ABSTRACT
The structure of DNA within CLDCs used for gene delivery is controversial. Previous studies using CD have been interpreted to indicate that the DNA is converted from normal B to C form in complexes. This investigation reexamines this interpretation using CD of model complexes, FTIR as well as Raman spectroscopy and molecular dynamics simulations to address this issue. CD spectra of supercoiled plasmid DNA undergo a significant loss of rotational strength in the signal near 275 nm upon interaction with either the cationic lipid dimethyldioctadecylammonium bromide or 1,2-dioleoyltrimethylammonium propane. This loss of rotational strength is shown, however, by both FTIR and Raman spectroscopy to occur within the parameters of the B-type conformation. Contributions of absorption flattening and differential scattering to the CD spectra of complexes are unable to account for the observed spectra. Model studies of the CD of complexes prepared from synthetic oligonucleotides of varying length suggest that significant reductions in rotational strength can occur within short stretches of DNA. Furthermore, some alteration in the hydrogen bonding of bases within CLDCs is indicated in the FTIR and Raman spectroscopy results. In addition, alterations in base stacking interactions as well as hydrogen bonding are suggested by molecular dynamics simulations. A global interpretation of all of the data suggests the DNA component of CLDCs remains in a variant B form in which base/base interactions are perturbed.
Subject(s)
Lipids/chemistry , Liposomes/chemistry , Models, Molecular , Plasmids/chemistry , Spectrum Analysis/methods , Cations , Circular Dichroism/methods , Computer Simulation , DNA/chemistry , Fatty Acids, Monounsaturated/chemistry , Gels/chemistry , Macromolecular Substances , Motion , Nucleic Acid Conformation , Particle Size , Quaternary Ammonium Compounds/chemistry , Rotation , Solutions/chemistry , Spectroscopy, Fourier Transform Infrared/methods , Spectrum Analysis, Raman/methodsABSTRACT
The thermal stabilities of supercoiled (SC) and linear/open circular (LIN/OC) forms of plasmid DNA when complexed with cationic lipids or cationic polymers used for cellular transfection were assessed using differential scanning calorimetry. Differences in the stability of SC DNA produced by the cationic lipids DOTAP (1,2-dioleoyltrimethyl ammoniumpropane chloride), DSTAP (1,2-distearyltrimethyl ammoniumpropane chloride), and DDAB (dimethyldioctadecylammonium bromide) upon complexation suggest possible effects of headgroup structure on the stability of SC DNA and minimal effects of lipid acyl chain saturation/unsaturation. Complexation of DNA with the cationic polymers polyethylenimine (PEI) or poly-L-lysine (PLL) (but not poly-L-arginine) resulted in a decreased stability of SC DNA when the DNA was in charge excess, although all polymers stabilized SC DNA when the polymer was in charge excess. The effects of these cationic polymers on the stability of SC DNA can be explained by changes produced in the tertiary structure of SC DNA upon binding and may reflect the importance of the topological constraint of supercoiling upon the stability of the resulting complexes.
