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 transfectant poly((2-dimethylamino)ethyl methacrylate) was studied. The formed polyplexes were also evaluated for their transfection properties in vitro in two different cell lines. Anion-exchange chromatography was used for the separation of supercoiled and open-circular plasmid from a plasmid stock solution. Linear plasmids were prepared by endonucleases that cleaved the plasmid either in the promoter region or in a region not specific for expression (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 oligomeric forms. The transfection of two different cell lines was dependent on the topology of the DNA in the order supercoiled>open-circular approximately heat-denatured>linear DNA prepared by cleaving in the nonspecific region>linear DNA prepared by cleaving in the promoter region. No differences in the size of the complexes or in the quenching of the DNA-intercalating fluorophore acridine orange were found as function of the topology. However, circular dichroism spectroscopy revealed differences between the topological plasmid species, both in the free form and in the presence of excess of cationic polymer.
Cherng
J Y
JY
Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, P.O. Box 80.082, 3508 TB, Utrecht, The Netherlands.
Schuurmans-Nieuwenbroek
N M
NM
Jiskoot
W
W
Talsma
H
H
Zuidam
N J
NJ
Hennink
W E
WE
Crommelin
D J
DJ
D000165:Acridine Orange / Q000737:chemistry; D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002852:Chromatography, Ion Exchange; D004247:DNA / Q000737:chemistry*; D004587:Electrophoresis, Agar Gel; D004720:Endonucleases / Q000494:pharmacology; D005471:Fluoroscopy; D000882:Haplorhini; D008689:Methacrylates / Q000737:chemistry*; D010316:Particle Size; D010957:Plasmids / Q000737:chemistry*; D011108:Polymers / Q000138:chemical synthesis / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D014162:Transfection
D008689:Methacrylates; D011108:Polymers; D004247:DNA; D004720:Endonucleases; D000165:Acridine Orange