DOTAP cationic liposomes prefer relaxed over supercoiled plasmids.
Cationic liposomes and DNA interact electrostatically to form complexes called lipoplexes. The amounts of unbound (free) DNA in a mixture of cationic liposomes and DNA at different cationic lipid:DNA molar ratios can be used to describe DNA binding isotherms; these provide a measure of the binding efficiency of DNA to different cationic lipid formulations at various medium conditions. In order to quantify the ratio between the various forms of naked DNA and supercoiled, relaxed and single-stranded DNA, and the ratio between cationic lipid bound and unbound DNA of various forms we developed a simple, sensitive quantitative assay using agarose gel electrophoresis, followed by staining with the fluorescent cyanine DNA dyes SYBR Green I or SYBR Gold. This assay was compared with that based on the use of ethidium bromide (the most commonly used nucleic acid stain). Unlike ethidium bromide, SYBR Green I DNA sensitivity and concentration-dependent fluorescence intensity were identical for supercoiled and nicked-relaxed forms. DNA detection by SYBR Green I in solution is approximately 40-fold more sensitive than by ethidium bromide for double-stranded DNA and approximately 10-fold for single-stranded DNA, and in agarose gel it is 16-fold more sensitive for double-stranded DNA compared with ethidium bromide. SYBR Gold performs similarly to SYBR Green I. This study shows that: (a) there is no significant difference in DNA binding isotherms to the monocationic DOTAP (DOTAP/DOPE) liposomes and to the polycationic DOSPA (DOSPA/DOPE) liposomes, even when four DOSPA positive charges are involved in the electrostatic interaction with DNA; (b) the helper lipids affect DNA binding, as DOTAP/DOPE liposomes bind more DNA than DOTAP/cholesterol; (c) in the process of lipoplex formation, when the DNA is a mixture of two forms, supercoiled and nicked-relaxed (open circular), there is a preference for the binding to the cationic liposomes of plasmid DNA in the nicked-relaxed over the supercoiled form. This preference is much more pronounced when the cationic liposome formulation is based on the monocationic lipid DOTAP than on the polycationic lipid DOSPA. The preference of DOTAP formulations to bind to the relaxed DNA plasmid suggests that the binding of supercoiled DNA is weaker and easier to dissociate from the complex.
Even-Chen
S
S
Laboratory of Membrane and Liposome Research, Department of Biochemistry, The Hebrew University - Hadassah Medical School, P.O. Box 12272, 91120, Jerusalem, Israel.
Barenholz
Y
Y
D052160:Benzothiazoles; D002784:Cholesterol / Q000737:chemistry; D004278:DNA, Superhelical / Q000032:analysis / Q000737:chemistry*; D003959:Diamines; D004587:Electrophoresis, Agar Gel; D004996:Ethidium; D005229:Fatty Acids, Monounsaturated / Q000737:chemistry*; D005456:Fluorescent Dyes; D020404:Glycerophospholipids / Q000737:chemistry; D008081:Liposomes / Q000737:chemistry*; D009930:Organic Chemicals; D010316:Particle Size; D010714:Phosphatidylethanolamines; D010957:Plasmids / Q000032:analysis / Q000737:chemistry*; D000644:Quaternary Ammonium Compounds / Q000737:chemistry*; D011804:Quinolines
C410262:1,2-dioleoyl-glycero-3-phosphatidyl ethanolamine; D052160:Benzothiazoles; D004278:DNA, Superhelical; D003959:Diamines; D005229:Fatty Acids, Monounsaturated; D005456:Fluorescent Dyes; D020404:Glycerophospholipids; D008081:Liposomes; D009930:Organic Chemicals; D010714:Phosphatidylethanolamines; D000644:Quaternary Ammonium Compounds; D011804:Quinolines; C098022:SYBR Green I; D002784:Cholesterol; D004996:Ethidium; C070046:1,2-dioleoyloxy-3-(trimethylammonium)propane