Cholesterol-doped Gear DNA origami: Self-assembly, characterization and lipid membranous interactions

In this study, we report the assembly and imaging of Gear-shape DNA Origami (DO) with nine cholesterol-terminated oligonucleotide sequences for anchoring to a lipid membrane, and six fluorescent dye molecules for visualization in confocal microscopy. Following the folding process using an established protocol, transmission electron microscopy (TEM) confirmed the consistent creation of DO structures and established that the inclusion of cholesterol and fluorescent tags does not hinder the assembly of DO structures. To assess DO adsorption on lipid membranes using confocal microscopy, we mixed DO structures, both with and without cholesterol anchors, with giant unilamellar vesicles (GUVs). In the absence of cholesterol anchors, the dye molecules exhibited a uniform distribution of fluorescence in the surrounding solution, whereas GUVs had dim interiors and minimal localized green fluorescence at their outer membrane boundary. In contrast, in the presence of cholesterol-anchored DO there was a sharp increase toward the outer limits of the GUVs. Based on this visual evidence, it can be inferred that the DO, along with their cholesterol anchors, exhibit a significant propensity to adsorb onto the GUVs in substantial quantities. Moreover, it can be observed that the interiors of the GUVs lack illumination, which suggests that DO does not permeate through the membrane. In parallel, calorimetry and electrophysiology techniques are used to quantify cholesterol-doped DO interactions with lipid bilayers.