High temperature stabilization of DNA in complexes with cationic lipids.
The influence on the melting of calf thymus and plasmid DNA of cationic lipids of the type used in gene therapy was studied by ultraviolet spectrophotometry and differential scanning calorimetry. It was found that various membrane-forming cationic lipids are able to protect calf thymus DNA against denaturation at 100 degrees C. After interaction with cationic lipids, the differential scanning calorimetry melting profile of both calf thymus and plasmid DNA revealed two major components, one corresponding to a thermolabile complex with transition temperature, T(m(labile)), close to that of free DNA and a second corresponding to a thermostable complex with a transition temperature, T(m(stable)), at 105 to 115 degrees C. The parameter T(m(stable)) did not depend on the charge ratio, R(+/-). Instead, the amount of thermostable DNA and the enthalpy ratio Delta H((stable))/Delta H((labile)) depended upon R(+/-) and conditions of complex formation. In the case of O-ethyldioleoylphosphatidylcholine, the cationic lipid that was the main subject of the investigation, the maximal stabilization of DNA exceeded 90% between R(+/-) = 1.5 and 3.0. Several other lipids gave at least 75% protection in the range R(+/-) = 1.5 to 2.0. Centrifugal separation of the thermostable and thermolabile fractions revealed that almost all the transfection activity was present at the thermostable fraction. Electron microscopy of the thermostable complex demonstrated the presence of multilamellar membranes with a periodicity 6.0 to 6.5 nm. This periodic multilamellar structure was retained at temperatures as high as 130 degrees C. It is concluded that constraint of the DNA molecules between oppositely charged membrane surfaces in the multilamellar complex is responsible for DNA stabilization.
Tarahovsky
Yury S
YS
Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, Illinois 60208, USA.
Rakhmanova
Vera A
VA
Epand
Richard M
RM
MacDonald
Robert C
RC
D000818:Animals; D001665:Binding Sites; D002152:Calorimetry, Differential Scanning; D002417:Cattle; D004247:DNA / Q000737:chemistry* / Q000648:ultrastructure; D006358:Hot Temperature; D007700:Kinetics; D009691:Nucleic Acid Denaturation; D010713:Phosphatidylcholines / Q000737:chemistry; D010743:Phospholipids / Q000737:chemistry*; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics; D013950:Thymus Gland
D010713:Phosphatidylcholines; D010743:Phospholipids; D004247:DNA