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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., (© 2021 Wiley-VCH GmbH.)

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., (© 2020 Wiley-VCH GmbH.)

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., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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.

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., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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., Competing Interests: The authors declare no conflict of interest.

Nucleic acids are biomolecules of amazing versatility. Beyond their function for information storage they can be used for building nano-objects. We took advantage of loop-loop or kissing interactions between hairpin building blocks displaying complementary loops for driving the assembly of nucleic acid nano-architectures. It is of interest to make the interaction between elementary units dependent on an external trigger, thus allowing the control of the scaffold formation. To this end we exploited the binding properties of structure-switching aptamers (aptaswitch). Aptaswitches are stem-loop structured oligonucleotides that engage a kissing complex with an RNA hairpin in response to ligand-induced aptaswitch folding. We demonstrated the potential of this approach by conditionally assembling oligonucleotide nanorods in response to the addition of adenosine.

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., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.

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., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

RNA molecules uniquely form a complex through specific hairpin loops, called a kissing complex. The kissing complex is widely investigated and used for the construction of RNA nanostructures. Molecular switches have also been created by combining a kissing loop and a ligand-binding aptamer to control the interactions of RNA molecules. In this study, we incorporated two kinds of RNA molecules into a DNA origami structure and used atomic force microscopy to observe their ligand-responsive interactions at the single-molecule level. We used a designed RNA aptamer called GTPswitch, which has a guanosine triphosphate (GTP) responsive domain and can bind to the target RNA hairpin named Aptakiss in the presence of GTP. We observed shape changes of the DNA/RNA strands in the DNA origami, which are induced by the GTPswitch, into two different shapes in the absence and presence of GTP, respectively. We also found that the switching function in the nanospace could be improved by using a cover strand over the kissing loop of the GTPswitch or by deleting one base from this kissing loop. These newly designed ligand-responsive aptamers can be used for the controlled assembly of the various DNA and RNA nanostructures.

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., (Copyright © 2015 Elsevier Inc. All rights reserved.)

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).

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., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

To gain new insights into G-quadruplex-drug interactions, we captured the solution-state structures of the complexes between a drug-like small molecule and a G-hairpin/G-triplex. Our results indicated that the ligand initially binds to the intermediates and induces stepwise folding into a quadruplex.

DNA dendrimers consisting of several branched DNA units connected to each other using DNA ligase were quite effective for the delivery of immunostimulatory CpG DNA to immune cells. Therapeutic application of such DNA dendrimers, however, is hampered by the use of the ligase. Here, we report that self-assembling DNA dendrimers with high immunostimulatory potency can be prepared without DNA ligases. Annealing of DNA consisting of DNA units with elongated adhesive ends resulted in the formation of DNA dendrimers. Atomic force microscopy revealed that the several preparations of DNA dendrimers resulted in dendritic structures as designed. The cellular uptake of DNA dendrimers by mouse macrophage-like RAW264.7 cells and subsequent release of tumor necrosis factor-α were dependent on the structural complexity of the dendrimers. These results indicate that the ligation-free, self-assembling DNA dendrimers are a potent system for the delivery of immunostimulatory CpG DNA to immune cells.

A photofunctionalized square bipyramidal DNA nanocapsule (NC) was designed and prepared for the creation of a nanomaterial carrier. Photocontrollable open/close system and toehold system were introduced into the NC for the inclusion and release of a gold nanoparticle (AuNP) by photoirradiation and strand displacement. The reversible open and closed states were examined by gel electrophoresis and atomic force microscopy (AFM), and the open behavior was directly observed by high-speed AFM. The encapsulation of the DNA-modified AuNP within the NC was carried out by hybridization of a specific DNA strand (capture strand), and the release of the AuNP was examined by addition of toehold-containing complementary DNA strand (release strand). The release of the AuNP from the NC was achieved by the opening of the NC and subsequent strand displacement., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Controllable fabrication of DNA origami structures was achieved using cationic comb-type copolymers (CCCs) as locks and polyvinyl sulphonic acid (PVS) as a key. A CCC binds to the phosphate backbone of either M13mp18/staples alone or both together and restricts origami folding, while PVS unlocks the CCC, restoring the formation of origami structures.

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., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.

