
authors.affiliation: CIS Biointernational, HTRF/Bioassays, BP 84175, F-30204 Bagnols-sur-Ceze, France.
authors.forename: Manuel
authors.initials: M
authors.lastname: Gabourdes
authors.forename: Valérie
authors.initials: V
authors.lastname: Bourgine
authors.forename: Gérard
authors.initials: G
authors.lastname: Mathis
authors.forename: Hervé
authors.initials: H
authors.lastname: Bazin
authors.forename: Béatrice
authors.initials: B
authors.lastname: Alpha-Bazin
mesh_terms: D000880:Anthraquinones / Q000494:pharmacology; D003857:Deoxyuridine / Q000031:analogs & derivatives* / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000494:pharmacology; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D020014:K562 Cells; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D009942:Organometallic Compounds / Q000737:chemistry; D010798:Phycocyanin / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D019098:Telomerase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000880:Anthraquinones; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; C420282:K-11-dUTP; D009838:Oligodeoxyribonucleotides; D009942:Organometallic Compounds; C002497:allophycocyanin; D010798:Phycocyanin; D019098:Telomerase; D003857:Deoxyuridine