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Biophys J, June 2002, p. 3105-3117, Vol. 82, No. 6

Biophysical and Transfection Studies of the diC14-Amidine/DNA Complex

Vadim Cherezov,* Hong Qiu,* Veronique Pector,dagger Michel Vandenbranden,dagger Jean-Marie Ruysschaert,dagger and Martin Caffrey*

 *Biochemistry, Biophysics, and Chemistry, The Ohio State University, 100 West 18th Avenue, Columbus, Ohio 43210 USA; and  dagger Structure et Fonction des Membranes Biologiques, Universite Libre de Bruxelles, Campus Plaine CP 206/2, B-1050 Brussels, Belgium

Liposomes of the synthetic cationic lipid, N-t-butyl-N'-tetradecylamino-propionamidine (diC14-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 diC14-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 (Lc, d001 = 41.7 Å)-to-lamellar liquid crystal (L<UP><SUB>&agr;</SUB><SUP>c</SUP></UP>, d001 depends on hydration and T) transition at ~40°C. Sonicated lipid vesicles with a reported transition temperature of ~23°C complex with DNA. Complex formation is complete at a DNA/lipid mole ratio (rho ) of 0.8. Adding DNA to the lipid causes d001 of the multilayered complex to drop from 52 to 49 Å as rho  rises from 0.03 to 1.64. The minimal DNA-DNA duplex separation observed is 26 Å, consistent with the close packing of B-DNA. Lipid bilayers in the complex undergo a lamellar gel (L<UP><SUB>&bgr;</SUB><SUP>c</SUP></UP>)-to-L<UP><SUB>&agr;</SUB><SUP>c</SUP></UP> (superscript c refers to complex) transition at ~23°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.

Biophys J, June 2002, p. 3105-3117, Vol. 82, No. 6
© 2002 by the Biophysical Society   0006-3495/02/06/3105/13  $2.00


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