Sorting sub-150-nm liposomes of distinct sizes by DNA-brick-assisted centrifugation

Abstract

In cells, myriad membrane-interacting proteins generate and maintain curved membrane domains with radii of curvature around or below 50 nm. To understand how such highly curved membranes modulate specific protein functions, and vice versa, it is imperative to use small liposomes with precisely defined attributes as model membranes. Here, we report a versatile and scalable sorting technique that uses cholesterol-modified DNA 'nanobricks' to differentiate hetero-sized liposomes by their buoyant densities. This method separates milligrams of liposomes, regardless of their origins and chemical compositions, into six to eight homogeneous populations with mean diameters of 30-130 nm. We show that these uniform, leak-resistant liposomes serve as ideal substrates to study, with an unprecedented resolution, how membrane curvature influences peripheral (ATG3) and integral (SNARE) membrane protein activities. Compared with conventional methods, our sorting technique represents a streamlined process to achieve superior liposome size uniformity, which benefits research in membrane biology and the development of liposomal drug-delivery systems.

Figure 1.

DNA-brick-assisted liposome sorting scheme and results. (a) Schematic diagrams of cholesterol-labeled DNA bricks. Note the absence of sticky ends on DNA bricks. (b) Steps for brick-assisted liposome sorting — liposome coating by DNA bricks, separation of DNA-coated liposomes by isopycnic centrifugation, and removal of DNA bricks from the sorted liposomes. A monochromic fluorescence image of 12 fractions recovered after centrifugation (Step II) shows the spread of liposomes in the density gradient. (c) A plot showing buoyant densities of naked and DNA-brick coated liposomes of various sizes. The theoretical values were calculated assuming the buoyant density, footprint, and molecular weight of a six-helix bundle DNA brick to be 1.7 g/cm³, 189 nm² and 189 kD, respectively, and only meant to illustrate the general trends of liposome density versus size in the presence and absence of DNA coating. (d) Negative-stain TEM images (top) and size distributions (bottom, shown as D=mean±SD, n=390, 251, 1350 from left to right) of liposomes produced by extrusion through polycarbonate filters with 200 nm and 50 nm pores as well as by sonication. (e) A 1:1:1 mixture of extruded (through 200 nm and 50 nm pores) and sonicated liposomes are sorted into distinct sizes with the help of the six-helix-bundle DNA bricks. Representative negative-stain TEM images are shown above the corresponding histograms (shown as D=mean±SD, n=164, 353, 515, 1690, 1240, 1375 from left to right) fitted by Gaussian functions. Liposomes are made of ~59.2% 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 30% 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 10% 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), and 0.8% 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (rhodamine-DOPE). Scale bars: 100 nm.

Figure 2.

Sorting liposomes containing self-cleaving deoxyribozymes. (a) A schematic drawing of the leakage assay used to assess membrane permeability. Fluorescein-labeled deoxyribozymes undergo site-specific hydrolysis when exposed to Zn²⁺ outside of the liposomes. (b) Representative TEM images of sorted liposomes containing deoxyribozymes. Fraction numbers (e.g., F6) and liposome diameters (mean±SD, n=212, 436, 391, 376, 555, 621 from left to right) are noted above the corresponding images. Scale bar: 100 nm. (c) A plot showing the deoxyribozyme-to-lipid ratios in sorted liposomes fitted via linear regression (dashed line). (d) Permeability of liposomes characterized by SDS-polyacrylamide gel electrophoresis following the deoxyribozyme-based leakage assay. Prior to the leakage assay, free deoxyribozymes were removed from unsorted liposomes by isopycnic centrifugation without DNA-brick coating. Pseudo-colors: Cy5 (on DNA bricks) = yellow; fluorescein (on deoxyribozymes) = blue; rhodamine (on liposomes) = red. Liposomes are made of 59.2% DOPC, 30% DOPE, 10% DOPS, and 0.8% rhodamine-DOPE. Liposomes prepared in the same experiment were loaded in two gels that were run and processed in parallel. This experiment was repeated 3 times with similar results.

