Science.
(d) Single chemical reaction on DNA origami. Reactive groups (azido, amino, and alkyne groups) were incorporated into the DNA origami by conjugation with staple DNA strands. The coupling reactions were then performed using the biotin‐attached functional groups. The completion of the reactions was visualized by the binding of streptavidin. (e) AFM images of the three individual reactions and three successive reactions by the treatment of three biotin‐attached functional groups. Yields are presented below the AFM images.
Source: Voigt et al. [49]/with permission of Springer Nature.
Figure 1.8 (a) Schematic illustration of pinching of a target molecule.
Source: Kuzuya et al. [50]/with permission of Springer Nature.
(b) AFM images for streptavidin (SA) pinching by biotinylated DNA pliers. The dominant form of DNA pliers in Mg2+ solution before SA addition (left) was a cross. After SA addition (right), DNA pliers selectively pinched one SA tetramer and closed into the parallel closed form. Scale bars 300 nm.
Source: Kuzuya et al. [50]/with permission of Springer Nature.
(c) Mechanochemical sensing in optical tweezers using DNA origami nanostructures. A connected seven‐tile DNA origami with six probes is tethered between two optically trapped beads through dsDNA handles. Each tile has 39.5 nm × 27 nm in dimension. The adjacent tiles are locked (marked 1–6) by an aptamer DNA and its complementary strand. Unlocking of tiles by the target binding to an aptamer lock. (d) Real‐time observation of the target binding events in the constant‐force detection strategy. Upon switching to the target solution, the binding of the target unlocked the tiles, leading to the extension jumps (arrowheads).
Source: Koirala et al. [51]/with permission of John Wiley & Sons, Inc.
1.7 Application to Single Biomolecule AFM Imaging
1.7.1 High‐Speed AFM‐Based Observation of Biomolecules
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.
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
