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Papers In Press, published online ahead of print July 3, 2001
Dept of Biochemistry, Case Western Reserve University, Cleveland, OH 44106-4935
Corresponding Author: rwh{at}po.cwru.edu
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 (EM), 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 upon 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 EM. Using our condensation procedure, cooperative binding of poly-L-lysine to DNA is a necessary prerequisite for preparation of condensed DNA having a spherical shape and a diameter of 15-30nm. Condensed DNA, containing galactosylated poly-L-lysine, was further evaluated 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 15-30nm; 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.
J. Biol. Chem, 10.1074/jbc.M105250200
Submitted on June 7, 2001
Revised on July 3, 2001
Accepted on July 3, 2001
Biological properties of poly-L-lysine/DNA complexes generated by cooperative binding of the polycation
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