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J. Biol. Chem., Vol. 276, Issue 37, 34379-34387, September 14, 2001
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,§,,
From the 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.
Department of Biochemistry, Case Western
Reserve University School of Medicine, Cleveland, Ohio 44106-4935 and
¶ Copernicus Therapeutics, Inc., Cleveland, Ohio 44106
The on-line version of this article (available at
http://www.jbc.org) contains the EM images from which data are derived.
§
Current address: Departament de Ciencies Fisiologiques,
Universitat de Barcelona, Feixa Llarga s/n, L'Hospitalet 08907, Spain.
To whom correspondence should be addressed: Dept. of
Biochemistry, Case Western Reserve University, School of Medicine,
Cleveland, OH 44106-4935. Tel.: 216-368-5302; Fax: 216-368-4544;
E-mail: rwh@po.cwru.edu.
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