Figure 2.

Characterization of DNA origami-templated SUVs containing v-tethers.

(A) SDS-agarose gel for the templated vesicles. Gel runs from the top to the bottom as in the images. Fractions obtained after equilibrium density gradient separation (from the Fraction 1, “F1” on the top layer of the density gradient to the Fraction 12 on the bottom layer) were loaded on the gel. As shown in the images, Fraction 1 contained liposomes without ring complexes, Fraction 2 contained the ring-templated SUV, and later fractions contained little amount of detectable substances. Aggregates were absent in all the fractions. Limited by the space, gel images were shown till fraction 4 for each preparation. Fraction 2 (“F2” in gel image) was collected for further measurement in the fusion assay. The color of the product bands gradually changed from pure blue to pure red, with the number of VAMP2 (conjugated to anti-handle i-Alexa647, coded as red) increasing from 0 to 16, and the number of balancing lipid anchors (conjugated to anti handle ii-Alexa488, coded as blue) decreasing from 16 to 0. (B–C) Transmission Electronic Microscopy (TEM) characterization of the final templated vesicles. (B) Representative TEM images with a selection of zoomed-in images (100 nm × 100 nm). Scale bar, 100 nm. (C) Size distribution of the vesicles on ring-templates. The measured mean size of vesicles was 39.1 ± 7.7 nm in diameter (N = 110 vesicles). The solid black line represents a Gaussian fit for the size-distribution histogram. The solid blue line represents a Gaussian fit for the size-distribution histogram for vesicles prepared by standard detergent-dialysis method that contained v-tethers and v-SNAREs with a measured mean diameter of 54.5 ± 16.3 nm (N = 370 vesicles).