LITMIR.BIZ

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the rotating motion itself, single‐molecule HS‐AFM imaging has revealed a variety of transient states appearing under thermal fluctuations which are sometimes difficult to identify from static AFM images.

3.5 Two‐Dimensional Self‐Assembly Processes

      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.

3.6 Sequential Self‐Assembly

One of the applications of the ordered DNA origami arrays is its use as a “scaffold” to organize a variety of particles such as proteins [58, 64, 66], metal inorganic particles [53], and other nanoscale objects. In the example shown in Figure 3.5, the internal cavities of a preassembled two‐dimensional DNA origami lattice were used to incorporate “supplemental” DNA origami nanostructures with shapes that were complementary to those of the cavities [67]. The lattice was first self‐assembled from cross‐shaped DNA origamis on a mica‐supported DOPC lipid bilayer membrane and subsequently exposed to square‐shaped origamis. Successive time‐lapse AFM at 10 mM Mg²⁺ revealed the squares have a dynamic adsorption/desorption behavior, wherein arrangements in the assembled lattice contentiously changed, suggesting that they indeed accessed the cavities, but were not stably trapped in them (Figure 3.5a).

      This dynamic feature revealed by HS‐AFM provided a clue that allowed a chequerboard‐like pattern to be derived from the lattice structure via sequential self‐assembly (Figure 3.5b). To realize this pattern derivation, a two‐dimensional lattice wherein every other cavity has polyT strands was first self‐assembled from two types of cross‐shaped DNA origamis. Then, the square origami carrying polyA strands at its four corners was loaded onto the preassembled lattice. The polyA‐modified squares that entered correct positions (cavities with 8T strands) could be docked in the cavity by sticky‐ended cohesion despite insufficient adsorption onto the membrane surface, whereas those that entered false positions (cavities without 8T linkers) could desorb from the cavity. Hence, the square origami would be finally incorporated only in the correct positions to make a chequerboard‐like pattern as demonstrated in HS‐AFM images (Figure 3.5c).

Figure 3.5 Placing square‐shaped DNA origami into lipid bilayer‐supported 2D lattices. (a) Dynamic docking events of square‐shaped origamis into the 2D lattice cavities recorded at a scan rate of 0.2 fps. Scale bar = 200 nm. (b) Schematic representation of the sequential self‐assembly and directed placement of square‐shaped origamis to produce a chequerboard‐like pattern. The streptavidin‐modified cross A and cross B were self‐assembled into a framework structure. Topographic AFM images of the assembled chequerboard‐like pattern is also shown. (c) Dynamic rearrangement of square‐shaped origami on the false positions (dashed circles) and docking into a correct position (dashed square). Edge reorganization was also occurred in the area enclosed in orange dotted lines. Scale

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