Figure 3.

Atg3-catalyzed GL1 lipidation reaction studied using uniform-size liposomes. (a) Schematic illustrations of GL1-DOPE conjugate (left) and the expected lipidation outcomes on liposomes with differential membrane curvatures (right, radii of curvature = 70 nm, 43 nm, and 18 nm). (b) GL1-lipidation efficiencies on extruded, sonicated and sorted liposomes (59.2% DOPC, 30% DOPE, 10% DOPS, and 0.8% rhodamine-DOPE) characterized by gel electrophoresis (top row, stained by Coomassie Blue) and immunoblot against GL1 with an antibody that preferentially recognizes the GL1-PE conformation (bottom row). The numbers (in nm) above lanes represent the nominal pore size of the filters (extruded liposomes) or measured mean diameters (sorted liposomes). Reaction mixtures from the same experiment were loaded in two gels that were run and processed in parallel. This experiment was repeated 5 times with similar results (see Supplementary Fig. 28).

Figure 4.

SNARE-mediated membrane fusion studied using uniform-size liposomes. (a) A schematic illustration of the lipid-mixing assay used to monitor membrane fusion. Initially quenched NBD dyes (green) fluoresce following membrane fusion due to a decrease in FRET with rhodamine dyes (magenta). SNARE proteins are shown as blue, yellow (t-SNAREs) and green (VAMP2, v-SNARE) ribbons on the membranes. Models of liposomes, proteins and dyes are not drawn to scale. (b) Left: representative fluorescence traces showing the kinetics of fusion between unsorted liposomes bearing t-SNAREs and unsorted (red) or sorted (different shades of blue, diameters marked as mean±SD, n=603, 488, 345, 477 from top to bottom) liposomes bearing v-SNAREs. Protein-free liposomes are mixed with v-SNARE bearing liposomes as a negative control (black). The solid curves are a guide to the eye. Right: representative TEM images of sorted and unsorted v-SNARE-bearing liposomes. Scale bar: 200 nm. Liposomes with v-SNAREs are reconstituted with 82% 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 12% DOPS, 1.5% Rhodamine-DOPE, 1.5% 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2–1,3-benzoxadiazol-4-yl) (NBD-DOPE), and a lipid:protein molar ratio of 200:1. Liposomes with t-SNAREs are reconstituted with 58% POPC, 25% DOPS, 15% 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 2% phosphatidylinositol 4,5-bisphosphate (PIP2) and a lipid:protein molar ratio of 400:1. (c) v-SNARE density on sorted and unsorted liposomes reconstituted with lipid:VAMP2 molar ratios of 200:1 (two trials, labeled with a and b), 300:1, and 400:1. Mean liposome diameters are measured from TEM images for unsorted liposomes and all fractions of sorted liposomes with initial lipid:VAMP2 ratio of 200:1, but only for 3 (out of 7) fractions of those with initial lipid:VAMP2 ratio of 300:1 or 400:1. Sizes of other fractions are interpolated. (d) Lipid mixing after 2 hours of fusion reactions (measured by NBD fluorescence, as shown in (b)) plotted against the average diameters of sorted v-SNARE-bearing liposomes. (e) Correlation between NBD fluorescence and v-SNARE density. (f) Rounds of fusion that sorted v-SNARE-bearing liposomes undergo in 2 hours (see Methods for details). (g) v-SNARE copy numbers per liposome measured from sorted liposomes. (h) Correlation between rounds of fusion and v-SNARE copy number per liposome. (i) The rates for rounds of fusion (between 30 and 60 min) normalized by surface area of v-SNARE-bearing liposomes and the corresponding v-SNARE copy numbers, as a function of mean diameter of sorted liposomes. (j) Rounds of fusion normalized by surface area of v-SNARE-bearing liposomes and the corresponding v-SNARE copy numbers, as a function of the mean curvature (1/radius) of sorted liposomes.