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., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Site-specific recombination involves reciprocal exchange between defined DNA sites. The reaction initiates from the formation of a recombinase-DNA synaptic complex, in which two recombination sites arrange in an appropriate configuration. However, there is incomplete information about how the topological state of the substrate influences the synapsis and outcome of the reaction. Here, we show that Cre-mediated recombination can be regulated by controlling the orientation and topology of the loxP substrate in a DNA frame nanoscaffold. High-speed atomic force microscopy analyses revealed that the loxP-containing substrate strands in the antiparallel orientation can be recombined only through formation of synaptic complexes. By tethering Holliday junction (HJ) intermediates to DNA frames in different connection patterns and using them as a starting substrate, we found that the topological state of the HJ intermediates dictates the outcome of the resolution. Our approach should provide a new platform for structural-functional studies of various DNA targeting enzymes, especially which require formation of synaptic complexes.

Guanine-rich oligonucleotides often show a strong tendency to form supramolecular architecture, the so-called G-quadruplex structure. Because of the biological significance, it is now considered to be one of the most important conformations of DNA. Here, we describe the direct visualization and single-molecule analysis of the formation of a tetramolecular G-quadruplex in KCl solution. The conformational changes were carried out by incorporating two duplex DNAs, with G-G mismatch repeats in the middle, inside a DNA origami frame and monitoring the topology change of the strands. In the absence of KCl, incorporated duplexes had no interaction and laid parallel to each other. Addition of KCl induced the formation of a G-quadruplex structure by stably binding the duplexes to each other in the middle. Such a quadruplex formation allowed the DNA synapsis without disturbing the duplex regions of the participating sequences, and resulted in an X-shaped structure that was monitored by atomic force microscopy. Further, the G-quadruplex formation in KCl solution and its disruption in KCl-free buffer were analyzed in real-time. The orientation of the G-quadruplex is often difficult to control and investigate using traditional biochemical methods. However, our method using DNA origami could successfully control the strand orientations, topology and stoichiometry of the G-quadruplex.

Using the RNA transcript as a template, RNA-templated DNA origami structures were constructed by annealing with designed DNA staple strands. RNA-templated DNA origami structures were folded to form seven-helix bundled rectangular structures and six-helix bundled tubular structures. The chemically modified RNA-DNA hybrid origami structures were prepared by using RNA templates containing modified uracils.

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.

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., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.

We demonstrate a novel strategy for visualizing sequence-selective alkylation of target double-stranded DNA (dsDNA) using a synthetic pyrrole-imidazole (PI) polyamide in a designed DNA origami scaffold. Doubly functionalized PI polyamide was designed by introduction of an alkylating agent 1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole (seco-CBI) and biotin for sequence-selective alkylation at the target sequence and subsequent streptavidin labeling, respectively. Selective alkylation of the target site in the substrate DNA was observed by analysis using sequencing gel electrophoresis. For the single-molecule observation of the alkylation by functionalized PI polyamide using atomic force microscopy (AFM), the target position in the dsDNA (∼200 base pairs) was alkylated and then visualized by labeling with streptavidin. Newly designed DNA origami scaffold named "five-well DNA frame" carrying five different dsDNA sequences in its cavities was used for the detailed analysis of the sequence-selectivity and alkylation. The 64-mer dsDNAs were introduced to five individual wells, in which target sequence AGTXCCA/TGGYACT (XY = AT, TA, GC, CG) was employed as fully matched (X = G) and one-base mismatched (X = A, T, C) sequences. The fully matched sequence was alkylated with 88% selectivity over other mismatched sequences. In addition, the PI polyamide failed to attach to the target sequence lacking the alkylation site after washing and streptavidin treatment. Therefore, the PI polyamide discriminated the one mismatched nucleotide at the single-molecule level, and alkylation anchored the PI polyamide to the target dsDNA.

A transcription regulation system initiated by DNA nanostructure changes was designed and constructed. Using the toehold system, specific DNA strands induced the opening of the tubular structure. A transcription product from the purified tube-attached dsDNA template was observed by addition of DNA strands that were specific for opening the tubular structure.

Synthetic molecular motors can be fuelled by the hydrolysis or hybridization of DNA. Such motors can move autonomously and programmably, and long-range transport has been observed on linear tracks. It has also been shown that DNA systems can compute. Here, we report a synthetic DNA-based system that integrates long-range transport and information processing. We show that the path of a motor through a network of tracks containing four possible routes can be programmed using instructions that are added externally or carried by the motor itself. When external control is used we find that 87% of the motors follow the correct path, and when internal control is used 71% of the motors follow the correct path. Programmable motion will allow the development of computing networks, molecular systems that can sort and process cargoes according to instructions that they carry, and assembly lines that can be reconfigured dynamically in response to changing demands.

