A homogeneous time-resolved fluorescence detection of telomerase activity.
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.
Gabourdes
Manuel
M
CIS Biointernational, HTRF/Bioassays, BP 84175, F-30204 Bagnols-sur-Ceze, France.
Bourgine
Valérie
V
Mathis
Gérard
G
Bazin
Hervé
H
Alpha-Bazin
Béatrice
B
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*
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