LITMIR.BIZ

Скачать книгу

and coworkers incorporated single‐stranded DNAs to detect target RNA molecules at the single‐molecule level on the surface of DNA origami (Figure 1.7a–c) [48]. Even though samples containing large amounts of cell‐derived RNAs were used, the binding of the target RNA could only be visualized by AFM, and nonspecific binding was not observed. Because DNA origami tiles carry different types of complementary DNAs and corresponding hairpin DNA markers, binding of target RNAs could be identified from the specific hairpin markers on the DNA origami even though the different origami tiles were mixed. In this study, the detection limit of RNA molecules was approximately 1000 molecules, meaning that the target RNA could be directly detected from a single cell without using polymerase chain reaction (PCR) amplification.

      1.6.2 Single‐Molecule Detection of Chemical Reactions

      Gothelf and coworkers detected selective bond cleavage and bond formation reactions on a DNA origami surface. Target molecules having specific reactivity were introduced at specific positions on DNA origami. Reductive cleavage of disulfide bonds and oxidative cleavage of an olefin by singlet oxygen were carried out on the DNA origami surface, and the reactions proceeded quantitatively at the single‐molecule level [49]. In addition, amide bond formation and click reactions were performed with 80–90% yield, and three successive reactions were also performed (Figure 1.7d,e). These chemical reactions were monitored by the cleavage of biotin‐attached chemical linkers and bond formation with biotin‐tethered functional groups, which can be labeled with streptavidin for visualization by AFM.

      1.6.3 Single‐Molecule Detection using Mechanical DNA Origami

Kuzuya and coworkers developed a versatile sensing system to detect a variety of chemical and biological targets at molecular resolution by using nanomechanical DNA devices [50]. They designed functional nanomechanical DNA origami devices that can be used as “single‐molecule beacons” and function as pinching devices (Figure 1.8a). Using “DNA origami pliers” and “DNA origami forceps,” which consist of two levers ~170 nm long connected at a fulcrum, various single‐molecule targets ranging from metal ions to proteins could be detected by observing a shape transition of the DNA origami devices using AFM (Figure 1.8b). Any detection mechanism suitable for the target of interest, pinching, zipping, or unzipping can be chosen and used orthogonally with differently shaped origami devices in the same mixture using a single platform.

      1.6.4 Single‐Molecule Sensing using Mechanical DNA Origami

Mao and coworkers developed a strategy for the detection of a single biomolecule using a DNA origami nanostructure as a mechanochemical platform [51]. A connected seven‐tile DNA origami was designed and six sensing probes were incorporated at different locations on the tiles (Figure 1.8c). Platelet‐derived growth factor (PDGF) was used as a target molecule, and binding of the target to the aptamer induces dehybridization of the complementary strand, opens the lock, and finally induces an expansion of approximately 15 nm (Figure 1.8d). Binding of a target molecule to these probes induces rearrangement of the origami nanostructure, which is monitored in real time using optical tweezers. Without PDGF, no recognition events were observed. This platform can detect 10 pM PDGF within 10 minutes, while the PDGF and a DNA target were differentiated and identified in a multiplexing fashion. The results show that this mechanochemical platform could offer a solution for high‐throughput sensing at the single molecular level.

Figure 1.7 Detection of target RNA by hybridization with probe DNA strands introduced on the DNA origami. (a) Method for imaging the hybridization of target RNA to a probe DNA on the DNA origami. (b) Multiple DNA probes complementary to the target RNAs were introduced onto the DNA origami, and hairpin DNAs were also introduced as an index for identifying the probe strand. (c) AFM images of binding of target RNA to the probe strands. Specific DNA probes can be identified by the corresponding index.

      Source: Ke et al. [48]/with permission of American Association for the Advancement of 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.

Скачать книгу