A novel DNA scaffold, called a DNA slit, was designed for the programmed positioning of Au nanoparticles (AuNPs), and various patterns of thiolated DNA slits were constructed. AuNPs were correctly placed at the predesigned positions in the thiolated DNA slits, indicating that the thiolated staples and the slit cavities guide the correct assembly of AuNPs., (This journal is © The Royal Society of Chemistry 2011)

Heat tolerance of DNA origami structures has been improved about 30 °C by photo-cross-linking of 8-methoxypsoralen. To demonstrate one of its applications, the cross-linked origami were used for higher-temperature self-assembly, which markedly increased the yield of the assembled product when compared to the self-assembly of non-cross-linked origami at lower-temperature. By contrast, at higher-temperature annealing, native non-cross-linked tiles did not self-assemble to yield the desired product; however, they formed a nonspecific broken structure.

A cuboid structure was constructed using a DNA origami design based on a square prism structure. The structure was characterized by atomic force microscopy (AFM) and dynamic light scattering. The real-time opening event of the cuboid was directly observed by high-speed AFM.

Two-dimensional self-assembly of DNA origami structures was carried out using a connector that has connection sites at all four edges. By utilizing this four-way connector, five and eight origami monomers were assembled to form a cruciate and a hollow square structure, respectively.

We demonstrate a novel strategy of self-assembly to scale up origami structures in two-dimensional (2D) space using multiple origami structures, named "2D DNA jigsaw pieces", with a specially designed shape. For execution of 2D self-assembly along the helical axis (horizontal direction), sequence-programmed tenon and mortise were introduced to promote selective connections via π-stacking interaction, sequence-complementarity, and shape-complementarity. For 2D self-assembly along the helical side (vertical direction), the jigsaw shape-complementarity in the top and bottom edges and the sequence-complementarity of single-stranded overhangs were used. We designed and prepared nine different jigsaw pieces and tried to obtain a 3 × 3 assembly. The proof of concept was obtained by performing the assembly in four different ways. Among them, the stepwise self-assembly from the three vertical trimer assemblies gave the target 2D assembly with ∼35% yield. Finally, the surfaces of jigsaw pieces were decorated with hairpin DNAs to display the letters of the alphabet, and the self-assembled 2D structure displayed the word "DNA JIG SAW" in nanoscale. The method can be expanded to create self-assembled modules carrying various functional molecules for practical applications.

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.

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.

Novel multiarm DNA structures were designed using two-dimensional DNA origami scaffolds, and these structures were folded into hollow three-dimensional (3D) structures by introducing connection strands into the arms. The opening of the prism structures was examined by high-speed AFM imaging, which showed the dissociation of the connecting arms in the 3D structures.

We established a method to determine the butyrylcholinesterase genotype associated with a BCHE deficiency directly using multiple PCR from stored serum, which was stored at -70 degrees C for more than 30 years. PCR products from sera of six propositi were used for DNA sequence analysis. All of these BChE variants were characterized by a single nucleotide substitution. Four of them were homozygotes and demonstrated a C-->T single nucleotide point mutation at codon 100 from CCA (Pro) to TCA (Ser). The fifth case was a heterozygote of this mutation. The remaining one was a compound heterozygote showing a T-->C transition mutation at codon 203 from TCA (Ser) to CCA (Pro) and a G-->C transversion mutation at codon 365 from GGA (Gly) to CGA (Arg). Furthermore we developed a method to determine the ABO genotype from the same serum. These results indicated that serum is useful as a starting material for amplification of genomic DNA when fresh blood samples are not available.

Using plasmids containing the genes for thymidine kinase (tk) and neomycin resistance (neo), we have shown that DNA-mediated genotypic transformation of L and Chinese hamster ovary (CHO) cells is increased several-fold by the presence of the sterol-binding polyene antibiotic, amphotericin. Transformation into the same host cells, using genomic DNA, was also enhanced by amphotericin. Phenotypic expression of beta-galactosidase activity of a plasmid containing the gene for the enzyme was also markedly elevated when the antibiotic was added at transfection. Other sterol-binding polyene antibiotics also showed activity in these DNA-mediated gene transfer assays.