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Both ligand binding and nanocavity can increase the stability of a biomolecular structure. Using mechanical unfolding in optical tweezers, here we found that a DNA origami nanobowl drastically increased the stability of a human telomeric G-quadruplex bound with a pyridostatin (PDS) ligand. Such a stability change is equivalent to >4 orders of magnitude increase (upper limit) in binding affinity (Kd: 490 nM → 10 pM (lower limit)). Since confined space can assist the binding through a proximity effect between the ligand-receptor pair and a nanoconfinement effect that is mediated by water molecules, we named such a binding as mechanochemical binding. After minimizing the proximity effect by using PDS that can enter or leave the DNA nanobowl freely, we attributed the increased affinity to the nanoconfinement effect (22%) and the proximity effect (78%). This represents the first quantification to dissect the effects of proximity and nanoconfinement on binding events in nanocavities. We anticipate these DNA nanoassemblies can deliver both chemical (i.e. ligand) and mechanical (i.e. nanocavity) milieus to facilitate robust mechanochemical binding in various biological systems.

Stimuli-responsive switching molecules have been widely investigated for the purpose of the mechanical control of biomolecules. Recently developed arylazopyrazole (AAP) shows photoisomerization activity, displaying a faster response to light-induced conformational changes and unique absorption spectral properties compared with those of conventionally used azobenzene. Herein, it is demonstrated that AAP can be used as a photoswitching molecule to control photoinduced assembly and disassembly of DNA origami nanostructures. An AAP-modified DNA origami has been designed and constructed. It is observed that the repeated assembly and disassembly of AAP-modified X-shaped DNA origami and hexagonal origami with complementary strands can be achieved by alternating UV and visible-light irradiation. Closed and linear assemblies of AAP-modified X-shaped origami were successfully formed by photoirradiation, and more than 1 μm linear assemblies were formed. Finally, it is shown that the two photoswitches, AAP and azobenzene, can be used in tandem to independently control different assembly configurations by using different irradiation wavelengths. AAP can extend the variety of available wavelengths of photoswitches and stably result in the assembly and disassembly of various DNA origami nanostructures.

The modular architecture of naturally occurring ribozymes makes them a promising class of structural platform for the design and assembly of three-dimensional (3D) RNA nanostructures, into which the catalytic ability of the platform ribozyme can be installed. We have constructed and analyzed RNA nanostructures with polygonal-shaped (closed) ribozyme oligomers by assembling unit RNAs derived from the Tetrahymena group I intron with a typical modular architecture. In this study, we dimerized ribozyme trimers with a triangular shape by introducing three pillar units. The resulting double-decker nanostructures containing six ribozyme units were characterized biochemically and their structures were observed by atomic force microscopy. The double-decker hexamers exhibited higher catalytic activity than the parent ribozyme trimers.

The micrometer-scale assembly of various DNA nanostructures is one of the major challenges for further progress in DNA nanotechnology. Programmed patterns of 1D and 2D DNA origami assembly using specific DNA strands and micrometer-sized lattice assembly using cross-shaped DNA origami were performed on a lipid bilayer surface. During the diffusion of DNA origami on the membrane surface, the formation of lattices and their rearrangement in real-time were observed using high-speed atomic force microscopy (HS-AFM). The formed lattices were used to further assemble DNA origami tiles into their cavities. Various patterns of lattice–tile complexes were created by changing the interactions between the lattice and tiles. For the control of the nanostructure formation, the photo-controlled assembly and disassembly of DNA origami were performed reversibly, and dynamic assembly and disassembly were observed on a lipid bilayer surface using HS-AFM. Using a lipid bilayer for DNA origami assembly, it is possible to perform a hierarchical assembly of multiple DNA origami nanostructures, such as the integration of functional components into a frame architecture.

Ribozymes with modular structures are attractive platforms for the construction of nanoscale RNA objects with biological functions. We designed group I ribozyme dimers as unit ribozyme dimers (Urds), which self-assembled to form their polymeric states and also oligomeric states with defined numbers of Urds. Assembly of Urds yielded catalytic ability of a pair of distinct ribozyme units to cleave two distinct substrates. The morphologies of the assembled ribozyme structures were observed directly by atomic force microscopy (AFM).

We report the use of DNA origami nanostructures, functionalized with aptamers, as a vehicle for delivering the antibacterial enzyme lysozyme in a specific and efficient manner. We test the system against Gram‐positive (Bacillus subtilis) and Gram‐negative (Escherichia coli) targets. We use direct stochastic optical reconstruction microscopy (dSTORM) and atomic force microscopy (AFM) to characterize the DNA origami nanostructures and structured illumination microscopy (SIM) to assess the binding of the origami to the bacteria. We show that treatment with lysozyme‐functionalized origami slows bacterial growth more effectively than treatment with free lysozyme. Our study introduces DNA origami as a tool in the fight against antibiotic resistance, and our results demonstrate the specificity and efficiency of the nanostructure as a drug delivery vehicle., Antibiotic resistance is a growing health issue that is now rendering humans vulnerable once again to infections that have been treatable for decades. Various approaches have been proposed to overcome this threat and effectively treat bacterial infections. DNA nanostructures, functionalized with aptamers, were used as a vehicle for delivering the antibacterial enzyme lysozyme in a specific and efficient manner, to destroy bacterial targets.

