Effect of DNA topology on the transfection efficiency of poly((2-dimethylamino)ethyl methacrylate)-plasmid complexes

In this paper the effect of the topology of plasmid DNA (supercoiled, open- circular and linear) on its binding characteristics with the polymeric tran sfectant poly((2-dimerhylamino)ethyl methacrylate) was studied. The formed polyplexes were also evaluated for their transfection properties in vitro i n two different cell lines. Anion-exchange chromatography was used for the seperation of supercoiled and open-circular plasmid from a plasmid stock so lution. Linear plasmids were prepared by endonucleases that cleaved the pla smid either in the promoter region or in a region not specific for expressi on (ampicillin resistance region). Plasmid DNA was also heat-denatured for 6 h at 70 degrees C, resulting in DNA mainly in the open-circular and oligo meric forms. The transfection of two different cell lines was dependent on the topology of the DNA in the order supercoiled>open-circular= heat-denatu red>Linear DNA prepared by cleaving in the nonspecific region>linear DNA pr epared by cleaving in the promoter region. No differences in the size of th e complexes or in the quenching of the DNA-intercalating fluorophore acridi ne orange were found as function of the topology. However, circular dichroi sm spectroscopy revealed differences between the topological plasmid specie s, both in the free form and in the presence of excess of cationic polymer. (C) 1999 Elsevier Science B.V. All rights reserved.