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DNA origami has enabled the construction of a wide variety of 2D structures approximately 100 nm in size, including rectangles, triangles, and even a smiley face and five‐pointed star (Figure 1.3) [6]. In this method, a long single‐stranded DNA (M13mp18; 7249 nucleotides) and sequence‐designed complementary strands (called “staple strands”; most of which are 32‐mer) are mixed and then annealed from 95 °C to room temperature over two hours, resulting in the formation of target structures by self‐assembly (Figure 1.3a). The structure can be imaged by AFM, and the assembled structure formed according to a design. To create 2D DNA origami structures, adjacent dsDNAs should be connected to each other via a crossover. In this design, the geometry of the double helices involved has three helical rotations for 32 base pairs (Figure 1.3b). For example, two neighboring crossovers of the central dsDNA in an arrangement of three adjacent dsDNAs should be located at the opposite sites (rotated at 180°, 0.5 turns); therefore, the crossovers should be separated by 16 base pairs (1.5 turns). This rule should be preserved to maintain stable planar structure when placing multiple staple strands on the scaffold. DNA origami structures are formed using many different staple strands, so DNA hairpins can be placed as markers at any position on the surface of the DNA structure. A hairpin DNA (dumbbell‐type) used as a topological marker was observed as a dot by AFM imaging (Figure 1.3c). In this case, hairpins are placed perpendicular to the surface of the origami; therefore, each hairpin should be placed at a position eight base pairs from the crossover (270° rotation). The distance between the centers of the adjacent staples is approximately 6 nm, so the adjacent hairpins can be observed as different spots according to the spatial resolution of AFM. Using the hairpin markers, patterns, such as the map of a hemisphere (Figure 1.3c), can be displayed precisely on the DNA origami surface.

Figure 1.2 DNA nanotechnology before the emergence of DNA origami. (a) DNA double helix structure, base pair, and double‐stranded DNA (dsDNA). (b) Holliday junction structure, four‐way junction, and conceptual diagram for construction of 2D structure.

Source: Modified from Seeman [1].

(c) Double crossover structure, in which two dsDNAs are connected by four‐way branched strands (crossover; arrows). Two‐dimensional periodic structure was formed by self‐assembly using two double‐crossover components (A‐tile and B‐tile* with hairpin) with sticky ends (complementary single‐stranded DNAs at the ends). AFM image of the self‐assembled 2D nanostructure.

Source: Modified from Winfree et al. [10]

(d) Dynamic open/close behavior of DNA tweezers operated by strand displacement using toehold containing DNA strands.

Source: Modified from Yurke et al. [8].

(e) PX‐JX₂ device to exchange the bottom part of by insertion and removal of the strands. The structures can be observed in AFM images.

Source: Yan et al. [9]/with permission of Springer Nature.

Figure 1.3 DNA origami. (a) Method to prepare a DNA origami structure from the template single‐stranded DNA and staple strands. (b) Design of a self‐assembled DNA origami structure and geometry of the incorporated double‐stranded DNAs. Colored strands and a gray/black strand represent staple strands and template single‐stranded DNA, respectively. Staple strands connect the adjacent duplexes with crossovers. Inset: Structure of hairpin DNA for a topological marker. (c) Design and AFM images of self‐assembled DNA origami structures. Drawing of a hemisphere on the DNA origami with hairpin DNAs (white dots) and an AFM image of the assembled DNA origami.

Source: Rothemund [6]/with permission of Springer Nature.