DNA nanostructures are expected to be applied for targeted drug delivery to immune cells. However, the structural properties of DNA nanostructures required for the delivery have not fully been elucidated. In this study, we focused on the DNA density that can be important for the their recognition and uptake by immune cells. To examine this, DNA nanostructures with almost identical molecular weights and structural flexibility, but with different shapes and DNA densities, were designed using DNA origami technology. We compared the following five types of DNA nanostructures, all of which consisted of ten DNA helices using an identical circular, single-stranded scaffold and staples. Rec180 had a rectangular-shaped, almost flat structure. Rec90, Rec50 and Rec0 were bent forms of Rec180 at the center by 90, 50 or 0 degrees, respectively. Rec50/50 has two bends of 50 degrees each so that the both ends stick together to form a triangular prism shape. The fluctuation, or flexibility, of these DNA nanostructures under solution conditions was estimated using CanDo software. The DNA density estimated from the average distance between any two of the ten DNA helices in the DNA nanostructures was different among them; Rec50, Rec0 and Rec50/50 had a higher density than Rec180 and Rec90. Agarose gel electrophoresis and atomic force microscopy showed that all of the nanostructures were prepared with high yield. Flow cytometry analysis revealed that the uptake of DNA nanostructures by murine macrophage-like RAW264.7 cells was higher for those with higher DNA density than those with low density. There was a positive correlation between the density and cellular uptake. These results indicate that DNA nanostructures with high DNA density are suitable for delivery to immune cells.

The nascent field of DNA nanotechnology has undergone rapid growth since its inception. By using DNA as a biologically "safe" material, DNA nanotechnology shows great promise in applications such as drug-delivery systems. Further progress, however, relies on understanding the different ways in which DNA nanostructures behave in and interact with cells, tissues and even whole organisms. Moreover, this knowledge must then be harnessed in innovative ways to improve existing DNA nanostructures and design new ones, so that they can perform more diverse functions more effectively. There have been many developments in this regard in the past few years, and herein some of these are highlighted, with respect to both works that improve our understanding of what happens to DNA nanostructures once they are at their target site, and those that utilise clever design to accomplish desired functions.

At the cellular level, numerous nanocues guide the cells to adhere, interact, proliferate, differentiate, etc. Understanding and manipulating the cellular functions in vitro, necessitates the elucidation of these nanocues provided to the cells by the extracellular matrix (ECM), neighbouring cells or in the form of ligands. DNA nanotechnology is a biocompatible, flexible and a promising molecular level toolkit for mimicking cell-cell and cell-matrix interactions. In this review, we summarize various advances in cell-matrix, cell-cell and cell receptor-ligand interactions using DNA nanotechnology as a tool. We also provide a brief outlook on the current challenges and the future potentials of these DNA-based nanostructures so as to inspire novel innovations in the field.

We demonstrated the photo-controlled sequence-selective dsDNA cleavage using a DNA origami structure with Cas9 nuclease. The activity of Cas9 incorporated inside the ring-shaped DNA origami was completely suppressed. After photoirradiation, Cas9 was released, and its activity for selective dsDNA cleavage was restored.

RNA-protein (RNP) complexes are promising biomaterials for the fields of nanotechnology and synthetic biology. Protein-responsive RNA sequences (RNP motifs) can be integrated into various RNAs, such as messenger RNA, short-hairpin RNA, and synthetic RNA nano-objects for a variety of purposes. Direct observation of RNP interaction in solution at high resolution is important in the design and construction of RNP-mediated nanostructures. Here we describe a method to construct and visualize RNP nanostructures that precisely arrange a target protein on the RNA scaffold with nanometer scale. High-speed AFM (HS-AFM) images of RNP nanostructures show that the folding of RNP complexes of defined sizes can be directly visualized at single RNP resolution in solution.

Ribozymes with modular architecture constitute an attractive class of structural platforms for design and construction of nucleic acid nanostructures with biological functions. Through modular engineering of the Tetrahymena ribozyme, we have designed unit RNAs (L-RNAs), assembly of which formed ribozyme-based closed trimers and closed tetramers. Their catalytic activity was dependent on oligomer formation. In this study, the structural variety of L-RNA oligomers was extended by tuning their structural elements, yielding closed pentamers and closed hexamers. Their assembly properties were analyzed by electrophoretic mobility shift assay (EMSA) and atomic force microscopy (AFM).

Biological macromolecular machines perform impressive mechanical movements. F-adenosine triphosphate (ATP) synthase uses a proton gradient to generate ATP through mechanical rotations. Here, a programmed hexagonal DNA nanomachine, in which a three-armed DNA nanostructure (TAN) can perform stepwise rotations in the confined nanospace powered by DNA fuels, is demonstrated. The movement of TAN can precisely go through a 60° rotation, which is confirmed by atomic force microscopy, and each stepwise directional rotating is monitored by fluorescent measurements. Moreover, the rotary nanomachine is used to spatially organize cascade enzymes: glucose oxidase (GOx) and horseradish peroxidase (HRP) in four different arrangements. The multistep regulations of the biocatalytic activities are achieved by employing TAN rotations. This work presents a new prototype of rotary nanodevice with both angular and directional control, and provides a nanoscale mechanical engineering platform for the reactive molecular components, demonstrating that DNA-based framework may have significant roles in futuristic nanofactory construction.

