observation of DNA structural change and enzyme reactions using high-speed AFM."/>
Figure 1.9 Direct observation of DNA structural change and enzyme reactions using high‐speed AFM. (a) Visualization of G‐quadruplex formation using the structural change of two dsDNAs placed in a DNA frame. In the presence of KCl, the separated state changes to the X‐shape by connection at the center of two dsDNAs via G‐quadruplex formation. Scanning rate 0.2 frame/s.
Source: Sannohe et al [59]/with permission of American Chemical Society.
(b) B–Z transition observed in the DNA frame. Two dsDNAs having a (5meCG)6 sequence (B–Z system; upper), and a random sequence (control; lower) with a flag marker were introduced in the DNA frame. HS‐AFM images of the flipping motion of the flag marker at the upper site (yellow arrow). Scanning rate 0.2 frame/s.
Source: Rajendran et al. [60]/with permission of American Chemical Society
(c) Cre‐mediated DNA recombination observed in the DNA frame. Successive HS‐AFM images of the dissociation of the Cre tetramer from the dsDNAs into four Cre monomers and the appearance of a recombinant product. Scanning rate 1.0 frame/s.
Source: Suzuki et al. [61]/with permission of American Chemical Society.
By using the robust DNA origami structure as a scaffold for AFM observation, we visualized and analyzed the movement of biomolecules including proteins and enzymes when the substrate dsDNAs are attached to the origami scaffold. In addition, the physical properties of a target dsDNA such as tension, rotation, and orientation of dsDNA can be controlled in the designed nanospace constructed in the DNA origami structures.
1.7.2 Visualization of DNA Structural Changes in the DNA Nanospace
The formation and disruption of a single G‐quadruplex structure were observed in nanospace [59]. To observe G‐quadruplex formation, two dsDNA strands containing single‐strand G‐rich overhangs in the middle of an interstrand G‐quadruplex were attached to a DNA frame (Figure 1.9a). Three G‐tracts were placed in the upper G‐strand, whereas the lower strand had a single G‐tract [63]. In the presence of K+, the formation of an interstrand G‐quadruplex was observed in 44% yield (X‐shape). The dynamic formation of the G‐quadruplex was directly observed in real time using HS‐AFM. During scanning of the sample in the presence of K+, the two G‐strands maintained a separated state for a given period, then spontaneously formed the G‐quadruplex which was observed as an X‐shape. In a similar fashion, we observed the disruption of G‐quadruplexes in the absence of K+. The X‐shape was unchanged for a period of time, then separated under AFM scanning.
In addition, we directly visualized the rotary motion of a B–Z conformational transition in the DNA frame [60]. To visualize the B–Z transition, dsDNA containing a 5‐methyl‐CG (5meCG) six‐repeat sequence (B–Z system; upper strand) and a flag marker containing three‐helix‐bundled DNA connected by crossovers was introduced to the DNA frame (Figure 1.9b). During the B–Z transition, the flag marker rotates around the dsDNA shaft, and the rotary motion can be observed by monitoring the position of the marker. By controlling the concentration of Mg2+ ions under equilibrium conditions for the B–Z transition, the movement of the flag marker in the B–Z system was observed during HS‐AFM scanning. The change in height of the flag marker could also be observed, further indicating that rotation around the dsDNA shaft occurred in the B–Z transition system.
1.7.3 Visualization of the Reaction Events of Enzymes and Proteins in the DNA Nanospace
DNA modification using enzymes often requires bending specific DNA strands to facilitate the reaction. Using tense and relaxed dsDNAs incorporated in the DNA frame, relaxed dsDNA can be a better substrate for the DNA methylation enzyme EcoRI methyltransferase, which requires bending of dsDNA for the methyl‐transfer reaction [62, 64]. Methylation preferentially occurred in the relaxed dsDNA, indicating the importance of structural flexibility in the bending of dsDNA during the methyl‐transfer reaction. Therefore, 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),