A method of gentle hydration to prepare oil-free giant unilamellar vesicles that can confine enzymatic reactions

Abstract

We report a new and improved method to prepare, by gentle hydration of lipid films, oil-free giant unilamellar vesicles (GUVs), in which enzymatic reactions can be encapsulated. The traditional method of gentle hydration requires very low concentrations of metal ions, whereas enzymatic reactions generally require mono- and divalent metal ions at physiological concentrations. In order to improve the production of oil-free GUVs that can confine enzymatic reactions, we developed a novel method also based on gentle hydration, but in which the precursor lipid film was doped with both 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEGylated lipid) and sugar. Close examination of the size, shape, and lamellarity of vesicles prepared in this manner demonstrated that the process improves the production of oil-free GUVs even at low temperatures and physiological salt concentrations. PEGylated lipid and sugar were found to synergistically improve GUV formation. Finally, we demonstrate the successful enzymatic synthesis of RNA within oil-free GUVs that were prepared on ice.

Keywords: Flow cytometry; GMV, giant multilamellar vesicle; GUV; GUV, giant unilamellar vesicle; Gentle hydration; Liposome; Membrane protein; Nile red, 9-(diethylamino)-5 H-benzo(α)phenoxazin-5-one; PEGylated lipid, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]; PSGH, PEGylated-lipid-and-sugar-doped gentle hydration; Synthetic biology.

Graphical abstract

Fig. 1

Outline of PEGylated lipid-and-sugar-doped gentle hydration (PSGH).

Fig. 2

Phase contrast (A) and fluorescence (B) microscopy of GVs prepared by PSGH. Arrowheads indicate myelin-like multilamellar vesicles. Scale bars are 20 μm.

Fig. 3

Plots of peripheral red fluorescence intensity versus diameter of thin-walled GVs. (A) A reference preparation containing uni-, bi-, and trilamellar vesicles. (B) A sample of vesicles prepared by PSGH. Error bars are standard error. Myelin-like multilamellar vesicles were excluded from analysis.

Fig. 4

Flow cytometry of GVs prepared by the PSGH method. (A) Phase contrast (left), and red (center) and green (right) fluorescence images of samples used in flow cytometry. Vesicle membranes and encapsulated aqueous pools were stained with a red (Nile red) and a green (Mag-Fluo-4) fluorescent dye, respectively. Scale bars are 10 μm. (B) Log–log density plot of fluorescence intensity, with red fluorescence intensity (I_Fred) on the horizontal axis and green fluorescence intensity (I_Fgreen) on the vertical axis. The slopes of the solid and dashed lines are 1.5 and 1.0, respectively.

Fig. 5

Phase contrast and fluorescence microscopy and flow cytometry of GV samples prepared by doping with either PEGylated lipid only (A, B, C) or sugar only (D, E, F). Scale bars are 20 μm. Horizontal and vertical axes are the red and the green fluorescence intensities, respectively, in arbitrary units. The slopes of the solid and dashed lines are 1.5 and 1.0, respectively.

Fig. 6

Encapsulated RNA synthesis in oil-free GUVs. (A) RNA synthesis was detected using the molecular beacon (MB) probe. F, green fluorescent dye; Q, quencher. (B) Reduction by RNAseH of fluorescence from unencapsulated reactions. RNA strands annealed to MB are specifically digested by RNaseH, and the probe spontaneously refolds back to the hairpin structure, in which fluorescence is quenched by FRET. (C) Phase contrast (left), red fluorescence (center) and green fluorescence (right) images of GUVs encapsulating transcription reactions. Scale bars are 20 μm. (D) Flow cytometry of vesicles with encapsulated enzymes. The slope of the red solid line is 1.5.