DNA Origami - Группа авторов

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used to demonstrate that fluorescent dye‐labeled staple strands immobilized at specific positions on a DNA origami exhibit a defined separation. Two dye‐modified strands were placed in a rectangular origami structure at a distance of 89.5 nm (Figure 1.10a). The immobilized samples were imaged using total internal reflection fluorescence (TIRF) microscopy. The emission patterns of the two fluorophores overlapped in the diffraction‐limited image (Figure 1.10a, left image). However, super‐resolution imaging using blink microscopy allowed for the identification of the positions of individual fluorophores (Figure 1.10a, right image), and the measured distance between the dyes was in good agreement with the original design (Figure 1.10b). This origami nanoscopic ruler was also used for single‐molecule imaging and calibration of other super‐resolution imaging techniques, such as SHRImP and dSTORM. In addition to the 2D origami ruler, a rigid 3D DNA origami ruler was developed for single‐molecule FRET spectroscopic analysis.

Schematic illustration of DNA-PAINT super-resolution imaging.

      Source: Steinhauer et al. [82]/with permission of American Chemical Society.

      (c) The design of origami tile with a dye at the corner and a docking strand in the middle. The binding of a red dye modified imager strand to the docking strand. The fluorescence intensity vs time trace of the binding and unbinding event is shown. (d) Diffraction‐limited TIRF and super‐resolved DNA‐PAINT images of triple labeled oligomers with 129.5 nm separation. Distance distribution histogram of triple‐labeled DNA origami (length scale, 120 nm).

      Source: Jungmann et al. [83]/with permission of American Chemical Society.

      (e) Super‐resolution fluorescent barcodes. Schematic illustration of barcodes for DNA‐PAINT super‐resolution imaging. The DNA nanorod consists of four binding zones (for binding of red, green or blue imager strand) separated by 113 nm. (f) A segment diagram of the nanorod monomers used to create five barcodes. (g) Super‐resolution image of the mixture containing five barcodes in (f). The inset shows the diffraction‐limited image of a barcode.

      Source: Lin et al. [84]/with permission of American Chemical Society.

      1.8.2 Kinetics of Binding and Unbinding Events and DNA‐PAINT

      Fluorescence microscopy was successfully applied to study the kinetics of dynamic DNA binding and dissociation [82]. For kinetic analysis, a long rectangular DNA origami structure was used to incorporate a green label dye (ATTO532) at a corner and a docking strand was positioned in the middle of the rectangle (Figure 1.10c) [83]. The addition of a red dye (ATTO655)‐modified imager strand led to the formation of a duplex with the complementary docking strand. The formation of the duplex structure was monitored and the kinetics of the binding and unbinding events were determined. The association rate was calculated to be 2.3 × 106 M/s (for 600 mM NaCl), which is comparable with the results of bulk measurements. In contrast, the dissociation rate was independent of the concentration, but strongly dependent on the length of the duplex formed by the imager and docking strands. The dissociation rate was estimated at 1.6 and 0.2 s–1 for 9 and 10 base pairs, respectively. The distance between multiple fluorescence dyes on DNA origami was precisely measured using DNA‐PAINT (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.

      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

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