Photochem. Photobiol., 81, 1450-1459 (2005)

Photophysical behavior of a dimeric cyanine dye (BOBO-1) within cationic liposomes

Madeira, C., Loura, L., Aires-Barros, R., Fedorov, A., Prieto, M.

Abstract

This study is aimed at establishing optimal conditions for the use of 2,29-[1,3-propanediylbis[(dimethyliminio)-3,1- propanediyl-1(4H)-pyridinyl-4-ylidenemethy-lidyne]]bis[3- methyl]-tetraiodide (BOBO-1) as a fluorescent probe in the characterization of lipid/DNA complexes (lipoplexes). The fluorescence spectra, anisotropy, fluorescence lifetimes and fluorescence quantum yields of this dimeric cyanine dye in plasmid DNA (2694 base pairs) with and without cationic liposomes (1,2-dioleoyl-3-trimethylammonium-propane [DOTAP]), are reported. The photophysical behavior of the dye in the absence of lipid was studied for several dye/DNA ratios using both supercoiled and relaxed plasmid. At dye/DNA ratios (d/b) below 0.01 the fluorescence intensity increases linearly, whereas lifetime and anisotropy values of the dye are constant (t ~ 2.5 ns and 5 0.20). By agarose gel electrophoresis it was verified that up to d/b 5 0.01 DNA conformation is not considerably modified, whereas for d/b 5 0.05–0.06 a single heavy band appears on the gel. For these and higher dye/DNA ratios the fluorescence intensity, anisotropy and average lifetime values decrease with an increase in BOBO-1 concentration. When cationic liposomes are added to the BOBO-1/DNA complex, an additional effect is noticed: The difference in the environment probed by BOBO-1 bound to DNA leads to a decrease in quantum yield and average lifetime values, and a redshift is apparent in the emission spectrum. For fluorescence measurements including energy transfer (FRET), a d/b ratio of 0.01 seems to be adequate because no considerable change on DNA conformation is detected, a considerable fluorescent signal is still measured after lipoplex formation, and energy migration is not efficient.

Keywords: dimeric cyanine dye; BOBO-1; cationic lipid; plasmid DNA; fluorescent probe; lipoplex

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