Fig. 4

Experimental demonstration of lipid scrambling by DNA nanostructures in lipid vesicles and human cells. a Schematic illustration of dithionite reduction assay. POPC (gray) vesicles containing a fraction of NBD-labeled lipids (green) are incubated with DNA constructs tagged with Cy3 dye (red spheres). Upon dithionite addition, NBD fluorophores are irreversibly reduced (black). The nanostructure design containing two cholesterol modifications (2C, top) allows for membrane insertion to induce lipid scrambling whereas the constructs with one cholesterol (1C, bottom) do not. b Confocal fluorescence microscopy images of GUVs containing NBD fluorophores (green) and incubated with Cy3-labeled DNA nanostructures (red) showing the same vesicle before and 35 min after dithionite addition roughly at the equatorial plane. The third column displays a merged image of the red and green channels. Scale bars are 10 µm. c Graphs showing four representative fluorescence intensity traces of NBD fluorescence reduction over time for both 1C and 2C designs. Values have been normalized to the initial intensity per vesicle and aligned to the onset of dithionite reduction. d Histograms of residual NBD fluorescence intensity at 35 min after dithionite addition normalized to the initial intensity for each vesicle. Data were obtained from four (2C) and one (1C) experiments using the same GUV stock solution. Black arrows and dashed lines in c and d highlight the shifted fluorescence intensity values for 2C nanostructures in contrast to the 1C control. Data shown were collected from vesicles with a diameter above 6 µm (for smaller vesicles see Supplementary Fig. 12). e, g Schematic illustration of FITC-labeled annexin V binding assay without (e) and with membrane-inserted DNA nanostructures (g). Phosphatidylserine (PS) lipids are highlighted in light blue. f, h Confocal microscopy images of fixed cells after incubation with DNA folding buffer only (f) or with Cy3-labeled 2C DNA scramblases (h). While no annexin V binding occurs in the negative control, incubation with DNA scramblases showed cell attachment of the DNA nanostructures (red) associated with increased annexin V (green) binding (see merged signal of both channels) due to PS exposure. Corresponding bright field images illustrate cell locations (scale bar is 20 µm)