Fluorogenic dyes with modulated fluorescence.

CARLA C. SPAGNUOLO; LUCIANA GIORDANO; THOMAS M. JOVIN; ELIZABETH JARES-ERIJMAN

Rovinj, Croacia

Workshop; 18th International Summer School on Biophysics.; 2003

IUPAB

We are involved in the development of new synthetic fluorescent probes such as 5,6-carboxy-4´,5´-bis(1,3,2-dithioarsolan-2-yl)fluorescein-(1,2-ethanedithiol) bound covalently to a dithienylethene photocromic FRET acceptor. These probes are membrane permeable and specifically target recombinant proteins containing four cysteine residues such as CCPGCC inserted into a fusion protein. The probes are non-fluorescent until they are bound to the target peptide. The specific binding of the fluorescent probe to this 4 Cys motif enables fluorescent labeling inside cells by its extra-cellular administration. The high sensitivity of the fluorophore to its environment enables monitoring of the conformational changes of the proteins in live cells as changes in its fluorescence intensity. Modulation of its fluorescence by pc-FRET enables the subtraction of auto-fluorescence noise and greater stability of the dye. The reference form for noise subtraction can be obtained by irradiation at 366 nm. The UV irradiation is used to convert de dithienylethene to its closed "FRET acceptor" form and consequently enable the quenching of the emission of the dye.