Oxazole yellow homodimer YOYO-1-labeled DNA: a fluorescent complex that can be used to assess structural changes in DNA following formation and cellular delivery of cationic lipid DNA complexes.
To improve transfection efficiency following delivery of plasmid expression vectors using lipid-based carriers, it is crucial to define structural characteristics of the lipid/DNA complexes that optimize transgene expression. Due to its strong affinity for DNA and high quantum yield, the fluorescent DNA intercalator YOYO-1 was used as a tool to assess changes in DNA that occur following lipid binding and cell delivery. In this study, the stability of the dye/DNA complex following binding of poly-L-lysine or monocationic lipids is characterized. More than 98% of the fluorescence measured for a defined DNA/YOYO-1 complex was lost when DNA was condensed using poly-L-lysine. This loss in fluorescence could be attributed to displacement of bound dye. In contrast, more than 30% of the fluorescence of the dye-labeled DNA was retained after formation of cationic lipid/DNA complexes. Significantly, the results illustrate differences in structural changes cationic lipids and PLL exert on plasmid DNA. The fluorescent lipid/DNA complex was used to assess DNA delivery to murine B16/BL6 cells in vitro. An assay relying on fluorescence resonance energy transfer between bound YOYO-1 and propidium iodide was used to distinguish between DNA attached to the cell surface and internalized DNA.
Wong
M
M
Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, Canada.
Kong
S
S
Dragowska
W H
WH
Bally
M B
MB
D001583:Benzoxazoles / Q000378:metabolism*; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000378:metabolism*; D050356:Lipid Metabolism; D046911:Macromolecular Substances; D010957:Plasmids / Q000235:genetics*; D011107:Polylysine / Q000378:metabolism; D011806:Quinolinium Compounds / Q000378:metabolism*
D001583:Benzoxazoles; D004269:DNA, Bacterial; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D011806:Quinolinium Compounds; C075296:1,1'-((4,4,7,7-tetramethyl)-4,7-diazaundecamethylene)bis-4-(3-methyl-2,3-dihydro(benzo-1,3-oxazole)-2-methylidene)quinolinium; D011107:Polylysine