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.
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