Despite the importance of the interaction between DNA and cells for its biological activity, little is known about exactly how DNA interacts with cells. To elucidate the relationship between the structural properties of DNA and its cellular uptake, a single-stranded circular DNA of 1801 bases was designed and folded into a series of rectangular DNA (RecDNA) nanostructures with different rigidities using DNA origami technology. Interactions between these structures and cells were evaluated using mouse macrophage-like RAW264.7 cells. RecDNA with 50 staple DNAs, including four that were Alexa Fluor 488-labeled, was designed. RecDNA with fewer staples, down to four staples (all Alexa Fluor 488-labeled), was also prepared. Electrophoresis and atomic force microscopy showed that all DNA nanostructures were successfully obtained with a sufficiently high yield. Flow cytometry analysis showed that folding of the single-stranded circular DNA into RecDNA significantly increased its cellular uptake. In addition, there was a positive correlation between uptake and the number of staples. These results indicate that highly folded DNA nanostructures interact more efficiently with RAW264.7 cells than loosely folded structures do. Based on these results, it was concluded that the interaction of DNA with cells can be controlled by folding using DNA origami technology.

This work demonstrates single-molecule imaging of metal-ion induced double-stranded DNA formation in DNA nanostructures. The formation of the metal ion-mediated base pairing in a DNA origami frame was examined using C-Ag-C and T-Hg-T metallo-base pairs. The target DNA strands containing consecutive C or T were incorporated into the DNA frame, and the binding was controlled by the addition of metal ions. Double-stranded DNA formation was monitored by observing the structural changes in the incorporated DNA strands using high-speed atomic force microscopy (AFM). Using the T-Hg-T base pair, the dynamic formation of unique dsDNA and its dissociation were observed. The formation of an unusual shape of dsDNA with consecutive T-Hg-T base pairs was visualized in the designed nanoscale structure.

Recombinase A (RecA) is central to homologous recombination. However, despite significant advances, the mechanism with which RecA is able to orchestrate a search for homology remains elusive. DNA nanostructure-augmented high-speed AFM offers the spatial and temporal resolutions required to study the RecA recombination mechanism directly and at the single molecule level. We present the direct in situ observation of RecA-orchestrated alignment of homologous DNA strands to form a stable recombination product within a supporting DNA nanostructure. We show the existence of subtle and short-lived states in the interaction landscape, which suggests that RecA transiently samples micro-homology at the single RecA monomer-level throughout the search for sequence alignment. These transient interactions form the early steps in the search for sequence homology, prior to the formation of stable pairings at >8 nucleotide seeds. The removal of sequence micro-homology results in the loss of the associated transient sampling at that location., Graphical Abstract Graphical AbstractA schematic diagram depicting the homologous recombination reaction scheme within the DNA origami frame and associated HS-AFM micrographs. Left to right: the layout of the internal suspended DNA molecules (control, NPF and reaction) is shown in reference to an inbuilt polarity marker (black triangle). The RecA NPF (nucleoprotein filament) is formed on the 30 nt ssDNA region of the NPF DNA molecule and is connected into the DNA frame via a UV photocleavable linker (yellow star). Exposure to UV releases the NPF from its tether initiating homologous recombination. This culminates in the formation of a synaptic joint at the region of sequence homology (green) on the reaction DNA. HS-AFM image scale bar = 40 nm.

It is important to create new, specifically designed and controlled nanomaterials that can be used as molecular delivery systems for cells. Here, we describe a method for creating a nanosized DNA capsule (NC) using a photocaged unlocking system as a carrier for cell delivery. The photocaged NC (caged-NC) was designed and constructed to control the opening of the closed NC by photoirradiation. The opening of the NC was observed by atomic force microscopy, and the dynamic opening of the caged-NC was characterized by fluorescence quenching and recovery processes. The caged-NC was then introduced into the cytoplasm of a cell, where the photoinduced opening of the caged-NC was observed. The selective opening of the caged-NC in a single cell was successfully achieved by laser irradiation of individual cells. The caged-NC system could be used as a delivery system for relatively large nanomaterials in cells, similar to a native virus system.

We demonstrate direct observation of the dynamic opening and closing behavior of photocontrollable DNA origami nanoscissors using high-speed atomic force microscopy (HS-AFM). First the conformational change between the open and closed state controlled by adjustment of surrounding salt concentration could be directly observed during AFM scanning. Then light-responsive moieties were incorporated into the nanoscissors to control these structural changes by photoirradiation. Using photoswitchable DNA strands, we created a photoresponsive nanoscissors variant and were able to distinguish between the open and closed conformations after respective irradiation with ultraviolet (UV) and visible (Vis) light by gel electrophoresis and AFM imaging. Additionally, these reversible changes in shape during photoirradiation were directly visualized using HS-AFM. Moreover, four photoswitchable nanoscissors were assembled into a scissor-actuator-like higher-order object, the configuration of which could be controlled by the open and closed switching induced by irradiation with UV and Vis light.

Various DNA-based nanodevices have been developed on the nanometer scale using light as regulation input. However, the programmed controllability is still a major challenge for these artificial nanodevices. Herein, we demonstrate a rotary DNA nanostructure in which the rotations are controlled by light. A bar-shaped DNA rotor, fabricated as a stiff double-crossover molecule, was placed on the top of a rectangular DNA tile. The photoresponsive oligonucleotides modified with azobenzenes were employed as switching motifs to release/trap the rotor at specific angular position on DNA tile by switching photoirradiations between ultraviolet and visible light. As a result, two reconfigurable states (perpendicular and parallel) of rotor were obtained, in which the angular changes were characterized by AFM and fluorescence quenching assays. Moreover, the reversible rotary motions during the photoirradiation were directly visualized on the DNA tile surface in a nanometer-scale precision using a second-scale scanning of the high-speed AFM.

