Characterization of DNA/lipid complexes by fluorescence resonance energy transfer.
Fluorescence resonance energy transfer (FRET) is a potential method for the characterization of DNA-cationic lipid complexes (lipoplexes). In this work, we used FRET models assuming a multilamellar lipoplex arrangement. The application of these models allows the determination of the distance between the fluorescent intercalator on the DNA and a membrane dye on the lipid, and/or the evaluation of encapsulation efficiencies of this liposomal vehicle. The experiments were carried out in 1,2-dioleoyl-3-trimethylammonium-propane/pUC19 complexes with different charge ratios. We used 2-(3-(diphenylhexatrienyl)propanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (DPH-PC) and 2-(4,4-difluoro-5-octyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (BODIPY-PC) as membrane dyes, and ethidium bromide (EtBr) and BOBO-1 as DNA intercalators. In cationic complexes (charge ratios (+/-) >or= 2), we verified that BOBO-1 remains bound to DNA, and FRET occurs to the membrane dye. This was also confirmed by anisotropy and lifetime measurements. In complexes with all DNA bound to the lipid (charge ratio (+/-) = 4), we determined 27 A as the distance between the donor and acceptor planes (half the repeat distance for a multilamellar arrangement). In complexes with DNA unbound to the lipids (charge ratio (+/-) = 0.5 and 2), we calculated the encapsulation efficiencies. The presented FRET methodology is, to our knowledge, the first procedure allowing quantification of lipid-DNA contact.
Centro de Engenharia Biológica e Química, Instituto Superior Técnico, Lisbon, Portugal.
Catarina
C
Madeira
Luís M S
LM
Loura
M Raquel
MR
Aires-Barros
Aleksander
A
Fedorov
Manuel
M
Prieto
D001665:Binding Sites; D004269:DNA, Bacterial / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes; D008081:Liposomes / Q000737:chemistry*; D046911:Macromolecular Substances; D008563:Membrane Lipids / Q000737:chemistry*; D008968:Molecular Conformation; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods
D004269:DNA, Bacterial; D005456:Fluorescent Dyes; D008081:Liposomes; D046911:Macromolecular Substances; D008563:Membrane Lipids