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Interlocked DNA nanostructures perform programmable movements in nanoscales such as sliding, contraction, and expansion. However, utilizing nanoscaled interlocked movements to regulate the functions of larger length scaled matrix and developing their applications has not yet been reported. Herein we describe the assembly of DNA-based daisy chain rotaxane nanostructure (DNA-DCR) composed of two hollow DNA nanostructures as macrocycles, two interlocked axles and two triangular prism-shaped DNA structures as stoppers, in which three mechanical states─fixed extended state (FES), sliding state (SS), and fixed contracted state (FCS)─are characterized by using toehold-mediated strand displacement reaction (SDR). The DNA-DCRs are further used as nanocomposites and introduced into hydrogel matrix to produce interlocked hydrogels, which shows modulable stiffness by elongating the interlocked axles to regulate the hydrogel swelling with hybridization chain reaction (HCR) treatment. Then the DCR-hydrogels are employed as dynamic biointerfaces for human mesenchymal stem cells (hMSCs) adhesion studies. First, hMSCs showed lower cell density on bare DCR-hydrogel treated with HCR-initiated swelling for stiffness decreasing. Second, the cell adhesion ligand (RGD) modified DNA-DCRs are constructed for hydrogel functionalization. DCR

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.

The extracellular matrix (ECM) in which the cells reside provides a dynamic and reversible environment. Spatiotemporal cues are essential when cells are undergoing morphogenesis, repair and differentiation. Emulation of such an intricate system with reversible presentation of nanoscale cues can help us better understand cellular processes and can allow the precise manipulation of cell function in vitro. Herein, we formulated a photoswitchable DNA mechanical nanostructure containing azobenzene moieties and dynamically regulated the spatial distance between adhesion peptides using a photoswitchable DNA polymer with photoirradiation. We found that the DNA polymer reversibly forms two different structures, a relaxed linear and shrunken compact form, observed by AFM. Using the mechanical properties of this DNA polymer, UV and visible light irradiation induced a significant morphology change of the cells between a round shape and spindle shape, thus providing a tool to decipher the language of the ECM better.

Cellular environments such as nanoconfinement and molecular crowding can change biomolecular properties. However, in nanoconfinement, it is extremely challenging to investigate effects of crowding cosolutes on macromolecules. By using optical tweezers, here, we elucidated the effects of hexaethylene glycol (HEG) on the mechanical stability of a telomeric G-quadruplex (GQ) in a zeptoliter DNA origami reactor (zepto-reactor). When HEG molecules were introduced in the GQ-containing zepto-reactor at different positions, we found that the GQ species split into two equilibrated populations, reflecting diverse effects of the oligoethylene glycol on the GQ via either a long-range dehydration effect or direct interactions. When the number of HEG molecules was increased, the stability of the GQ unexpectedly decreased, suggesting that the direct destabilizing interaction between the GQ and HEG is dominating over the long-range stabilizing dehydration effects of the HEG in hydrophilic nanocavities. These findings indicate that a nanoconfined environment can alter regular effects of cosolutes on biomacromolecules.

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 nucleosome is one of the most fundamental units involved in gene expression and consequent cell development, differentiation, and expression of cell functions. We report here a method to place reconstituted nucleosomes into a DNA origami frame for direct observation using high-speed atomic-force microscopy (HS-AFM). By using this method, multiple nucleosomes can be incorporated into a DNA origami frame and real-time movement of nucleosomes can be visualized. The arrangement and conformation of nucleosomes and the distance between two nucleosomes can be designed and controlled. In addition, four nucleosomes can be placed in a DNA frame. Multiple nucleosomes were well accessible in each conformation. Dynamic movement of the individual nucleosomes were precisely monitored in the DNA frame, and their assembly and interaction were directly observed. Neither mica surface modification nor chemical fixation of nucleosomes is used in this method, meaning that the DNA frame not only holds nucleosomes, but also retains their natural state. This method offers a promising platform for investigating nucleosome interactions and for studying chromatin structure.

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.

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.

