Kumi Hidaka

Direct observation of stepwise movement of a synthetic molecular transporter

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

A DNA-based molecular motor that can navigate a network of tracks

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.

Visualization of dynamic conformational switching of the G-quadruplex in a DNA nanostructure.

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.

Photo-Cross-Linking-Assisted Thermal Stability of DNA Origami Structures and Its Application for Higher-Temperature Self-Assembly

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.

DNA Prism Structures Constructed by Folding of Multiple Rectangular Arms

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.

A Versatile DNA Nanochip for Direct Analysis of DNA Base-Excision Repair†

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

Photo-controllable DNA origami nanostructures assembling into predesigned multiorientational patterns.

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.

Design and development of nanosized DNA assemblies in polypod-like structures as efficient vehicles for immunostimulatory CpG motifs to immune cells.

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.

Direct and Single‐Molecule Visualization of the Solution‐State Structures of G‐Hairpin and G‐Triplex Intermediates

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.

Single-Molecule Visualization of the Hybridization and Dissociation of Photoresponsive Oligonucleotides and Their Reversible Switching Behavior in a DNA Nanostructure†

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.