Cationic Lipid Binding to DNA: Characterization of Complex Formation
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- 作者：Frances M. P. Wong ; Dorothy L. Reimer ; and Marcel B. Bally
- 刊名：Biochemistry
- 出版年：1996
- 出版时间：May 7, 1996
- 年：1996
- 卷：35
- 期：18
- 页码：5756 - 5763
- 全文大小：407K
- 年卷期：v.35,no.18(May 7, 1996)
- ISSN：1520-4995
文摘
We recently demonstrated that cationic lipids, added in monomer ormicellar form, bind toDNA, resulting in the formation of a hydrophobic complex. Thiscomplex can serve as a well-definedintermediate in the preparation of DNA-lipid particles (DLPs) withmany potential applications for deliveryof polynucleotides in vitro and in vivo. Todevelop a better understanding of the factors governingcomplexformation, we have characterized the cationic lipid/DNA bindingreaction. This was evaluated by measuringDNA and cationic lipid (DODAC) complex formation using the Bligh andDyer extraction procedure.Efficient recovery of DNA (>95%) in the organic phase wasachieved when sufficient monocationiclipids interact with DNA phosphate groups. The rate of bindingdepends on the amount of DNA orcationic lipid present in the system. The time required togenerate the hydrophobic complex was increasedwhen <10 
g of DNA or <40 nmol of DODAC was present.Surprisingly, the rate of complex formationwas contingent on the incubation period after partitioning theDNA/lipid mixture into organic and aqueousphases. These results suggest that the cationic lipid/DNA complexforms at the aqueous/organic interfaceand that DNA/lipid binding is dependent on multivalent interactions atthis interface. A Scatchard analysisof DNA/DODAC binding demonstrated that the binding reaction exhibits ahigh degree of positivecooperativity. The apparent dissociation constant(Kn), using data obtained under conditions whereDODACbinding to DNA approached saturation, indicated a high-affinityreaction (Kn > 10-11mol L-1). At thispoint, ~8400 mol of DODAC was bound per mole of DNA, which isequivalent to a charge ratio (+/-)of 0.585 for the 7.2 kb plasmid used and suggests that formation of thehydrophobic complex occurs ata stage prior to charge neutralization. The influence of otherlipids on DNA/cationic lipid binding at theaqueous/organic interface was also studied. Cholesterol and DOPChad little effect on DNA/DODACbinding while the anionic lipids LPI, DOPS, and DMPG inhibited complexformation. The zwitterioniclipid DOPE, however, had a concentration-dependent effect on cationiclipid binding that was also dependenton the mixing order. We believe that this approach for evaluatinglipid/DNA binding provides an effectiveprocedure for assessing factors which control the dissociation oflipids from DNA and may be beneficialin the selection of lipids for effective use in gene transfectionstudies.
g of DNA or <40 nmol of DODAC was present.Surprisingly, the rate of complex formationwas contingent on the incubation period after partitioning theDNA/lipid mixture into organic and aqueousphases. These results suggest that the cationic lipid/DNA complexforms at the aqueous/organic interfaceand that DNA/lipid binding is dependent on multivalent interactions atthis interface. A Scatchard analysisof DNA/DODAC binding demonstrated that the binding reaction exhibits ahigh degree of positivecooperativity. The apparent dissociation constant(Kn), using data obtained under conditions whereDODACbinding to DNA approached saturation, indicated a high-affinityreaction (Kn > 10-11mol L-1). At thispoint, ~8400 mol of DODAC was bound per mole of DNA, which isequivalent to a charge ratio (+/-)of 0.585 for the 7.2 kb plasmid used and suggests that formation of thehydrophobic complex occurs ata stage prior to charge neutralization. The influence of otherlipids on DNA/cationic lipid binding at theaqueous/organic interface was also studied. Cholesterol and DOPChad little effect on DNA/DODACbinding while the anionic lipids LPI, DOPS, and DMPG inhibited complexformation. The zwitterioniclipid DOPE, however, had a concentration-dependent effect on cationiclipid binding that was also dependenton the mixing order. We believe that this approach for evaluatinglipid/DNA binding provides an effectiveprocedure for assessing factors which control the dissociation oflipids from DNA and may be beneficialin the selection of lipids for effective use in gene transfectionstudies.