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The development of the structural DNA nanotechnology has enabled the construction of almost arbitrarily shaped 2D and 3D DNA nanostructures, which can be further assembled into micrometer‐sized higher order structures based on sticky‐ended interactions and blunt‐ended stacking interactions [35, 49–55]. However, assembling flexible structures such as 2D DNA origami structures into a uniform large‐scale structure in the solution phase is not always an easy task and often causes the formation of undesired aggregates. An effective solution for this problem is a surface‐assisted self‐assembly, in which molecules to be assembled are adsorbed onto the substrate surface [56, 57]. Adsorption to the surface suppresses the flexibility of the structures and increases their effective concentration, promoting large‐scale self‐assembly along the two‐dimensional surface. The key to the success of this surface‐assisted self‐assembly is ensuring intricate “not too strong but not too weak” adsorption conditions that allow two‐dimensional diffusion of molecules on the substrate surface.

DNA origami is generally prepared in a buffer solution containing Mg²⁺ at about 10–20 mM. However, in such solution conditions, DNA origami is strongly adsorbed onto the surface of the mica substrate, which is standard substrate for AFM observation, and does not show 2D diffusion. Therefore, it is necessary to moderately weaken the sample–surface interaction by adding several hundred millimolars of NaCl to the buffer solution [58–60].

As an alternative approach, changing the substrate properties while keeping the buffer conditions is also conceivable. Two‐dimensionally expanded lipid bilayer membranes offer a flat surface and also have attractive features for supporting the surface‐assisted self‐assembly. Their fluidities and surface charges can be purpose‐tuned by adjusting the composition of lipid molecules. It is also noteworthy that the orientation of a DNA origami structure on the lipid membrane can be predefined by modifying a specific site of the DNA origami structure with hydrophobic groups [61–63].

Figure 3.4 shows an example of a 2D‐DNA origami lattice assembled from cross‐shaped DNA origami structures as components. In this example, a lipid bilayer membrane of dioleoyl phosphatidylcholine (DOPC) is first formed on a mica substrate by the vesicle fusion method [65] and then the DNA origami structures are electrostatically adsorbed on the membrane surface [64] (Figure 3.4a). The cross‐shaped origamis concentrated on the surface bind to each other by stacking interactions at the ends of the cross structure and assemble into a micrometer‐sized 2D lattice. Successive time‐lapse AFM images revealed a variety of fundamental processes of the lattice growth (Figure 3.4b,c). In particular, it was observed that dynamic rearrangement of origami occurred near each boundary of adjacent sublattices. The association and dissociation of monomers or small oligomers took place repeatedly at gaps between lattices to produce “matched” boundaries. Meanwhile, excess origamis located in between those boundaries were expelled to allow complete connection. The compensation and repair of point defects in the lattice were also captured. These direct observations clearly demonstrate the involvement of dynamic trial‐and‐error interactions in the growing process.

Figure 3.4 Dynamic events involved in a lipid bilayer‐assisted self‐assembly of cross‐shaped DNA origami into 2D lattices and close‐packed 2D crystalline structures. (a) Schematic drawing of the lipid‐bilayer‐assisted self‐assembly. (b–d) Time‐lapsed AFM images depicting lattice fusion (b), defect filling (c), and defect diffusion (d). Defects are indicated with yellow triangles. Scale bar = 200 nm.

Source: Suzuki et al. [64]/Springer Nature/CC BY 4.0.

Other interesting events revealed by HS‐AFM imaging were defect diffusion and defect healing occurring in close‐packed 2D crystalline structures assembled on the DOPC lipid bilayer. Figure 3.4d shows successive AFM images of a crystalline structure obtained from close packing of the cross‐shaped DNA origami structure whose blunt ends were inactivated by adding polyT tails. The defect arose at around 105 seconds, exhibited diffusion in the crystalline structure, and seemed to be filled up probably with a monomer in the observation buffer solution, demonstrating how the 2D crystalline structures are maintained at the interface between the fluid and lipid membrane.