Bio-inspired functional microcapsules have attracted increasing attention in many fields from physical/chemical science to artificial-cell engineering. Although particle-stabilised microcapsules are advantageous for their stability and functionalisation potential, versatile methods for their functionalisation are desired to expand their possibilities. This study reports a water-in-oil microdroplet stabilised with amphiphilic DNA origami nanoplates. By utilising DNA nanotechnology, DNA nanoplates were designed as a nanopore device for ion transportation and to stabilise the oil-water interface. Microscopic examination revealed the microcapsule formed by the accumulation of amphiphilic DNA nanoplates at the oil-water interface. Ion current measurements revealed the nanoplate pores functioned as channel to transport ions. These findings provide a general strategy for the programmable design of microcapsules to engineer artificial cells and molecular robots.

Bimolecular ribozymes derived by physical dissection of unimolecular ribozymes consisting of two structural modules are promising platforms for the design and construction of assembled RNA nanostructures. Unit RNAs to be assembled intermolecularly into one-dimensional (1D) oligomers are designed by reconnecting the two structural modules in a manner different from the parent ribozymes. This strategy was applied to the Tetrahymena group I ribozyme. We constructed 1D ribozyme oligomers the assembly of which was observed by atomic force microscopy (AFM) and also controlled rationally to design a heterooctamer by differentiating the interface between the two modules.

In the present study, we demonstrate single-molecule imaging of triple helix formation in DNA nanostructures. The binding of the single-molecule third strand to double-stranded DNA in a DNA origami frame was examined using two different types of triplet base pairs. The target DNA strand and the third strand were incorporated into the DNA frame, and the binding of the third strand was controlled by the formation of Watson-Crick base pairing. Triple helix formation was monitored by observing the structural changes in the incorporated DNA strands. It was also examined using a photocaged third strand wherein the binding of the third strand was directly observed using high-speed atomic force microscopy during photoirradiation. We found that the binding of the third strand could be controlled by regulating duplex formation and the uncaging of the photocaged strands in the designed nanospace.

Direct visualization of the biomolecules of interest is a straightforward way to elucidate the physical properties of individual molecules and their reaction processes. Atomic force microscopy (AFM) enables direct imaging of biomolecules in suitable solution conditions. As AFM visualizes the molecules at a nanometer-scale spatial resolution, a versatile observation platform is required for precise imaging of the molecules in action. The DNA origami technology allows precise placement of target molecules in a designed nanostructure, enabling their detection at the single-molecule level. We used DNA origami technology for visualizing the detailed movement of target molecules in reactions using high-speed AFM (HS-AFM), which enables the analysis of dynamic movement of biomolecules with a subsecond time resolution. By combining the DNA origami system and HS-AFM, DNA conformational changes, including G-quadruplex formation and disruption and B-Z transition, were visualized. In addition, enzyme-based reactions such as DNA recombination were also visualized at the single-molecule level using this combined observation system. Moreover, the enzyme-based reaction could be directly regulated in the DNA origami frame by imposing structural stress on the substrate DNAs to elucidate the reaction mechanism. These target-orientated observation systems should contribute to a detailed analysis of biomolecular motions in real time at molecular resolution.

Due to the small size of a nanoconfinement, the property of water contained inside is rather challenging to probe. Herein, we measured the amount of water molecules released during the folding of individual G-quadruplex and i-motif structures, from which water activities are estimated in the DNA nanocages prepared by 5 × 5 to 7 × 7 helix bundles (cross-sections, 9 × 9 to 15 × 15 nm). We found water activities decrease with reducing cage size. In the 9 × 9-nm cage, water activity was reduced beyond the reach of regular cosolutes such as polyethylene glycol (PEG). With this set of nanocages, we were able to retrieve the change in water molecules throughout the folding trajectory of G-quadruplex or i-motif. We found that water molecules absorbed from the unfolded to the transition states are much fewer than those lost from the transition to the folded states. The overall loss of water therefore drives the folding of G-quadruplex or i-motif in nanocages with reduced water activities.

DNA can assemble various molecules and nanomaterials in a programmed fashion and is a powerful tool in the nanotechnology and biology research fields. DNA also allows the construction of desired nanoscale structures via the design of DNA sequences. Structural nanotechnology, especially DNA origami, is widely used to design and create functionalized nanostructures and devices. In addition, DNA molecular machines have been created and are operated by specific DNA strands and external stimuli to perform linear, rotational, and reciprocating movements. Furthermore, complicated molecular systems have been created on DNA nanostructures by arranging multiple molecules and molecular machines precisely to mimic biological systems. Currently, DNA nanomachines, such as molecular motors, are operated on DNA nanostructures. Dynamic DNA nanostructures that have a mechanically controllable system have also been developed. In this review, we describe recent research on new DNA nanomachines and nanosystems that were built on designed DNA nanostructures.

Genomic integrity, when compromised by accrued DNA lesions, is maintained through efficient repair via homologous recombination. For this process the ubiquitous recombinase A (RecA), and its homologues such as the human Rad51, are of central importance, able to align and exchange homologous sequences within single-stranded and double-stranded DNA in order to swap out defective regions. Here, we directly observe the widely debated mechanism of RecA homology searching at a single-molecule level using high-speed atomic force microscopy (HS-AFM) in combination with tailored DNA origami frames to present the reaction targets in a way suitable for AFM-imaging. We show that RecA nucleoprotein filaments move along DNA substrates via short-distance facilitated diffusions, or slides, interspersed with longer-distance random moves, or hops. Importantly, from the specific interaction geometry, we find that the double-stranded substrate DNA resides in the secondary DNA binding-site within the RecA nucleoprotein filament helical groove during the homology search. This work demonstrates that tailored DNA origami, in conjunction with HS-AFM, can be employed to reveal directly conformational and geometrical information on dynamic protein-DNA interactions which was previously inaccessible at an individual single-molecule level.

