Partitioning of Membrane-Anchored DNA between Coexisting Lipid Phases

Abstract

The partitioning of different cholesterol-modified single-stranded DNA molecules (chol-DNAs) between the domains of phase-separated lipid vesicles is investigated by laser-scanning confocal fluorescence microscopy. All chol-DNAs studied preferentially localized into the fluid phase of giant vesicles in liquid-solid phase coexistence (1:1 DLPC:DPPC, 1:1 DLPC:DMPE). Partitioning behavior of chol-DNAs into liquid-liquid phase-separated vesicles (DOPC/DPPC/cholesterol) was found to be less straightforward. Single-cholesterolanchored DNA molecules partitioned roughly equally between coexisting domains, whereas chol-DNAs with two cholesterol anchors were seen to be enriched in the liquid-ordered domains with apparent surface concentrations up to double that of the liquid-disordered phase. Quantitative analysis of the fluorescence intensity of DNA between the two phases also revealed a weaker dependence of the apparent partitioning on the initial lipid composition of the vesicles. We rationalize these observations by proposing a simple partitioning model based on the conformational entropy of insertion of a cholesterol anchor into each phase.

Application of nucleic acid–lipid conjugates for the programmable organisation of liposomal modules

“…This leaves an overhanging single-stranded region of DNA to bind anti-sense strands in solution, while having two cholesterol moieties to reinforce the hydrophobic anchoring into a lipid bilayer. DNA strands with two parallel cholesterol modifications attached through a Y-shaped modification on one end of the DNA have also been reported for enhanced hydrophobic association with membrane surfaces [56,57].…”

“…In fact, the cholesteryl-TEG anchors have been shown not to have the same lipid condensing behaviour as cholesterol in POPC membranes [78]. However it is suggested that the rigid fused ring structure of the cholesteryl anchor imposes an unfavourable entropic penalty on the conformational degrees of freedom moderate enhancement within the L o phase [56].…”

“…The partitioning behaviour of cholesteryl modified DNAs in phase separated GUVs has also been characterized [56]. Single and double anchored chol-DNAs predictably partition into the L phase of the various liquid-solid phase separated mixtures that were tested.…”

“…The proportion of DNA in the liquid ordered domains could be enhanced by up to a factor of two when doubly-anchored chol-DNA was used instead. While cholesterol is known to be enriched in the L o domains of GUVs, chemical modification of the cholesterol group means that these molecules cannot be assumed to have similar partitioning behaviour [56]. In fact, the cholesteryl-TEG anchors have been shown not to have the same lipid condensing behaviour as cholesterol in POPC membranes [78].…”

Thermal-driven domain and cargo transport in lipid membranes

“…Similarly, species that preferentially locate within the domains [for example, some integral membrane proteins prefer to reside in cholesterol-rich regions (20)] can be moved selectively to heated regions. We use the transport of DNA constructs with cholesterol anchors, which preferentially locate in the cholesterol-rich Lo phase (21), to demonstrate this thermal manipulation mechanism.…”