DNA Origami. Группа авторов

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3 Capturing Structural Switching and Self‐Assembly Events Using High‐Speed Atomic Force Microscopy

       Yuki Suzuki

       Frontier Research Institute for Interdisciplinary Sciences, Tohoku University, Sendai, Japan

3.1 Introduction

      Atomic force microscopy (AFM) is a powerful tool for imaging individual bio‐macromolecules [1, 2]. In particular, its range of operation has been suitable for the morphological analysis of nucleic acid molecules ranging from a single DNA fragment (several tens of nanometers) to a large assembly of DNA nanostructures (several hundred nanometers to a few micrometers). Initial attempts to use the instrument for imaging DNA molecules date back to the early days of AFM. Throughout the late 1980s to the 1990s, it was notably applied to observe double‐stranded DNA [3–6], and new preparation methods for nucleic acid specimens amenable to AFM imaging were developed [7, 8]. These achievements greatly encouraged investigation of various DNA–protein assemblies [9, 10] and artificially designed DNA nanostructures [11]. In particular, in the field of structural nucleic acid nanotechnology, AFM has been routinely used to visualize various types of artificial DNA nanostructures, such as DNA tile motifs [11], DNA origamis [12], and DNA bricks [13], and it has become an almost indispensable tool.

Compared with electron microscopy, AFM offers the advantage of being able to measure the surface topography of specimens without the need for chemical staining or fixation. Furthermore, its capability to scan samples in an aqueous solution offers the potential to directly image dynamic movements of biomolecules at nanoscale resolution under user‐defined buffer solutions; however, this type of application had been limited for a long time due to the inherently slow scanning rate of conventional AFM, which takes several minutes to produce a single image. It should be mentioned that although the conventional instrument cannot capture rapid molecular motions, dynamic events involving structural changes of the molecule of interest can often be interpreted even with the slow scanning rate by comparing “before” and “after” images. However, it is clearly more ideal to see a single molecule in action or dynamic events involving numbers of molecules in the same imaging area in real time. High‐speed AFM (HS‐AFM) introduced by Ando realized a scan rate of >1 frame/second (fps) [14], and it has been applied to unravel structural–function relationships of a variety of biological molecules, including proteins, protein–protein complexes, and protein–nucleic acid complexes [15–17]. HS‐AFM is now also employed in the field of structural DNA nanotechnology for the development of DNA nanomachines and studies of dynamic processes of artificial self‐assembly systems made up of DNA nanostructures. This chapter focuses on how the time‐lapse AFM technique has been utilized to study structural changes of DNA origami nanomachines, dynamic processes of two‐dimensional (2D) DNA origami lattice self‐assembly, and morphological changes of other DNA origami‐based dynamic systems.

3.2 DNA Origami Nanomachines

      Although a variety of DNA‐based molecular machines had already been realized before the invention of the scaffolded DNA origami method [18–21], its excellent shape designability [12, 22, 23] brought remarkable progress in the development of DNA nanodevices and DNA nanorobots [24]. Representative examples include a nano‐sized box capable of being opened by reacting with a specific “key” DNA strand [25], pH‐ or photoresponsive nanocapsules [26, 27], and a capsule‐shaped nanorobot that recognizes specific proteins on the specific cell surface to expose its cargos [28]. In addition to these container‐like structures, researchers have attempted to imitate normal‐sized mechanical parts, such as bearings, sliders, and hinges with DNA origami [29–31]. Coordinated operations that combine multiple mechanical units have also been examined [30]. These mechanical motions are often regulated by strand displacement reactions [18], DNAzyme‐mediated cleavage [32], triplex formations [27], and quadruplex formations such as a guanine quadruplex (G‐quadruplex) and i‐Motifs [29, 33]. It is noteworthy that all of these can, in principle, be realized with natural nucleobases, exhibiting the great advantages of DNA as a material that not only enables the design of arbitrarily shaped nanostructures but also allows us to design responses to stimuli such as ions, pH changes, or small molecules, using only four fundamental nucleotides.

3.3 Ion‐Responsive Mechanical DNA Origami Devices

HS‐AFM is often applied to monitor quick mechanical motions triggered by ions and small molecules. One successful example is visualizing the configuration switching of DNA origami nanoscissors, which has blunt ends on the self‐shape‐complementary recession–protrusion patterns on the surface [34] (Figure 3.1). Since DNA is a negatively charged polymer, there is electrostatic repulsion between DNA helices; however, this repulsion can be weakened by increasing the concentration of divalent cations (such as Mg²⁺), enabling interaction between the shape‐complementary interfaces, which are further stabilized by blunt‐end stacking. Based on this mechanism, the multilayer DNA origami nanoscissors exhibit switching behavior from open to closed and vice versa were developed [35] (Figure 3.1a). For the direct visualization of Mg²⁺ concentration‐dependent changes, the open nanoscissors were first adsorbed onto a mica surface and imaged at an Mg²⁺ concentration of 5 mM. While the scanned area was visualized, buffer containing a high concentration of MgCl₂ was injected into the observation system, so that the final concentration of MgCl₂ was 20 mM. The increase in Mg²⁺ concentration led the nanoscissors to switch from an open to a closed formation (Figure 3.1b). The reverse change was subsequently investigated by decreasing Mg²⁺ concentration by adding Mg‐free buffer solution to the observation buffer, resulting in the nanoscissors opening again. This example demonstrates the potential of time‐lapse AFM to directly visualize continuous reversible structural changes on the single nanostructure level.

Figure 3.1 DNA origami nanoscissors exhibiting open/closed switching in response to Mg²⁺ concentration. (a) Schematic drawing of the nanoscissors with shape‐complementary recession‐protrusion patterns. (b) Time‐lapsed AFM images of nanoscissors depicting Mg²⁺ concentration‐dependent configuration switching. The concentration of MgCl₂ was changed while keeping the same area scanned: Left, 5 mM; middle, 20 mM; right, 7 mM. Note that 5 mM of NaCl was also added to the observation buffer to weaken the electrostatic interaction between the nanoscissors and mica surface. The dashed‐circled NSs exhibited reversible switching from open to close and vice versa.

      Source: Willner et al. [34]/with permission from John Wiley & Sons, Inc.

Na⁺‐ or K⁺‐responsive DNA origami nanodevices are often realized by employing G‐quadruplex formations [29]. The bending molecular actuator that undergoes large deformation in response to K⁺ was constructed by designing serially