DNA has recently emerged as a promising material for the construction of nanosized architectures. Chemically modified DNA has been suggested to be an important component of such architectural building blocks. We have designed and synthesized a novel H-shaped DNA oligonucleotide dimer that is cross-linked with a structurally rigid linker composed of phenylene and ethynylene groups. A rotatable DNA unit was constructed through the self-assembly of this H-shaped DNA component and two complementary DNA oligonucleotides. In addition to the rotatable unit, a locked DNA unit containing two H-shaped DNA components was also constructed. As an example of an extended locked structure, a hexagonal DNA origami dimer and oligomer were constructed by using H-shaped DNA as linkers.

In nucleic acid nanotechnology, designed RNA molecules are widely explored because of their usability originating from RNA's structural and functional diversity. Herein, a method to design and prepare RNA nanostructures by employing DNA origami strategy was developed. A single-stranded RNA scaffold and staple RNA strands were used for the formation of RNA nanostructures. After the annealing of the mixtures, 7-helix bundled RNA tile and 6-helix bundled RNA tube structures were observed as predesigned shapes. These nanostructures were easily functionalized by introducing chemical modification to the RNA scaffolds. The DNA origami method is extended and utilized to construct RNA nanostructures.

In synthetic biology, the control of gene expression requires a multistep processing of biological signals. The key steps are sensing the environment, computing information and outputting products1. To achieve such functions, the laborious, combinational networking of enzymes and substrate-genes is required, and to resolve problems, sophisticated design automation tools have been introduced2. However, the complexity of genetic circuits remains low because it is difficult to completely avoid crosstalk between the circuits. Here, we have made an orthogonal self-contained device by integrating an actuator and sensors onto a DNA origami-based nanochip that contains an enzyme, T7 RNA polymerase (RNAP) and multiple target-gene substrates. This gene nanochip orthogonally transcribes its own genes, and the nano-layout ability of DNA origami allows us to rationally design gene expression levels by controlling the intermolecular distances between the enzyme and the target genes. We further integrated reprogrammable logic gates so that the nanochip responds to water-in-oil droplets and computes their small RNA (miRNA) profiles, which demonstrates that the nanochip can function as a gene logic-chip. Our approach to component integration on a nanochip may provide a basis for large-scale, integrated genetic circuits.

Molecular machines composed of RNA–protein (RNP) complexes may expand the fields of molecular robotics, nanomedicine, and synthetic biology. However, constructing and directly visualizing a functional RNP nanostructure to detect and control living cell function remains a challenge. Here we show that RNP nanostructures with modular functions can be designed and visualized at single-RNP resolution in real time. The RNP structural images collected in solution through high-speed atomic force microscopy showed that a single RNP interaction induces a conformational change in the RNA scaffold, which supports the nanostructure formation designed. The specific RNP interaction also improved RNA nanostructure stability in a serum-containing buffer. We developed and visualized functional RNPs (e.g., to detect human cancer cells or knockdown target genes) by attaching a protein or RNA module to the same RNA scaffold of an optimal size. The synthetic RNP architecture may provide alternative materials to detect and control functions in target mammalian cells.

Holliday junction (HJ) resolution is a fundamental step for completion of homologous recombination. HJ resolving enzymes (resolvases) distort the junction structure upon binding and prior cleavage, raising the possibility that the reactivity of the enzyme can be affected by a particular geometry and topology at the junction. Here, we employed a DNA origami nano-scaffold in which each arm of a HJ was tethered through the base-pair hybridization, allowing us to make the junction core either flexible or inflexible by adjusting the length of the DNA arms. Both flexible and inflexible junctions bound to Bacillus subtilis RecU HJ resolvase, while only the flexible junction was efficiently resolved into two duplexes by this enzyme. This result indicates the importance of the structural malleability of the junction core for the reaction to proceed. Moreover, cleavage preferences of RecU-mediated reaction were addressed by analyzing morphology of the reaction products.

Biological membranes fulfill many important tasks within living organisms. In addition to separating cellular volumes, membranes confine the space available to membrane-associated proteins to two dimensions (2D), which greatly increases their probability to interact with each other and assemble into multiprotein complexes. We here employed two DNA origami structures functionalized with cholesterol moieties as membrane anchors—a three-layered rectangular block and a Y-shaped DNA structure—to mimic membrane-assisted assembly into hierarchical superstructures on supported lipid bilayers and small unilamellar vesicles. As designed, the DNA constructs adhered to the lipid bilayers mediated by the cholesterol anchors and diffused freely in 2D with diffusion coefficients depending on their size and number of cholesterol modifications. Different sets of multimerization oligonucleotides added to bilayer-bound origami block structures induced the growth of either linear polymers or two-dimensional lattices on the membrane. Y-shaped DNA origami structures associated into triskelion homotrimers and further assembled into weakly ordered arrays of hexagons and pentagons, which resembled the geometry of clathrin-coated pits. Our results demonstrate the potential to realize artificial self-assembling systems that mimic the hierarchical formation of polyhedral lattices on cytoplasmic membranes.

