Patent ID: 8470523

Claim:
A method of detecting interactions between intracellular biomolecules or a translocation or conformational change of intracellular biomolecules in living cells, comprising the following steps: a) labeling a first biomolecule, in a living cell, with a first fluorescent compound having a long fluorescence lifetime, selected from the group consisting of a rare earth chelate and a rare earth cryptate, to produce a first fluorescently labeled biomolecule; b) labeling at least one second biomolecule, in said living cell, with a second fluorescent compound, to produce a second fluorescently labeled biomolecule; c) subjecting the living cells to a specific stimulation adapted to the biological response to be studied, in the presence or absence of a compound belonging to a bank of test molecules; d) subjecting the living cells to a light source whose wavelength excites said first long-lived fluorescent compound; e) measuring the intensity of the fluorescence emitted by said first and second fluorescent compounds; f) calculating the ratio between the fluorescence intensities of said first fluorescent compound and said second fluorescent compound, or measuring the lifetime of the first or second fluorescent compound; and g) comparing the measured signals with those obtained before stimulation of the cells, wherein said first and at least second fluorescent compounds are time-resolved FRET (TR-FRET) partners; said first and/or the second biomolecule are each expressed as a fusion protein with a suicide enzyme, and step a) and/or b) are achieved by extracellularly contacting the cell with a substrate for the suicide enzyme, said substrate being covalently bonded to the first or second fluorescent compound, and at least one of the fluorescent compounds (i) is not naturally membrane-permeant (ii) is conjugated with a unit that enables it to cross the plasma membrane into the living cell, and (iii) is added to the extracellular medium for incubation with the living cells, whereby said the labeling of said intracellular biomolecule with a fluorescent compound that is not naturally membrane-permeant is effected.