Elucidation of the details of the associating mode is one of the major concerns for the precise design of DNA-binding molecules that are used for gene regulation. Pyrrole-imidazole polyamide (PIP) is a well-established synthetic DNA-binding molecule that has sequence-specificity for duplex DNA. By the design of the sequence of pyrrole, imidazole, and other synthetic units, PIP is bound to the target DNA sequence selectively. Here, we report the X-ray crystal structure of newly synthesized chiral cyclic PIP (cPIP) complexed with DNA at 1.5 A resolution and reveal that cPIP binds in the reverse orientation in the DNA minor groove. Analysis of the crystal structure revealed that the positions of the hydrogen bonds between the bases and the pyrrole-imidazole moieties of cPIP were similar for both forward- and reverse-binding orientations and that the distortion of the B-form DNA structure caused by cPIP binding was also similar for both orientations. We further found that new hydrogen bonds formed between the amino groups on the γ-turn units and DNA at both ends of the cPIP molecule. Additionally, by comparing the reverse PIP orientation with the forward orientation, we could clarify that the cause of the preference toward the reverse orientation in the S-form cPIP as used in this study is the overall conformation of the cPIP-DNA complex, particularly the configuration of hydrogen bonds. These results thus provide an explanation for the different stereoselectivity of cPIP binding in the minor groove.

For proteins and DNA secondary structures such as G-quadruplexes and i-motifs, nanoconfinement can facilitate their folding and increase structural stabilities. However, the properties of the physiologically prevalent B-DNA duplex have not been elucidated inside the nanocavity. Using a 17-bp DNA duplex in the form of a hairpin stem, here, we probed folding and unfolding transitions of the hairpin DNA duplex inside a DNA origami nanocavity. Compared to the free solution, the DNA hairpin inside the nanocage with a 15 × 15 nm cross section showed a drastic decrease in mechanical (20 → 9 pN) and thermodynamic (25 → 6 kcal/mol) stabilities. Free energy profiles revealed that the activation energy of unzipping the hairpin DNA duplex decreased dramatically (28 → 8 kcal/mol), whereas the transition state moved closer to the unfolded state inside the nanocage. All of these indicate that nanoconfinement weakens the stability of the hairpin DNA duplex to an unexpected extent. In a DNA hairpin made of a stem that contains complementary telomeric G-quadruplex (GQ) and i-motif (iM) forming sequences, formation of the Hoogsteen base pairs underlining the GQ or iM is preferred over the Watson-Crick base pairs in the DNA hairpin. These results shed light on the behavior of DNA in nanochannels, nanopores, or nanopockets of various natural or synthetic machineries. It also elucidates an alternative pathway to populate noncanonical DNA over B-DNA in the cellular environment where the nanocavity is abundant.

DNA methylation and demethylation play a key role in the epigenetic regulation of gene expression; however, a series of oxidation reactions of 5-methyl cytosine (5mC) mediated by ten-eleven translocation (TET) enzymes driving demethylation process are yet to be uncovered. To elucidate the relationship between the oxidative processes and structural factors of DNA, we analysed the behavior of TET-mediated 5mC-oxidation by incorporating structural stress onto a substrate double-stranded DNA (dsDNA) using a DNA origami nanochip. The reactions and behaviors of TET enzymes were systematically monitored by biochemical analysis and single-molecule observation using atomic force microscopy (AFM). A reformative frame-like DNA origami was established to allow the incorporation of dsDNAs as 5mC-containing substrates in parallel orientations. We tested the potential effect of dsDNAs present in the tense and relaxed states within a DNA nanochip on TET oxidation. Based on enzyme binding and the detection of oxidation reactions within the DNA nanochip, it was revealed that TET preferred a relaxed substrate regardless of the modification types of 5-oxidated-methyl cytosine. Strikingly, when a multi-5mCG sites model was deployed to further characterize substrate preferences of TET, TET preferred the fully methylated site over the hemi-methylated site. This analytical modality also permits the direct observations of dynamic movements of TET such as sliding and interstrand transfer by high-speed AFM. In addition, the thymine DNA glycosylase-mediated base excision repair process was characterized in the DNA nanochip. Thus, we have convincingly established the system's ability to physically regulate enzymatic reactions, which could prove useful for the observation and characterization of coordinated DNA demethylation processes at the nanoscale.

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.

Compaction of bulky DNA is a universal issue for all DNA-based life forms. Chloroplast nucleoids (chloroplast DNA-protein complexes) are critical for chloroplast DNA maintenance and transcription, thereby supporting photosynthesis, but their detailed structure remains enigmatic. Our proteomic analysis of chloroplast nucleoids of the green alga Chlamydomonas reinhardtii identified a protein (HBD1) with a tandem repeat of two DNA-binding high mobility group box (HMG-box) domains, which is structurally similar to major mitochondrial nucleoid proteins transcription factor A, mitochondrial (TFAM), and ARS binding factor 2 protein (Abf2p). Disruption of the HBD1 gene by CRISPR-Cas9-mediated genome editing resulted in the scattering of chloroplast nucleoids. This phenotype was complemented when intact HBD1 was reintroduced, whereas a truncated HBD1 with a single HMG-box domain failed to complement the phenotype. Furthermore, ectopic expression of HBD1 in the mitochondria of yeast Δabf2 mutant successfully complemented the defects, suggesting functional similarity between HBD1 and Abf2p. Furthermore, in vitro assays of HBD1, including the electrophoretic mobility shift assay and DNA origami/atomic force microscopy, showed that HBD1 is capable of introducing U-turns and cross-strand bridges, indicating that proteins with two HMG-box domains would function as DNA clips to compact DNA in both chloroplast and mitochondrial nucleoids.

