Biophysical and transfection studies of the diC(14)-amidine/DNA complex.
Liposomes of the synthetic cationic lipid, N-t-butyl-N'-tetradecylamino-propionamidine (diC(14)-amidine), efficiently ports DNA into mammalian cells in the absence of other (neutral) lipids. The compositional simplicity of this transfection mix makes it attractive from a formulation perspective. We have used low- and wide-angle x-ray diffraction and polarized light microscopy to characterize the thermotropic phase behavior and microstructure of diC(14)-amidine and of the lipid/DNA (circular plasmid, 5.4 kb) complex with a view to understanding the structure of the complex and its role in transfection. Upon heating, the lipid in buffer undergoes a lamellar crystalline (L(c), d(001) = 41.7 A)-to-lamellar liquid crystal (L(c)(alpha), d(001) depends on hydration and T) transition at approximately 40 degrees C. Sonicated lipid vesicles with a reported transition temperature of approximately 23 degrees C complex with DNA. Complex formation is complete at a DNA/lipid mole ratio (rho) of 0.8. Adding DNA to the lipid causes d(001) of the multilayered complex to drop from 52 to 49 A as rho rises from 0.03 to 1.64. The minimal DNA-DNA duplex separation observed is 26 A, consistent with the close packing of B-DNA. Lipid bilayers in the complex undergo a lamellar gel (L(c)(beta))-to-L(c)(alpha) (superscript c refers to complex) transition at approximately 23 degrees C. Transfection efficiency was maximized at rho = 0.4. The structure and transfection data combined suggest that densely packaged DNA in a net positively charged complex is essential for transfection.
Cherezov
Vadim
V
Biochemistry, Biophysics, and Chemistry, The Ohio State University, 100 West 18th Avenue, Columbus, Ohio 43210, USA.
Qiu
Hong
H
Pector
Veronique
V
Vandenbranden
Michel
M
Ruysschaert
Jean-Marie
JM
Caffrey
Martin
M
D016475:3T3 Cells; D000578:Amidines / Q000737:chemistry*; D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D004274:DNA, Recombinant / Q000737:chemistry* / Q000235:genetics*; D046911:Macromolecular Substances; D051379:Mice; D008859:Microscopy, Polarization; D008958:Models, Molecular; D013816:Thermodynamics; D014162:Transfection; D014961:X-Ray Diffraction
D000578:Amidines; D004274:DNA, Recombinant; D046911:Macromolecular Substances; C089665:N-t-butyl-n'-tetradecyl-3-tetradecylaminopropionamidine