DNA Origami. Группа авторов

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СКАЧАТЬ DNA methylation can be regulated using tension‐controlled dsDNAs constructed in the DNA frame. DNA base excision repair enzymes, 8‐oxoguanine glycosylase [65], and T4 pyrimidine dimer glycosylase [66], also show that the relaxed substrate dsDNAs were preferable for the reactions. The DNA frame system and direct observation serve to elucidate the detailed properties of the modifying enzymes and these events.

Direct observation of DNA recombination was carried out by incorporating the substrate sequences into the DNA frame (Figure 1.9c) [61, 67, 68]. Using Cre recombinase, the Cre–DNA complex and recombinant products were clearly observed in the DNA frame, demonstrating that recombination occurred in the nanospace. Lapsed HS‐AFM images showed that the Cre–DNA complex formed first, followed by the complex dissociating into four monomers, and the simultaneous appearance of the recombinant product. In addition, the structural stress imposed on the Holliday junction (HJ) intermediates in the DNA frame can regulate the direction of recombination.

      Using the HJ‐containing DNA frame and Rec U resolvase, the resolution event was visualized in the DNA frame [69]. We also visualized the binding preference and the activity of the HJ‐resolvase monokaryotic chloroplast 1 (MOC1 in Arabidopsis thaliana) using HS‐AFM [70]. The interaction of MOC1 with the center of the HJ and symmetric cleavage of the HJ structure were observed in the DNA frame. Observation of geometric arrangements of substrate dsDNAs using DNA frames is valuable for studying recombination events.

      Using a DNA origami scaffold and HS‐AFM system, important DNA conformational changes including G‐quadruplex formation [59, 71], photo‐induced duplex formation [72], triple helix formation [73], G‐quadruplex/i‐motif formation [74], and B–Z transition [60] have been successfully imaged. This method can be extended to the direct observation of various enzymes and reaction events, such as DNA‐modifying enzyme [62], repair enzymes [75], recombinase [61, 76], resolvase [69, 71], Cas9 [70, 77], TET [78], DNA recognition [79, 80], and RNA interactions [81]. Using the DNA frame for the incorporation of substrates in various arrangements, the enzyme reactions can be visualized and regulated in the DNA frame to study the reaction mechanisms. The observation system can be used as a general strategy for investigating various DNA structural changes and molecular switches working at the single‐molecule level.

1.8 Single‐Molecule Fluorescence Studies

      The ability to study single‐molecule events using DNA origami extends beyond chemical and biochemical reactions. DNA origami allows for control of the distance between fluorescence dyes, which can be applied for the precise labeling of molecules and read‐out by super‐resolution microscopy. A method called DNA‐PAINT (DNA Points Accumulation for Imaging in Nanoscale Topography) has been developed using DNA origami as a scaffold for positioning fluorescent dyes.

      1.8.1 Nanoscopic Ruler for Single‐Molecule Imaging

Recent developments in fluorescence microscopy have enabled the resolution of images below the diffraction limit of the sub‐200 nm scale for optical analysis. For precise measurement of the distance between the fluorescent dyes, the optical resolution of super‐resolution microscopic techniques needs to be calibrated. Actin filaments, microtubules, and short duplex DNA molecules are used to demonstrate optical resolution, but they are disadvantageous because of their flexibility. DNA origami offers a novel nanostructure with a defined size and can be easily immobilized on a surface in a fixed orientation for single‐molecule analysis. These features turn DNA origami structures into a nanoscopic ruler for the calibration of super‐resolution imaging techniques [82]. Different super‐resolution methods, such as single‐molecule high‐resolution imaging with photobleaching (SHRImP), direct stochastic optical reconstruction microscopy (dSTORM), and blink microscopy, have been 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.

Figure 1.10 DNA‐PAINT super‐resolution imaging. (a) DNA origami tile with two fluorescent dye‐labeled staple strands (F in black circle) (left). TIRF image of origami tiles each containing two ATTO655‐labeled staple strands (middle). Super‐resolution image of the same region using blink microscopy (right). Scale bar: 500 nm. (b) Intensity vs time profile (left) and the statistical distribution of the measured distance (right).

      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 СКАЧАТЬ