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For construction of a large‐sized DNA nanostructure by self‐assembly, rigid DNA building blocks are required. The first DNA building block, the double‐crossover (DX) motif, is one of the most essential and important inventions in DNA nanotechnology [7]. In the DX motif, two double‐stranded DNAs (dsDNA) are connected at two crossover points in parallel and antiparallel arrangements, which reduces the flexibility of the single dsDNA (Figure 1.2c). The two crossover points are separated by a defined number of base pairs. Using these DX tiles as building blocks, large nanostructures can be constructed via hybridization of the four sticky ends introduced to the DX tiles, which directs the self‐assembly into 2D nanostructures [10]. By using this strategy, 2D building blocks have been further developed for the preparation of various 2D tiles, such as triple‐crossover [11], triangular [12, 13], and 4 × 4 tiles [14]. This concept has also been extended to double‐helix bundled building blocks designed for the construction of tubular structures [15]. All the structures were constructed by simply using defined numbers of unmodified DNA strands. For further extension of the nanostructures, a more complicated design of the building blocks and sequences with larger numbers of DNA strands are needed.

In addition, mechanical DNA nanomachines with a controllable molecular system were developed. An extra sequence called a “toehold” is attached to the end of the DNA strand. Using this toehold, the DNA molecular machines are operated by adding and removing specific DNA strands for complex movements. When a DNA strand fully complementary to a toehold‐containing DNA is added, the initial complementary strand without the toehold is selectively removed by strand displacement [16]. The thermodynamic stabilization energy for hybridization works as “fuel” to provide the mechanical motion of the DNA molecular machine. Using this strategy, DNA tweezers that perform open–close motions were constructed (Figure 1.2d) [8]. Seeman and coworkers created a molecular machine combining DNA nanostructures. Using the helical rotation of dsDNA during the B–Z transition, in which the dsDNA conformation changes from a right‐handed (B‐form DNA) to a left‐handed (Z‐form DNA) conformation, a reciprocating motion of the DNA nanostructure was observed [17]. In addition, they developed molecular machines that perform 180° rotation at the ends of two adjacent dsDNAs, called PX‐JX₂ devices, by hybridization and removal of DNA strands (Figure 1.2e) [9]. Both the PX and JX₂ states were directly observed by atomic force microscopy (AFM). These dynamic systems were also introduced to DNA origami to operate DNA nanodevices (see Section 1.9).