Regnase-1-mediated mRNA decay (RMD), in which inflammatory mRNAs harboring specific stem–loop structures are degraded, is a critical part of proper immune homeostasis. Prior to initial translation, Regnase-1 associates with target stem–loops but does not carry out endoribonucleolytic cleavage. Single molecule imaging revealed that UPF1 is required to first unwind the stem–loops, thus licensing Regnase-1 to proceed with RNA degradation. Following translation, Regnase-1 physically associates with UPF1 using two distinct points of interaction: The Regnase-1 RNase domain binds to SMG1-phosphorylated residue T28 in UPF1; in addition, an intrinsically disordered segment in Regnase-1 binds to the UPF1 RecA domain, enhancing the helicase activity of UPF1. The SMG1-UPF1–Regnase-1 axis targets pioneer rounds of translation and is critical for rapid resolution of inflammation through restriction of the number of proteins translated by a given mRNA. Furthermore, small-molecule inhibition of SMG1 prevents RNA unwinding in dendritic cells, allowing post-transcriptional control of innate immune responses., 炎症が制御される新たなRNA分解メカニズムを解明 --新たな免疫賦活化法の開発に道筋--. 京都大学プレスリリース. 2019-07-25.

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.

We report a nanosized DNA capsule with a photoinducible mechanical unlocking system for creation of a carrier for delivery system to the cells. A photocage system was introduced into the nanocapsule (NC) for control of opening of the NC with photoirradiation. The opening of the NC was observed by atomic force microscopy (AFM), and the dynamic opening of the NC was examined by fluorescence recovery from the quenching. The photocaged NC was introduced to the cell without toxicity and observed in the cytoplasm, and the photoinduced opening of the NC was observed in the cell. The selective unlocking and opening of the caged-NC in a single cell was successfully achieved by a laser irradiation to individual cells.

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.

We report the direct observation of the formation and degradation of tensegrity triangle DNA crystals using atomic force microscopy (AFM). We observed the crystal surface by AFM and characterized the lattice coordination of the assembled triangle units at a molecular level. We visualized dynamic formation and degradation of the crystals and characterized them at nano-scale resolution.

Self-assembly is commonly seen in living systems and plays a central role in the construction of cellular structures and thus influences the functions of organized biological systems in the cell. In molecular science, the precise formation of structures via self-assembly processes attracts attention, because the functions of assembled molecules originate from the precise structure and arrangement of the molecules. The emergence of DNA origami technology allows for the design and construction of a desired DNA structure and size at nanoscales. Precise placement and integration of target molecules into the designed DNA nanostructures enables creation of advanced materials and molecular devices. The DNA origami scaffold can provide an analytical method to elucidate the physical properties of a target molecule and its reaction process when combined with high-speed atomic force microscopy (HS-AFM), which enables direct imaging of dynamic motions of biomolecules in physiological environments at both the subsecond time and nanometer-scale spatial resolutions. In addition, the DNA origami realizes to create designed nanoscale spaces for single-molecule analysis.

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.

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.

Resolution achieved Chloroplasts possess multiple copies of their own chloroplast DNA that are packaged into DNA-protein complexes known as nucleoids. The shape, number, and distribution of chloroplast nucleoids change markedly depending on the cell cycle, developmental stage, and nutritional environment. Kobayashi et al. identified Holliday junction resolvase as a key factor in the dynamism of chloroplast nucleoids in the unicellular green algae Chlamydomonas reinhardtii . The gene encoding the resolvase is ubiquitously conserved among green plants. Disruption or down-regulation of this gene also disturbed chloroplast nucleoid organization and segregation in the land plant Arabidopsis thaliana . Science , this issue p. 631

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.

We demonstrate the single-molecule operation and observation of the formation and resolution of double-stranded DNA (dsDNA) containing G-quadruplex (GQ)- and counterpart i-motif-forming sequences 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 the strand displacement and the addition and removal of K

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.

