title: 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.
abstract: 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.
authors: Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, Canada.
authors: M
authors: M
authors: Wong
authors: S
authors: S
authors: Kong
authors: W H
authors: WH
authors: Dragowska
authors: M B
authors: MB
authors: Bally
mesh_terms: 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*
chemical_list: 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