We demonstrate a novel strategy for constructing multidirectional programmed 2D DNA nanostructures in various unique patterns by introducing photoresponsive oligonucleotides (Azo-ODNs) into hexagonal DNA origami structures. We examined regulation of assembly and disassembly of DNA nanostructures reversibly by different photoirradiation conditions in a programmed manner. Azo-ODNs were incorporated to the hexagonal DNA origami structures, which were then employed as self-assembly units for building up nanosized architectures in regulated arrangements. By adjusting the numbers and the positions of Azo-ODNs in the hexagonal units, the specific nanostructures with face controlling can be achieved, resulting in construction of ring-shaped nanostructures. By combining DNA origami strategy with photoregulating system, remote controlling of assembly and disassembly of DNA nanostructures has been accomplished simply by photo irradiation.

Molecular simulations suggest that the stability of a folded macromolecule increases in a confined space due to entropic effects. However, due to the interactions between the confined molecular structure and the walls of the container, clear-cut experimental evidence for this prediction is lacking. Here, using DNA origami nanocages, we show the pure effect of confined space on the property of individual human telomeric DNA G-quadruplexes. We induce targeted mechanical unfolding of the G-quadruplex while leaving the nanocage unperturbed. We find that the mechanical and thermodynamic stabilities of the G-quadruplex inside the nanocage increase with decreasing cage size. Compared to the case of diluted or molecularly crowded buffer solutions, the G-quadruplex inside the nanocage is significantly more stable, showing a 100 times faster folding rate. Our findings suggest the possibility of co-replicational or co-transcriptional folding of G-quadruplex inside the polymerase machinery in cells.

A framed photo of DNA: A pair of photoresponsive oligonucleotides containing azobenzene moieties was introduced into double-stranded DNA within the cavity of a DNA nanostructure (see scheme). The two dsDNAs, in contact at the center, were dissociated using UV irradiation and hybridized with visible light; this was directly observed using high-speed atomic force microscopy.

Controlled motion at the nanoscale can be achieved by using Watson-Crick base-pairing to direct the assembly and operation of a molecular transport system consisting of a track, a motor and fuel, all made from DNA. Here, we assemble a 100-nm-long DNA track on a two-dimensional scaffold, and show that a DNA motor loaded at one end of the track moves autonomously and at a constant average speed along the full length of the track, a journey comprising 16 consecutive steps for the motor. Real-time atomic force microscopy allows direct observation of individual steps of a single motor, revealing mechanistic details of its operation. This precisely controlled, long-range transport could lead to the development of systems that could be programmed and routed by instructions encoded in the nucleotide sequences of the track and motor. Such systems might be used to create molecular assembly lines modelled on the ribosome.

Direct observation of enzymes interacting with DNA should be one of the ultimate technologies for investigating the mechanical behavior of the enzymes during the reactions. Atomic force microscopy (AFM) enables observation of biomolecules at a nanoscale spatial resolution; however, for a stable analysis, a scaffold to observe the reaction should be explored. DNA origami has recently been developed for the construction of a wide variety of multidimensional nanostructures, which can be used as scaffolds to incorporate various functionalities at specific positions. We intended to construct an AFM-based analysis system for DNA repair using a DNA origami scaffold carrying various substrate double-stranded DNAs (dsDNA). We employed DNA baseexcision repair (BER) enzymes 8-oxoguanine glycosylase (hOgg1) and T4 pyrimidine dimer glycosylase (PDG) to analyze the reaction on the DNA scaffold (Figure 1a). In the repair process, hOgg1 removes 8-oxoguanine (oxoG) to prevent G:C!T:A transversion during replication by DNA glycosylase activity and endonuclease activity for apurine– apyrimidine (AP) sites (AP-lyase activity) (Figure 1a). PDG removes photodamaged pyrimidine dimer including cis–syn cyclobutane thymine dimer (T T) by DNA glycosylase/AP-lyase activity. BER enzymes often require structural changes of the target DNA strands, such as DNA bending, for the reaction to proceed. The enzyme hOgg1 bends double-helix DNA by about 708 with flipping out of the oxoG for procession of the excision. The enzyme PDG bends double-helix DNA by 608 with flipping out of the 3’-side of A in the opposite strand of T T. The glycosylase/AP-lyase activity of these enzymes leads to single-strand scission at the damaged nucleotide (Figure 1b). In addition, the reaction intermediates of both reactions form a covalent bond with the enzyme by reduction with NaBH4. [5,10] We recently developed a framelike DNA origami scaffold to incorporate two different substrate dsDNAs. If the above-mentioned enzymatic and chemical reactions

A novel method for assembling multiple DNA origami structures has been developed by using designed 2D DNA origami rectangles, so-called "DNA jigsaw pieces" that have sequence-programmed connectors. Shape and sequence complementarity were introduced to the concavity and convex connectors in the DNA rectangles for selective connection with the help of nonselective pi-stacking interactions between the side edges of the DNA jigsaw piece structures. Single DNA jigsaw piece units were assembled into unidirectional nanostructures with the correct alignment and uniform orientation. Three and five different DNA jigsaw pieces were assembled into predesigned and ordered nanostructures in a programmed fashion. Finally, three-, four-, and five-letter words have been displayed by using this programmed DNA jigsaw piece system.

