rel="nofollow" href="#ulink_8e24605f-61e4-5314-a5ac-43c0f4bc7af4">Figure 1.10d). Images and distance distribution histograms revealed that the fitted distances for adjacent dyes and the outer dyes were 111 ± 27 and 212 ± 44 nm, respectively, which is in good agreement with the initial design but slightly lower than the expected distance. These results indicate that, in addition to static analyses, dynamic processes in the subsecond range can be investigated on DNA origami in real time and are comparable with the results of ensemble measurements.
1.8.3 DNA Barcode Imaged by DNA‐PAINT
The identification and differentiation of a large number of distinct molecular species with high temporal and spatial resolution is a major challenge in biomedical science. Fluorescence microscopy is a powerful tool, but its multiplexing ability is limited by the number of spectrally distinguishable fluorophores. Yin and coworkers demonstrated the construction of submicrometer nanorods that can be used as fluorescent barcodes [84]. Barcodes can be modified easily to display sequence‐specific attachment sites through simple DNA‐origami staple extensions. Spatial control over the positioning of fluorophores was achieved on the surface of a DNA nanorod (Figure 1.10e). The fluorescent dye‐conjugated DNA strands (imager strands) transiently bind their specific target sites on the DNA nanorod, which produces “blinking” for super‐resolution reconstruction, similar to a previous report [82]. Using epifluorescence or TIRF microscopy, 216 distinct barcodes were unambiguous decoded. On the other hand, barcodes with higher spatial information density were demonstrated via the construction of super‐resolution barcodes (Figure 1.10f,g). One type of barcode was used to tag yeast surface receptors, which suggests their potential applications as in situ imaging probes for diverse biomolecular and cellular entities in their native environments.
1.9 DNA Molecular Machines
A controllable molecular system operated by specific DNA strands has been realized for the construction of DNA‐based nanomachines. DNA molecular machines are operated by exchanging specific DNA strands to create complex movements. For this purpose, an additional sequence called a “toehold” is attached to the end of the DNA strand. When a DNA strand that is fully complementary to a toehold‐containing strand is added, the initial toehold‐containing strand is selectively removed by strand displacement. The thermodynamic stabilization energy works as “fuel” during hybridization to provide the mechanical motion of DNA machines. Using this strategy, DNA tweezers that perform open–close motions were constructed (Figure 1.2) [8]. Two examples of a DNA walking device have been created: a DNA walker with two legs that can control its direction of motion and a DNA motor that can move forward autonomously by cleavage of a DNA‐nicking enzyme [16].
Seeman and coworkers developed molecular machines that are capable of rotating 180° at the ends of two adjacent dsDNAs, termed PX‐JX2 devices, by hybridization and removal of DNA strands [9]. They also successfully captured triangular DNA nanostructures using the sequence specificity of four single‐stranded ends by introducing two devices onto DNA origami and rotating each triangle [85]. Using this method, four types of PX‐JX2 patterns could be operated by specific DNA strands, and four different types of nanostructures were selectively captured.
1.9.1 DNA Assembly Line Constructed on the DNA Origami
Seeman and coworkers created an assembly line in which a DNA walker was operated on the DNA origami and captured multiple gold nanoparticles (AuNPs) (Figure 1.11a) [86]. They arranged the three PX‐JX2 devices and operated a DNA walker that moved on a predesigned track (route). All device movements and DNA walker movements were controlled by specific DNA strands. AuNPs of different sizes were placed on the PX‐JX2 devices and were transferred to the DNA walker by rotating motion at specific positions. These results show that the DNA walker moved on the track unidirectionally that the delivery of AuNPs was controlled by the PX‐JX2 devices, and that they were transferred to the DNA walker. After the completion of this process, DNA walkers with three different AuNPs bound were obtained with a yield of 43%. As the PX‐JX2 device can control the ON–OFF switching of the transfer of AuNPs using specific DNA strands, the product was obtained in high yield (>90%), and the error rate was suppressed (1%). In addition, eight patterns of capture of AuNPs onto the DNA walker were achieved using three PX‐JX2 devices with the ON–OFF switching operation of these devices.
1.9.2 DNA Spider System Constructed on the DNA Origami
Stojanovic and coworkers created DNA nanomachines called “DNA spiders” on various path patterns constructed on DNA origami (Figure 1.11b) [87]. The DNA spiders consisted of three DNA strand legs and one capture DNA strand (capture leg). The three legs of the DNA spiders each contained a DNA enzyme (DNAzyme leg) that could hydrolyze RNA. Single‐stranded DNAs containing a cleavable RNA site were arranged on DNA origami as a track for the DNA spider to walk. The DNA spider was immobilized at a specific position on the DNA origami using a trapping DNA strand, followed by dissociation and initiation of walking on the track. The DNA spider bound to the ssDNAs on DNA origami, cleaved the RNA site in the ssDNAs using its DNAzyme function, and walked autonomously. The DNA spider moved forward along the predetermined track and finally stopped at a specific point on the DNA origami where ssDNAs did not have an RNA site for cleavage. All of these processes, including initiation, walking, and stopping, were controlled in a programmed manner. In addition, by measuring the position of the DNA spider on DNA origami using super‐resolution microscopy, it was found that the spider moved at a speed of 3 nm/min.
Figure 1.11 Assembly line with a DNA walker capturing gold particles and DNA spider molecule walking in a track on a DNA origami. (a) The DNA walker binds to the DNA strand on the DNA origami with three legs, and gold particles (AuNPs) are collected with three hands.
Source: Gu et al. [86]/with permission of Springer Nature.
The DNA walker stops at three places on DNA origami and receives AuNPs (C1–C3) to be transferred by rotating PX‐JX2 DNA devices. Multiple operation on DNA origami and corresponding AFM image. (b) The DNA spider binds onto the DNA origami using three legs hybridized to ssDNAs (cleavage site contains RNA) in the track. DNAzyme for cleavage of RNA site in the ssDNA is introduced to three legs.
Source: Lund et al. [87]/with permission of Springer Nature.
DNA strands in the track before and after cutting (gray and light gray circles) and stopping DNA strands (right side circles). The path for walking with instruction (start, follow, turn, and stop) can be programmed on the DNA origami. AFM image of DNA spider molecule walking on the DNA origami track. Start (top), walking (middle), and stop (bottom). (c) A DNA motor system created on the DNA origami. A motor‐track (gray ssDNAs) was constructed on the DNA origami and the movement of the DNA motor (black ssDNA) was examined.
Source: Wickham et al. [88]/with permission of Springer Nature.
Stepwise movement of a DNA motor observed in real time by high‐speed AFM.
1.9.3
