Photophysical behavior of a dimeric cyanine dye (BOBO-1) within cationic liposomes.
This study is aimed at establishing optimal conditions for the use of 2,2'-[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 (tau approximately 2.5 ns and <r> = 0.20). By agarose gel electrophoresis it was verified that up to d/b = 0.01 DNA conformation is not considerably modified, whereas for d/b = 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.
Madeira
Catarina
C
Centro de Engenharia Biológica e Química, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001, Portugal.
Fedorov
Aleksander
A
Aires-Barros
M Raquel
MR
Prieto
Manuel
M
Loura
Luís M S
LM
D052160:Benzothiazoles / Q000737:chemistry*; D002232:Carbocyanines / Q000737:chemistry*; D002412:Cations; D019281:Dimerization; D004587:Electrophoresis, Agar Gel; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D008081:Liposomes / Q000737:chemistry* / Q000378:metabolism; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000644:Quaternary Ammonium Compounds / Q000737:chemistry*
C510783:2,2'-(1,3-propanediylbis((dimethyliminio)-3,1-propanediyl-1(4H)-pyridinyl-4-ylidenemethylidyne))bis(3-methyl); D052160:Benzothiazoles; D002232:Carbocyanines; D002412:Cations; D005456:Fluorescent Dyes; D008081:Liposomes; D000644:Quaternary Ammonium Compounds