Kumi Hidaka

Kyoto University, Kioto, Kyōto, Japan

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Publications (36)325.27 Total impact

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    ABSTRACT: 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.
    Angewandte Chemie International Edition 04/2014; 53(16):4107–4112. · 13.73 Impact Factor
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    ABSTRACT: We report the use of atomic force microscopy (AFM) to study Sox2-Pax6 complex formation on the DC5 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.
    Nano Letters 03/2014; · 13.03 Impact Factor
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    ABSTRACT: 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.
    Chemical Communications 03/2014; · 6.38 Impact Factor
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    ABSTRACT: Among the approaches for DNA-based drug targeting, G-quadruplex-binding ligands are of particular interest because of high abundance of G-rich sequences in regions such as human chromosomal telomeres and promoters of several proto-oncogenes. Number of quadruplex-ligands has been reported, but their functions at single-molecule level have not been explored using direct and real-time methods. Here, we report on the direct observation of the formation of a four-stranded G-quadruplex induced by bisquinolinium pyridine dicarboxamide with a linker containing biotin at one end. We fabricated a DNA origami frame with incorporated duplex DNAs that contained 3-6 G–G mismatches in the middle. In the absence of ligand, the duplex DNAs of interest had no interaction, as visualized by their parallel-shape in high-speed atomic force microscopy (HS-AFM) image. Presence of ligand induced the formation of G-quadruplex structure, which was characterized by an X-shape. Addition of streptavidin to the ligand-induced quadruplex caused the protein to localize in the middle of X-shape, indicating that the ligand is bound to the quadruplex. A sequence of real-time images of the ligand-induced formation of a quadruplex and its reverse conformational switching by removing the ligand was captured by HS-AFM. Unprecedented intermediate-like states were recorded in our real-time analysis.
    RSC Advances 12/2013; In press. · 2.56 Impact Factor
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    ABSTRACT: 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 (AFM) analyses revealed that the loxP-containing substrate strands in the antiparallel orientation can be recombined only through formation of synaptic complexes. By tethering 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.
    Journal of the American Chemical Society 12/2013; · 10.68 Impact Factor
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    ABSTRACT: HIV-1 nucleocapsid proteins (NCps) facilitate remodeling of nucleic acids to fold thermodynamically stable conformations, and thus called nucleic acid chaperones. To date only little is known on the stoichiometry, NCp-NCp interactions, chaperone activity on G-quadruplex formation, and so on. We report here the direct and real-time analysis on such properties of proteolytic intermediate NCp15 and mature NCp7 using DNA origami. The protein particles were found to predominantly exist in monomeric form, while dimeric and multimeric forms were also observed both in free solution and bound to the quadruplex structure. The formation and the dissociation events of the G-quadruplexes were well documented in real-time and the intermediate-like states were also visualized. We anticipate that this pioneering study will strengthen our understanding on the chaperone activity of HIV-1 proteins which in turn will be helpful for the drug design based on G-quadruplex and also for the development of drugs against AIDS.
    Journal of the American Chemical Society 11/2013; 135(49):18575-18585. · 10.68 Impact Factor
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    ABSTRACT: B-Z transition: A constrained and rotatable double-stranded DNA, in which the rotational freedom was controlled by its placement into a DNA nanoscaffold, was designed. The Z α β protein bound preferentially to the rotatable strand rather than the constrained strand, even using the same CG sequence.
    Chemistry 11/2013; · 5.93 Impact Factor
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    ABSTRACT: 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.
    Nucleic Acids Research 07/2013; · 8.28 Impact Factor
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    Dataset: c2cc37257f
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    ABSTRACT: 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.
    Chemical Communications 02/2013; · 6.38 Impact Factor
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    ABSTRACT: Analogous to the biologically abundant protein-based linear molecular machines that translocate along their target surface, we have recently constructed the DNA-based synthetic molecular motors that effect linear movement or navigate a network of tracks on a DNA origami substrate. However, a DNA-based molecular machine with rotary function, analogous to rotary proteins, is still unexplored. Here, we report the construction of a rotary motor based on the B-Z conformational transition of DNA and the direct and real-time observation of its function within a frame-shaped DNA origami. The motor can be switched off by introducing conditions that stabilize B-DNA, while it can be fueled by adding Z-DNA-promoting high-saline buffer. When MgCl(2) was used as external stimulus, 70% of the motors rotated, while 76% of the stators/controls exhibited no rotation. Such a motor system could be successfully applied to perform multiple actions aimed for our benefit. Moreover, for the first time we have directly observed the B-Z conformational transition of DNA in real-time, which shed light on the fundamental understanding of DNA conformations.
    Journal of the American Chemical Society 01/2013; · 10.68 Impact Factor
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    ABSTRACT: We have developed a new strategy to control the two-dimensional (2D) crystallization of DNA origami by introducing loops on the surface and aligning them in various orientations. Among the orientations tested, vertically connected loops successfully produced the 2D crystal lattice on a micrometer scale, while all other orientations failed.
    Chemical Communications 12/2012; · 6.38 Impact Factor
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    ABSTRACT: We demonstrate a novel strategy for constructing multidirectional programmed two-dimensional (2-D) DNA nanostructures in various unique patterns by introducing photoresponsive oligonucleotides (Azo-ODNs) into hexagonal DNA origami structures, and examined the regulation of repeated assembly and disassembly of the DNA nanostructures by different photoirradiation conditions in a programmed manner. Hexagonal DNA units were designed and constructed that 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 with hexagonal units, specific face-controlling nanostructures can be achieved, which permitted the construction of curved and ring-shaped nanostructures. The strategy presented here not only affords a new method for structural DNA nanotechnology, but also shows functional potential for practical applications in nanoscience.
    Journal of the American Chemical Society 12/2012; · 10.68 Impact Factor
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    ABSTRACT: 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. 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.
    Angewandte Chemie International Edition 09/2012; 51(42):10518-22. · 13.73 Impact Factor
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    ABSTRACT: Snapshots of transcription: Movement of a single molecule of T7 RNA polymerase (RNAP) along a double-stranded DNA (dsDNA) template attached to a DNA origami platform (green, see scheme) was recorded using high-speed AFM. A one kilobase template dsDNA containing the T7 promoter was used to record AFM images of transcription of the template dsDNA by RNAP and the resulting biotinylated RNA product was detected by streptavidin-labeling.
    Angewandte Chemie International Edition 07/2012; 51(35):8778-82. · 13.73 Impact Factor
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    ABSTRACT: 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.
    ACS Nano 06/2012; 6(7):5931-40. · 12.06 Impact Factor
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    ABSTRACT: 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.
    Nature Nanotechnology 03/2012; 7(3):169-73. · 31.17 Impact Factor