DNA Origami. Группа авторов

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СКАЧАТЬ each cavity [45]. The Zn‐finger proteins directly bound to the cavity containing the target sequence with 50–80% yield. In addition, GFP‐fused Zn‐finger proteins retained sequence‐specific recognition ability, albeit with a lower binding affinity. These results show that proteins can be directly targeted to specific sites using sequence‐selective Zn‐finger proteins. In addition, a sequence‐specific DNA recognition molecule, pyrrole‐imidazole polyamide was able to recognize and bind to the target sequences on DNA origami [46, 47]. We used a DNA origami structure with five cavities, into which five different sequences were incorporated. We visualized the selective alkylation of a biotinylated polyamide to the target sequence with streptavidin labeling. Using this method, the polyamide was found to alkylate the target sequence in 88% yield by discriminating one‐base mismatches. Selective alkylation and subsequent streptavidin labeling revealed the sequence selectivity of the polyamide at the single‐molecule level.

      1.5.3 Distance‐Controlled Enzyme Reactions and Photoreactions

      Yan and coworkers created a distance‐dependent enzymatic cascade on a DNA origami surface. Individual pairs of glucose oxidase (GOx) and HRP were placed at specific positions on the DNA origami with controlled spacing (Figure 1.6c) [37]. The distances between the enzymes were systematically changed from 10 to 65 nm, and their activities were evaluated. Two different distance‐dependent kinetics were observed between the assembled enzyme pairs, and by incorporating the intermediate protein, the activity was enhanced due to the hydration shells.

      Stein and coworkers performed a combination of multistep energy transfer in a photonic wire‐like structure using an energy‐transfer cascade [38]. Fluorophores that allow alternative energy‐transfer pathways to proceed, depending on the incorporation of a jumper dye, were arranged on a DNA origami surface (Figure 1.6d). An input dye (ATTO488), two output dyes (red fluorophore ATTO647N and IR fluorophore Alexa 750), and two jumper dyes (ATTO565) were placed onto three helices to minimize fluorophore interactions throughout the DNA molecule. Single‐molecule four‐color FRET by laser excitation was used in this study. As designed, the energy‐transfer pathways from blue to red or blue to IR dyes were successfully controlled at the single‐molecule level by the presence of the jumper dyes, which directed the excited‐state energy from the input dye to the output dyes. These results indicate that DNA origami might serve as a circuit board for photonic devices beyond the diffraction limit and at the molecular scale.

      These studies show that molecules and nanoparticles can be selectively incorporated into DNA origami, and the enzymatic cascade reactions and energy transfer pathways were controlled in a distance‐ and position‐dependent manner. These systems are relatively easily constructed by the placement of proteins via a corresponding ligand and hybridization of DNA with functional molecules and nanoparticles onto the addressable DNA origami nanostructures.

1.6 Single‐Molecule Detection and Sensing using DNA Origami Structures

      1.6.1 Single‐Molecule RNA Detection

Yan 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.

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