Probing DNA-cationic lipid interactions with the fluorophore trimethylammonium diphenyl-hexatriene (TMADPH).
The aim of this study is to get a better understanding of DNA-cationic lipid complex formation and its characterization through the properties of the lipid assembly, using fluorescent probes known to have different locations in the vesicle bilayer, 1,6-diphenylhexa-1,3,5-triene (DPH) and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMADPH). The location of these two fluorescent probes in the membrane differs; the positive charge of TMADPH is localized close to the water/lipid interface and its fluorophore is present in the upper part of the acyl chain region while DPH (lacking polar group) is embedded deeper in the hydrophobic part of the bilayer. Unilamellar vesicles ( approximately 100 nm size) composed of N-(1-(2, 3-dioleoyloxy)-propyl)-N,N,N-trimethylammonium chloride (DOTAP) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) as a helper lipid (at 1 : 1 mole ratio) were used as a model of cationic liposomes. Both linear and circular DNA gave almost identical results. DNA-/L+ (mole charge ratio of DNA negatively-charged phosphate to positively-charged lipid) ratios have large effects on the measured parameters. The effects monitored through TMADPH are much more striking than those obtained through the use of DPH, suggesting that the major DNA-lipid interaction occurs at the lipid/water interface. The fact that DNA induced much larger changes in TMADPH fluorescence intensity in H2O than in D2O suggests that the changes in the exposure of TMADPH to water and solvent relaxation effects are involved in the interaction. At DNA-/L+>/=1, fluorescence intensity increased concomitantly with a small increase in TMADPH fluorescence anisotropy without much affect in the size of the complex. At DNA-/L+<0.6, fluorescence quenching proportional to DNA-/L+ occurred, as well as a large increase in TMADPH fluorescence anisotropy and in complex size. These results suggest that at low DNA-/L+, negatively-charged DNA condenses positively-charged lipid headgroups, thereby inducing formation of lipid-ordered domains. This phase separation results in membrane defects at the lipid/water interface and increased exposure of the hydrophobic upper parts of the acyl chains to water, as indicated by the quenching of TMADPH. This leads to instability and aggregation/fusion of the DNA-lipid complexes. On the other hand, at DNA-/L+>/=1, the condensing effect is smaller, involving homogeneous lateral condensation of all the lipids, leading to a reduction in water content near the probe, and the DNA-lipid complexes are relatively small and stable.
Hirsch-Lerner
D
D
Department of Biochemistry, Hadassah Medical School, The Hebrew University, P.O. Box 12272, Jerusalem 91120, Israel.
Barenholz
Y
Y
D000818:Animals; D002021:Buffers; D002152:Calorimetry, Differential Scanning; D002412:Cations; D002417:Cattle; D004247:DNA / Q000378:metabolism*; D004161:Diphenylhexatriene / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism; D005456:Fluorescent Dyes; D005614:Freeze Fracturing; D006531:HEPES / Q000737:chemistry; D050356:Lipid Metabolism; D008081:Liposomes / Q000737:chemistry; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D009391:Nephelometry and Turbidimetry; D013050:Spectrometry, Fluorescence
D002021:Buffers; D002412:Cations; D005456:Fluorescent Dyes; D008081:Liposomes; D046911:Macromolecular Substances; D004161:Diphenylhexatriene; C033628:1-(4-(trimethylamino)phenyl)-6-phenylhexa-1,3,5-triene; D004247:DNA; D006531:HEPES