New applications of PC-FRET: Towards an environment sensitive fluorogenic dye with modulated fluorescence.

CARLA SPAGNUOLO; LUCIANA GIORDANO; ELIZABETH JARES-ERIJMAN

Viña del mar, Chile.

Congreso; VII Encuentro Latinoamericano de Fotoquímica y Fotobiología.; 2003

The work describes the development of a new synthetic fluorescent probe, 5,6-carboxy-4’,5’-bis(1,3,2-dithioarsolan-2-yl) fluorescein-(1,2-ethanothiol) bond covalently to a dithienylethene photochromic FRET acceptor. This probe can be attached to recombinant proteins containing four cysteine residues at the i, i + i, i +4, and i +5 positions of an alpha-helix. The specific binding of the fluorescent probe to this 4 Cys motif enables fluorescent labeling inside cells by its extracellular administration. The high sensitivity of the fluorophore to its environment enables monitoring of the conformational changes of the proteins in live cells as change in its fluorescence intensity. Modulation of its fluorescence by pcFRET enables the subtraction of autofluorescence noise. The reference form for noise subtraction can be obtained by irradiation at 366 nm. The UV irradiation is used to convert the dithienylethene to its closed “FRET acceptor” form and consequently enable the quenching of the emission of the dye.