A novel strategy for regulation of an enzymatic DNA modification reaction has been developed by employing a designed nanoscale DNA scaffold. DNA modification using enzymes often requires bending of specific DNA strands to facilitate the reaction. The DNA methylation enzyme EcoRI methyltransferase (M.EcoRI) bends double helix DNA by 55 degrees-59 degrees during the reaction with flipping out of the second adenine in the GAATTC sequence as the methyl transfer reaction proceeds. In this study, two different double helical tensions, tense and relaxed states of double helices, were created to control the methyl transfer reaction of M.EcoRI and examine the structural effect on the methylation. We designed and prepared a two-dimensional (2D) DNA scaffold named the "DNA frame" using the DNA origami method that accommodates two different lengths of the double-strand DNA fragments, a tense 64mer double strand and a relaxed 74mer double strand. Fast-scanning atomic force microscope (AFM) imaging revealed the different dynamic movement of the double-strand DNAs and complexes of M.EcoRI with 64mer and 74mer double-strand DNAs. After treatment of the double strands in the DNA frame with M.EcoRI and the subsequent digestion with restriction enzyme EcoRI (R.EcoRI), AFM analysis revealed that the 74mer double-strand DNA was not effectively cleaved compared with the 64mer double-strand DNA, indicating that the methylation preferentially occurred in the relaxed 74mer double-strand DNA compared with that in the tense 64mer double strand. Biochemical analysis of the methylation and specific digestion using a real-time PCR also supported the above results. These results indicate the importance of the structural flexibility for bending of the duplex DNA during the methyl transfer reaction with M.EcoRI. Therefore, the DNA methylation can be regulated using the structurally controlled double-strand DNAs constructed in the DNA frame nanostructure.

We demonstrate the single-molecule operation and observation of the formation and resolution of double-stranded DNA (dsDNA) containing a G-quadruplex (GQ) forming and counterpart i-motif forming sequence in the DNA nanostructure. Sequential manipulation of DNA strands in the DNA frame was performed to prepare a topologically controlled GQ/i-motif dsDNA. Using strand displacement and the addition and removal of K(+), the topologically controlled GQ/i-motif dsDNA in the DNA frame was obtained in high yield. The dsDNA was resolved into the single-stranded DNA, GQ, and i-motif by the addition of K(+) and operation in acidic conditions. The dissociation of the dsDNA under the GQ and i-motif formation condition was monitored by high-speed atomic force microscopy. The results indicate that the dsDNA containing the GQ- and i-motif sequence is effectively dissolved when the duplex is helically loosened in the DNA nanoscaffold.

A light-driven artificial molecular nanomachine was constructed based on DNA scaffolding. Pyrene-modified walking strands and disulfide bond-connected stator strands, employed as anchorage sites to support walker movement, were assembled into a 2D DNA tile. Pyrene molecules excited by photoirradiation at 350 nm induced cleavage of disulfide bond-connected stator strands, enabling the DNA walker to migrate from one cleaved stator to the next on the DNA tile. The time-dependent movement of the walker was observed and the entire walking process of the walker was characterized by distribution of the walker-stator duplex at four anchorage sites on the tile under different irradiation times. Importantly, the light-fuelled mechanical movements on DNA tile were first visualized in real time during UV irradiation using high-speed atomic force microscopy (HS-AFM).

The effect of pyrenes introduced into a tobacco mosaic virus (TMV) coat protein monomer on the formation and stability of the TMV assembly was investigated. The possible arrangement of the pyrenes in the inner cavity of the TMV rod was also estimated. The pyrene derivative was introduced to four specific amino acids in the cavity of the TMV rod structure. Rod-structure formation was examined by atomic force microscopy (AFM). Two pyrene-attached mutants (positions 99 and 100) assembled to increase the length of the rod structures by 2.5 microm at pH 5.5. The interaction of the pyrene moieties in the TMV cavity was investigated by steady-state and time-resolved spectroscopic analysis. Strong excimer emission with significantly short wavelength (465 nm) was observed from the two mutants mentioned above. Excitation and UV-visible spectra indicate that the pyrene moieties form pi-stacked structures in the TMV cavity. Details of the pyrene interaction were investigated by analyzing the fluorescence lifetime of the excimer. Results suggest that the pyrenes formed preassociated rigid structures with partially overlapped geometry in the restricted space of the TMV cavity. The pyrenes effectively stabilize the TMV rod through a pi-stacking interaction in a well-ordered way, and the single pyrene moiety introduced into the monomer affects the overall formation of the TMV rod structure.

While single-molecule sensing offers the ultimate detection limit, its throughput is often restricted as sensing events are carried out one at a time in most cases. 2D and 3D DNA origami nanostructures are used as expanded single-molecule platforms in a new mechanochemical sensing strategy. As a proof of concept, six sensing probes are incorporated in a 7-tile DNA origami nanoassembly, wherein binding of a target molecule to any of these probes leads to mechanochemical rearrangement of the origami nanostructure, which is monitored in real time by optical tweezers. Using these platforms, 10 pM platelet-derived growth factor (PDGF) are detected within 10 minutes, while demonstrating multiplex sensing of the PDGF and a target DNA in the same solution. By tapping into the rapid development of versatile DNA origami nanostructures, this mechanochemical platform is anticipated to offer a long sought solution for single-molecule sensing with improved throughput.

To create a nanoscale dual switch, two responsive DNA motifs, azobenzene-modified DNAs and G-telomeric repeat sequences, were introduced together into the nanoframe system. The dual-switching behaviors controlled by photoirradiation and K(+) were successfully visualized in real time by high-speed atomic force microscopy.