Herein, the direct visualization of the dynamic interaction between a photoresponsive transcription factor fusion, GAL4–VVD, and DNA using high‐speed atomic force microscopy (HS‐AFM) is reported. A series of different GAL4–VVD movements, such as binding, sliding, stalling, and dissociation, was observed. Inter‐strand jumping on two double‐stranded (ds) DNAs was also observed. Detailed analysis using a long substrate DNA strand containing five GAL4‐binding sites revealed that GAL4–VVD randomly moved on the dsDNA using sliding and hopping to rapidly find specific binding sites, and then stalled to the specific sites to form a stable complex formation. These results suggest the existence of different conformations of the protein to enable sliding and stalling. This single‐molecule imaging system with nanoscale resolution provides an insight into the searching mechanism used by DNA‐binding proteins.

We demonstrate the single-molecule operation and observation of the formation and resolution of double-stranded DNA (dsDNA) containing G-quadruplex (GQ)- and counterpart i-motif-forming sequences 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 the 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 under acidic conditions. The dissociation of the dsDNA under the GQ and i-motif formation conditions 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.

DNA nanoassemblies have demonstrated wide applications in various fields including nanomaterials, drug delivery and biosensing. In DNA origami, single-stranded DNA template is shaped into desired nanostructure by DNA staples that form Holliday junctions with the template. Limited by current methodologies, however, mechanical properties of DNA origami structures have not been adequately characterized, which hinders further applications of these materials. Using laser tweezers, here, we have described two mechanical properties of DNA nanoassemblies represented by DNA nanotubes, DNA nanopyramids and DNA nanotiles. First, mechanical stability of DNA origami structures is determined by the effective density of Holliday junctions along a particular stress direction. Second, mechanical isomerization observed between two conformations of DNA nanotubes at 10–35 pN has been ascribed to the collective actions of individual Holliday junctions, which are only possible in DNA origami with rotational symmetric arrangements of Holliday junctions, such as those in DNA nanotubes. Our results indicate that Holliday junctions control mechanical behaviors of DNA nanoassemblies. Therefore, they can be considered as ‘mechanophores’ that sustain mechanical properties of origami nanoassemblies. The mechanical properties observed here provide insights for designing better DNA nanostructures. In addition, the unprecedented mechanical isomerization process brings new strategies for the development of nano-sensors and actuators.

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.

Immunoinhibitory oligodeoxynucleotides (INH-ODNs) are promising inhibitors of Toll-like receptor 9 (TLR9) activation. To efficiently deliver INH-ODNs to TLR9-positive cells, we designed a Takumi-shaped DNA (Takumi) consisting of two partially complementary ODNs as the main component of a DNA hydrogel. Polyacrylamide gel electrophoresis showed that Takumi-containing INH-ODNs (iTakumi) and iTakumi-based DNA hydrogel (iTakumiGel) were successfully generated. Their activity was examined in murine macrophage-like RAW264.7 cells and DC2.4 dendritic cells by measuring tumor necrosis factor-α and interleukin-6 release after the addition of a TLR9 ligand (CpG ODN). Cytokine release was efficiently inhibited by the iTakumiGel. Flow cytometry analysis and confocal microscopy showed that cellular uptake of INH-ODN was greatly increased by the iTakumiGel. These results indicate that a Takumi-based DNA hydrogel is useful for the delivery of INH-ODNs to immune cells to inhibit TLR9-mediated hyperinduction of proinflammatory cytokines. From the Clinical Editor: Toll-like receptor 9 activation has been reported to be associated with many autoimmune diseases. DNA inhibition using oligodeoxynucleotides is one of the potential treatments. In this article, the authors described hydrogel-based platform for the delivery of the inhibitory oligodeoxynucleotides for enhanced efficacy. The positive findings could indicate a way for the future.

We previously demonstrated that polypod-like structured DNA, or polypodna, constructed with three or more oligodeoxynucleotides (ODNs), is efficiently taken up by immune cells such as dendritic cells and macrophages, depending on its structural complexity. The ODNs comprising the polypodna should bend to form the polypod-like structure, and may do so by adopting either a bendtype conformation or a cross-type conformation. Here, we tried to elucidate the orientation and bending of ODNs in polypodnas using atomic force microscopy (AFM). We designed two types of pentapodnas (i.e., a polypodna with five pods) using 60- to 88-base ODNs, which were then immobilized on DNA origami frames. AFM imaging showed that the ODNs in the pentapodna adopted bend-type conformations. Tetrapodna and hexapodna also adopted bend-type conformations when they were immobilized on frames under unconstrained conditions. These findings provide useful information toward the coherent design of, and the structure–activity relationships for, a variety of DNA nanostructures.

