Minimizing Cholesterol-Induced Aggregation of Membrane-Interacting DNA Origami Nanostructures

Abstract

DNA nanotechnology provides methods for building custom membrane-interacting nanostructures with diverse functions, such as shaping membranes, tethering defined numbers of membrane proteins, and transmembrane nanopores. The modification of DNA nanostructures with hydrophobic groups, such as cholesterol, is required to facilitate membrane interactions. However, cholesterol-induced aggregation of DNA origami nanostructures remains a challenge. Aggregation can result in reduced assembly yield, defective structures, and the inhibition of membrane interaction. Here, we quantify the assembly yield of two cholesterol-modified DNA origami nanostructures: a 2D DNA origami tile (DOT) and a 3D DNA origami barrel (DOB), by gel electrophoresis. We found that the DOT assembly yield (relative to the no cholesterol control) could be maximised by reducing the number of cholesterols from 6 to 1 (2 ± 0.2% to 100 ± 2%), optimising the separation between adjacent cholesterols (64 ± 26% to 78 ± 30%), decreasing spacer length (38 ± 20% to 95 ± 5%), and using protective ssDNA 10T overhangs (38 ± 20% to 87 ± 6%). Two-step folding protocols for the DOB, where cholesterol strands are added in a second step, did not improve the yield. Detergent improved the yield of distal cholesterol configurations (26 ± 22% to 92 ± 12%), but samples re-aggregated after detergent removal (74 ± 3%). Finally, we confirmed functional membrane binding of the cholesterol-modified nanostructures. These findings provide fundamental guidelines to reducing the cholesterol-induced aggregation of membrane-interacting 2D and 3D DNA origami nanostructures, improving the yield of well-formed structures to facilitate future applications in nanomedicine and biophysics.

Promoting Cell Fusion by Polyvalent DNA Ligands

“…Anchoring DNA ligands onto cell membrane via the hydrophobic insertion of cholesterol has emerged as a widely used technique. − One big challenge associated with this technique is that cholesterol may quickly disassociate from the membrane due to the dynamic nature of hydrophobic interaction, leading to the loss of anchored ligands, , which largely hinders its subsequent applications. To circumvent this challenge, herein, we designed PDLs by using programmable DNA self-assembly to enhance the anchoring stability of DNA ligands on cell membrane and to strengthen ligand interaction by polyvalency, which in theory will lead to better clustering and fusion efficiencies.…”

Surfactant‐Assisted Purification of Hydrophobic DNA Nanostructures

“…Two methods have been reported to avoid the aggregation. One method involves the elongation of DNA strands from the vicinity of hydrophobic molecules [36–39] . Ohmann et al.…”

“…However, the ssDNA elongation approach is limited to certain elongation positions, which decreases the design variation of DNA nanostructures. Another method involves using surfactants [38,40–42] . Mixing surfactants, such as sodium dodecyl sulfate (SDS), [40,41] octyl‐β‐ d ‐glucopyranoside, [38] and n ‐octyl‐oligo‐oxyethylene, [42] to a solution with hydrophobic DNA nanostructures prevented the formation of aggregates.…”

“…Another method involves using surfactants [38,40–42] . Mixing surfactants, such as sodium dodecyl sulfate (SDS), [40,41] octyl‐β‐ d ‐glucopyranoside, [38] and n ‐octyl‐oligo‐oxyethylene, [42] to a solution with hydrophobic DNA nanostructures prevented the formation of aggregates. This suggests that using surfactants provides a method to purify DNA nanostructures with hydrophobic molecules.…”

DNA Nanotechnology on Bio-Membranes

“…Singh et al [ 1 ] optimized design parameters to minimize cholesterol-induced aggregation of membrane-interacting DNA origami nanostructures. Cholesterol-modified DNA origami has been widely used for bio-membrane engineering and modification.…”