CONSPECTUS: Direct imaging of molecular motions is one of the most fundamental issues for elucidating the physical properties of individual molecules and their reaction mechanisms. Atomic force microscopy (AFM) enables direct molecular imaging, especially for biomolecules in the physiological environment. Because AFM can visualize the molecules at nanometer-scale spatial resolution, a versatile observation scaffold is needed for the precise imaging of molecule interactions in the reactions. The emergence of DNA origami technology allows the precise placement of desired molecules in the designed nanostructures and enables molecules to be detected at the single-molecule level. In our study, the DNA origami system was applied to visualize the detailed motions of target molecules in reactions using high-speed AFM (HS-AFM), which enables the analysis of dynamic motions of biomolecules in a subsecond time resolution. In this system, biochemical properties such as the placement of various double-stranded DNAs (dsDNAs) containing unrestricted DNA sequences, modified nucleosides, and chemical functions can be incorporated. From a physical point of view, the tension and rotation of dsDNAs can be controlled by placement into the DNA nanostructures. From a topological point of view, the orientations of dsDNAs and various shapes of dsDNAs including Holliday junctions can be incorporated for studies on reaction mechanisms. In this Account, we describe the combination of the DNA origami system and HS-AFM for imaging various biochemical reactions including enzymatic reactions and DNA structural changes. To observe the behaviors and reactions of DNA methyltransferase and DNA repair enzymes, the substrate dsDNAs were incorporated into the cavity of the DNA frame, and the enzymes that bound to the target dsDNA were observed using HS-AFM. DNA recombination was also observed using the recombination substrates and Holliday junction intermediates placed in the DNA frame, and the direction of the reactions was controlled by introducing structural stress to the substrates. In addition, the movement of RNA polymerase and its reaction were visualized using a template dsDNA attached to the origami structure. To observe DNA structural changes, G-quadruplex formation and disruption, the switching behaviors of photoresponsive oligonucleotides, and B-Z transition were visualized using the DNA frame observation system. For the formation and disruption of G-quadruplex and double-helix DNA, the two dsDNA chains incorporated into the DNA frame could amplify the small structural change to the global structural change, which enabled the visualization of their association and dissociation by HS-AFM. The dynamic motion of the helical rotation induced by the B-Z transition was also directly imaged in the DNA frame. Furthermore, the stepwise motions of mobile DNA along the DNA track were visualized on the DNA origami surface. These target-orientated observation systems should contribute to the detailed analysis of biomolecule motions in real time and at molecular resolution.

We developed a novel method to design various helical tubular structures using the DNA origami method. The size-controlled tubular structures which have 192, 256, and 320 base pairs for one turn of the tube were designed and prepared. We observed the formation of the expected short tubes and unexpected long ones. Detailed analyses of the surface patterns of the tubes showed that the short tubes had mainly a left-handed helical structure. The long tubes mainly formed a right-handed helical structure and extended to the directions of the double helical axes as structural isomers of the short tubes. The folding pathways of the tubes were estimated by analyzing the proportions of short and long tubes obtained at different annealing conditions. Depending on the number of base pairs involved in one turn of the tube, the population of left-/right-handed and short/long tubes changed. The bending stress caused by the stiffness of the bundled double helices and the non-natural helical pitch determine the structural variety of the tubes.

Here, we report the direct visualization of the assembly/disassembly processes of photoresponsive DNA origami nanostructures which can be placed on a lipid bilayer surface. The observation relies on controlled interactions between the bilayer components and cholesterol moieties introduced to the hexagonal origami structures, one of whose outer edges carries Azo-ODNs. The bilayer-placed hexagonal dimer was disassembled into monomer units by UV irradiation, and reversibly assembled again during visible light irradiation. These dynamic processes were directly monitored with high-speed atomic force microscopy. The successful application of our approach should facilitate studies of interactive and functional behaviors of various DNA nanostructures.

We herein report the real-time observation of G-quadruplex formation by monitoring the G-quadruplex-induced global change of two duplexes incorporated in a DNA nanoscaffold. The introduced G-rich strands formed an interstrand (3 + 1) G-quadruplex structure in the presence of K(+), and the formed four-stranded structure was disrupted by removal of K(+). These conformational changes were visualized in a nanoscaffold in real-time with fast-scanning atomic force microscopy.

The immunostimulatory activity of phosphodiester DNA containing unmethylated cytosine-phosphate-guanine (CpG) dinucleotides, or CpG motifs, was significantly increased by the formation of Y-, X-, or dendrimer-like multibranched shape. These results suggest the possibility that the activity of CpG DNA is a function of the structural properties of branched DNA assemblies. To elucidate the relationship between them, we have designed and developed nanosized DNA assemblies in polypod-like structures (polypod-like structured DNA, or polypodna for short) using oligodeoxynucleotides (ODNs) containing CpG motifs and investigated their structural and immunological properties. Those assemblies consisting of three (tripodna) to eight (octapodna) ODNs were successfully obtained, but one consisting of 12 ODNs was not when 36-mer ODNs were annealed under physiological sodium chloride concentration. High-speed atomic force microscopy revealed that these assemblies were in polypod-like structures. The apparent size of the products was about 10 nm in diameter, and there was an increasing trend with an increase in ODN length or with the pod number. Circular dichroism spectral data showed that DNA in polypodna preparations were in the B-form. The melting temperature of polypodna decreased with increasing pod number. Each polypodna induced the secretion of tumor necrosis factor-α and interleukin-6 from macrophage-like RAW264.7 cells, with the greatest induction by those with hexa- and octapodna. Increasing the pod number increased the uptake by RAW264.7 cells but reduced the stability in serum. These results indicate that CpG DNA-containing polypodna preparations with six or more pods are a promising nanosized device with biodegradability and high immunostimulatory activity.