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.

The visualization of biomolecules is a straightforward way to elucidate the physical properties of molecules and their reaction processes. Atomic force microscopy (AFM) enables the direct imaging of biomolecules under physiological conditions at nanometer-scale spatial resolution. Because AFM visualizes all molecules in a scanning area, an observation scaffold is required for the target-specific imaging of molecules in the dynamic state. The DNA origami technology allows the precise placement of target molecules in a designed nanostructure, and the detection of the molecules at the single-molecule level. DNA origami is applied for visualizing the detailed motions of molecules using high-speed AFM (HS-AFM), which enables the analysis of the dynamic movement of biomolecules in a subsecond time resolution. Here, we describe the combination of the DNA origami system with HS-AFM for the imaging of DNA structural changes controlled by photoresponsive molecules. The hybridization and dehybridization of photoresponsive oligonucleotides were visualized directly using this observation system. These target-oriented observation systems should contribute to the detailed analysis of biomolecules in real time with molecular resolution.

The visualization of biomolecules is a straightforward way to elucidate the physical properties of molecules and their reaction processes. Atomic force microscopy (AFM) enables the direct imaging of biomolecules under physiological conditions at nanometer-scale spatial resolution. Because AFM visualizes all molecules in a scanning area, an observation scaffold is required for the target-specific imaging of molecules in the dynamic state. The DNA origami technology allows the precise placement of target molecules in a designed nanostructure, and the detection of the molecules at the single-molecule level. DNA origami is applied for visualizing the detailed motions of molecules using high-speed AFM (HS-AFM), which enables the analysis of the dynamic movement of biomolecules in a subsecond time resolution. Here, we describe the combination of the DNA origami system with HS-AFM for the imaging of DNA structural changes controlled by photoresponsive molecules. The hybridization and dehybridization of photoresponsive oligonucleotides were visualized directly using this observation system. These target-oriented observation systems should contribute to the detailed analysis of biomolecules in real time with molecular resolution.

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.

The most fundamental step in gene expression is transcription, during which DNA is transcribed to a corresponding RNA strand by the action of RNA polymerases (RNAPs). Over the past two decades, atomic force microscopy (AFM) has been used as one of the key tools in the investigation of transcriptional events at the single-molecule level. AFM studies have provided significant insights into the structure-function relationships of RNAP-DNA complexes in different stages of transcription. Here, we begin by illustrating the basic setup of AFM, followed by an introduction of the applications of AFM techniques, including high-speed AFM (HS-AFM) imaging, to investigate RNAP-DNA interactions with a special focus on promoter-search processes and open promoter formations. The combination of AFM with a newly developed experimental technique, DNA origami nanotechnology, will also be described.

We demonstrate the single-molecule imaging of the catalytic reaction of a Zn(2+)-dependent DNAzyme in a DNA origami nanostructure. The single-molecule catalytic activity of the DNAzyme was examined in the designed nanostructure, a DNA frame. The DNAzyme and a substrate strand attached to two supported dsDNA molecules were assembled in the DNA frame in two different configurations. The reaction was monitored by observing the configurational changes of the incorporated DNA strands in the DNA frame. This configurational changes were clearly observed in accordance with the progress of the reaction. The separation processes of the dsDNA molecules, as induced by the cleavage by the DNAzyme, were directly visualized by high-speed atomic force microscopy (AFM). This nanostructure-based AFM imaging technique is suitable for the monitoring of various chemical and biochemical catalytic reactions at the 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 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.

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.

We report the use of atomic force microscopy (AFM) to study Sox2-Pax6 complex formation on the regulatory DNA element at a single molecule level. Using an origami DNA scaffold containing two DNA strands with different levels of tensile force, we confirmed that DNA bending is necessary for Sox2 binding. We also demonstrated that two transcription factors bind cooperatively by observing the increased occupancy of Sox2-Pax6 on the DNA element compared to that of Sox2 alone.

We present the direct and single-molecule visualization of the in-pathway intermediates of the G-quadruplex folding that have been inaccessible by any experimental method employed to date. Using DNA origami as a novel tool for the structural control and high-speed atomic force microscopy (HS-AFM) for direct visualization, we captured images of the unprecedented solution-state structures of a tetramolecular antiparallel and (3+1)-type G-quadruplex intermediates, such as G-hairpin and G-triplex, with nanometer precision. No such structural information was reported previously with any direct or indirect technique, solution or solid-state, single-molecule or bulk studies, and at any resolution. Based on our results, we proposed a folding mechanism of these G-quadruplexes.