Biological properties of poly-L-lysine-DNA complexes generated by cooperative binding of the polycation.
We have evaluated the effect of NaCl concentration on the mode of binding of poly-L-lysine to DNA and the resulting structural and functional features of the condensed DNA particles using DNA precipitation, DNase I resistance, electron microscopy, and receptor-mediated gene transfer assays. At a high concentration of NaCl and in the presence of excess DNA, poly-L-lysine interacted with DNA cooperatively, fully condensing some of the DNA and leaving the rest of the DNA unbound. At low NaCl concentrations, poly-L-lysine molecules interacted with DNA in a noncooperative fashion, i.e. they bind randomly to the whole population of DNA molecules. Cooperative binding of poly-L-lysine to DNA occurred over a narrow range of NaCl concentrations, and the specific salt concentration depended on the length of the poly-L-lysine. The ability of condensed DNA to withstand digestion by DNase I was correlated with the structural features of the condensed DNA as determined by electron microscopy. Using our condensation procedure, cooperative binding of poly-L-lysine to DNA is a necessary prerequisite for the preparation of condensed DNA having a spherical shape and a diameter of 15-30 nm. Condensed DNA, containing galactosylated poly-L-lysine, was evaluated further for the extent and specificity of receptor-mediated gene transfer into HuH-7 human hepatoma cells via the asialoglycoprotein receptor. Efficient receptor-mediated transfection occurred only when condensed DNA complexes had a spherical shape with a diameter of 15-30 nm; asialofetuin, a natural ligand for the asialoglycoprotein receptor, inhibited this process by up to 90%. Our results support the importance of appropriate DNA condensation for the uptake and ultimate expression of DNA in hepatic cells.
Liu
G
G
Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4935, USA.
Molas
M
M
Grossmann
G A
GA
Pasumarthy
M
M
Perales
J C
JC
Cooper
M J
MJ
Hanson
R W
RW
D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000494:pharmacology; D006801:Humans; D008099:Liver / Q000378:metabolism; D008854:Microscopy, Electron; D011107:Polylysine / Q000378:metabolism*; D012965:Sodium Chloride / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D011107:Polylysine; D012965:Sodium Chloride; D004247:DNA; D003850:Deoxyribonuclease I