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Direct observation of target molecules is a straightforward approach to understanding the physical properties of biomolecules in living systems. To facilitate the observation of single biomolecules, a versatile DNA origami scaffold is often needed for the precise analysis of interactions and reactions [4, 5, 52]. Using this method, detailed dynamics of functional molecules can be visualized. In the past decade, visualization of molecular movements during biological reactions at the subsecond timescale has been achieved using HS‐AFM [53–58]. Combining DNA origami system and HS‐AFM imaging, dynamic movement of molecules during enzymatic reactions, DNA structural changes, DNA photoreactions, DNA catalytic reactions, and RNA interactions has been imaged at the single‐molecule level (Figure 1.9) [62]. Target‐oriented design of DNA origami nanostructures and improvements to HS‐AFM imaging techniques have allowed these imaging and detection systems to be extensively used to elucidate the physical properties of individual molecules, assemblies, and structures involved in both biological and non‐biological events.

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.