Phase Separation on a Phospholipid Membrane Inducing a Characteristic Localization of DNA Accompanied by Its Structural Transition

Abstract

We report the coupling of phase separation on a phospholipid membrane with a structural transition of giant DNA. We performed single-DNA measurements in the presence of cell-sized giant unilamellar vesicles (GUV) composed of dioleoylphosphatidylcholine (DOPC), phosphatidylethanolamine (PE), and cholesterol. We observed the effect of phase separation of the membrane by changing PE from dioleoylphosphatidylethanolamine (DOPE) to dipalmitoylethanolamine (DPPE), which corresponds to a change from a homogeneous single phase to two segregated phases of liquid-ordered and liquid-disordered states on the membrane. In the homogeneous membrane, DNA molecules exhibited a compact conformation in aqueous solution containing a physiological concentration of Mg2+ and polyamine, without attaching to the membrane surface. In contrast, in the membrane that was segregated into two domains, DNA molecules were adsorbed onto the liquid-ordered phase that was rich in DPPE by taking an elongated conformation.

Characteristic Behavior of Crowding Macromolecules Confined in Cell-Sized Droplets

“…To avoid the intermolecular aggregation of T4 DNA molecules due to the addition of spermine, we perform experiments using a very low concentration of DNA (0.1-1.0 ng/mL). Consequently, encapsulated DNA molecules vary their conformations and spatial arrangements according to the concentrations of Mg 2þ and spermine, droplet size, and the types of lipid coating the droplet (Kato et al, , 2010. Figure 7.2 shows the distributions of T4 DNA labeled with the fluorescent dye YOYO-1 inside droplets with diameter <60 mm covered with two types of lipids: DOPE (dioleoylphosphatidylethanolamine) and DOPC.…”

“…Such localization of DNA molecules on a DOPE membrane is confirmed using With a decrease in temperature, the ternary liposome separates into a DPPE-Chol-rich phase and a DOPC-rich phase. When we add DNA molecules exhibiting a compact conformation in aqueous solution, they are adsorbed onto the PE-rich phase in presence of the Mg 2þ by assuming an elongated conformation (Kato et al, 2010).…”

“…Here, we first describe the behavior of long DNA molecules encapsulated in droplets, where DNA shows different localizations and conformational transitions according to the droplet size and the types of lipids coating the droplet surface (Kato et al, , 2010. The unfold-fold transition of DNA triggers the on/off switching of the transcription (Tsuji and Yoshikawa, 2010a).…”

Validation of liposomal lipid composition by thin-layer chromatography

“…We had to substitute bPS by ePE because incorporation of bPS interferes with the preparation of GUVs by the electroformation method (Rodriguez et al, 2000;Veatch and Keller, 2003), and thus we were not successful at producing GUVs with these mixtures. However, mixtures containing PE are used in a number of studies, for example, DPPE/DPPC (Bagatolli and Gratton, 2000a) or DMPE/ DMPC (Bagatolli and Gratton, 2000b), DMPC/DOPE/ PS (Perrier-Cornet et al, 2005), DOPC/DOPE/Chol, and DOPC/DPPE/Chol (Kato et al, 2010). In our ePEcontaining LUVs, the relative fraction of ePE showed a decreased value (g = 0.84) and increased values for Chol (g = 1.08) and eSM (g = 1.22).…”

Cationic Liposomes in Different Structural Levels for Gene Delivery

“…Their data demonstrated that efficiency of endocytosis was regulated by shape coupling between lipoplex and membrane lipids to suggest that such a shape-dependent coupling regulated efficient formation of endocytic vesicles thus determining the success of internalization (Marchini et al, 2010). Kato et al (Kato et al, 2010) observed the effect of phase separation of the membrane by changing PE from DOPE to dipalmitoylethanolamine (DPPE), which corresponded to a change from a homogeneous single phase to two segregated phases of liquid-ordered and liquid-disordered states on the membrane. This study further proved that helper lipids could change the morphologies of lipoplexes through the mutual interaction with DNA based on their chemical structures.…”