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The geometry of double‐helical DNA allows for the design of 3D DNA origami structures by extending the 2D DNA origami system. Two strategies for preparing 3D DNA origami structures have been developed. One is the bundling of dsDNAs, where the relative positioning between adjacent dsDNAs is controlled by crossovers, and the other is the folding of 2D origami domains into 3D structures using interconnection strands. In the former method, developed by Shih and coworkers, the relative positioning of adjacent dsDNAs is geometrically controlled by the crossovers. By arranging the positions of the crossovers, tubular and multilayered structures were constructed (Figure 1.5a) [25]. By increasing or decreasing the number of base pairs between crossovers (in this case, 21 base pairs for two helical turns), the relative positional relationship between adjacent dsDNAs can be controlled. Using a rotational angle of 240° for seven base pairs, three adjacent dsDNAs are placed at a relative angle of ±120° with crossovers every 7 or 14 base pairs. By alternating this relative positioning between adjacent dsDNAs, duplexes form a pleated structure. When adjacent dsDNAs are placed to rotate in one direction, the contiguous duplexes finally form a six‐helix bundled tubular structure. Therefore, when some parts of the pleated structures are turned backward by the introduction of one‐directional rotation of adjacent dsDNAs, the structures fold to become a stacked layer structure (Figure 1.5a). In this case, to stabilize the 3D structures, adjacent layers of dsDNAs are further connected by crossovers of staple strands. Because of the complexity and high density of the introduced crossovers, accurate folding into the target 3D structure requires a long folding time. When the pleated structures were integrated as multilayered structures, the repeating units of the six‐helix bundled tubular structures formed a honeycomb lattice, which was viewed from the axial direction of the double helices. It was also possible to create more complex structures by perpendicularly joining these 3D structures (Figure 1.5b). In addition, a wireframe icosahedron structure was assembled from three double‐triangle monomers made of a six‐helix bundled tubular structure with connections. Importantly, caDNAno software, which is publicly available, has been developed to support the design of these 3D structures [28].

Furthermore, using the layered structures described above, new 3D structures were built by changing the helical twist from the average helical pitch of 10.5 bp/turn to either 10 or 11 bp/turn [29]. When dsDNAs with different helical pitches were bundled together, torque and repulsion between base pairs caused overall structural changes including twisting or 30–180° bending. Using these structures as building blocks, left‐handed or right‐handed helical ribbon structures were prepared. In addition, when angle‐controlled duplex bundles were connected to each other, a six‐tooth gear and a spherical wireframe capsule were created.

Figure 1.5 Design and construction of three‐dimensional DNA origami structures. (a) Scheme for folding the 2D pleated structure into a 3D multilayered structure using staple strands connecting adjacent layers. Sectional views of the positions of the crossovers in the multilayered structure sliced at seven‐base‐pair intervals. (b) Global twisted structures of six‐helix DNA bundles obtained by the selective deletion or insertion of nucleotides to change the helical turns from the normal 10.5 base pairs to 10 or 11 base pairs. TEM images of the polymerized ribbons containing 10.5‐, 10‐, and 11‐base‐pair helical pitches.

Source: Douglas et al. [25]/with permission of Springer Nature.

(c) DNA box structure by folding of six DNA origami rectangles using interconnection strands introduced at the edges of rectangles. The DNA box model reconstructed from cryo‐EM images.

Source: Andersen et al. [26]/with permission of Springer Nature.

(d) Spherical shells, ellipsoidal shells, and nanoflask DNA origami using combination of curved dsDNAs.

Source: Han et al. [27]/with permission of American Association for the Advancement of Science.

Using a different strategy, a DNA box structure was created by folding multiple 2D origami domains with interconnecting strands [26]. Six independent rectangles were sequentially linked and were designed to be folded using interconnection strands in a programmed fashion (Figure 1.5c). Analyses of the assembled structure by AFM, cryo‐electron microscopy, dynamic light scattering, and small‐angle X‐ray scattering indicated that the size was close to the original design. The lid of the box could be opened using a specific DNA strand to release the closing duplex by strand displacement, and the opening event was monitored by fluorescence resonance energy transfer (FRET). Other types of DNA boxes have been created using a similar method, which can control both the inside and outside by adjusting the directions of the crossovers at the connection edges [30, 31]. A tetrahedral structure was designed and constructed from four aligned origami triangles, which were preconnected with an M13 scaffold strand without folding independent 2D plates [32]. Using the strategy of folding 2D origami structures, we designed and prepared new hollow triangular, square, and hexagonal prism structures [33]. The opening event of these prism structures was observed in real‐time and characterized using high‐speed AFM.

Yan and coworkers [27] created more complex rounded 3D structures, such as spheres by using a combination of curved dsDNAs (Figure 1.5d). By designing and arranging the nanorings, positions of crossovers and helical pitches for preparing the curvatures of the nanoring structures were examined. For the preparation of planar curvature, concentric rings of DNA were prepared by rationally designed geometries and crossover networks. In addition, nonplanar curvatures were created by adjusting the position and pattern of crossovers between adjacent dsDNAs to change the helical pitches from the native B‐form twist. Finally, round‐shaped 3D nanostructures such as spherical shells, ellipsoidal shells, and a nanoflask were created.