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DNA Nanotechnology Group

ResearchCONCEPT

Research Topics

Biomolecules such as a protein and DNA are widely used to create a desired nano-scale structure and space. Our group is working on developing novel technologies applicable for imaging, sensing, creation of nanosystem and nanodevice, and delivery system by combining designable nanoscale structure & space (bioarchitecture) and chemistry. We are employing DNA origami technology to construct the nanostructure and put functions into it by integrating desired molecules in a nanoscale precision.


DNA is one of the most promising molecules for preparing various nanoscale structures and spaces. This allows us to integrate multiple functional molecules and nanomaterials onto a designed nanostructure and also to produce a large-scale complicated architecture. Using an innovative DNA self-assembly system “DNA origami”, our group focuses on the following research topics. (1) Design and construction of the novel 2D and 3D DNA nanostructures and spaces; (2) Programmed assembly of nanostructures into the 2D and 3D architectures; (3) Regulation of chemical and enzymatic reactions in a designed nanospace; (4) Visualization and analysis of biomolecules in a designed nanostructure; (5) Analysis of physical properties of biomolecules in a designed nanospace; (6) Development of novel DNA crystals; (7) Development of molecular nanodevices; (8) Applications for optical nanomaterials; (9) Applications for molecular machines and robots; (10) Development of novel delivery system for cellular applications; (11) Diagnosis and medical applications. We are using the high-speed AFM imaging system for visualizing the dynamics of molecules and nanostructures.



Overview of molecular technology using nanoscale DNA structures and spaces: (A) Flexible design and construction of structures, spaces, and assemblies at various size scales (nano to millimeter). (B) Development to construct analysis system using nanospace. (C) Development of novel molecular nanosystems. (D) Application to create functionalized molecular devices and materials. (E) Application to control of cellular functions, and diagnostics and medical applications.

Nanostructure Design and Functionalization



We are designing and preparing various 2D and 3D DNA nanostructures using DNA origami method. We are also developing the techniques for selective positioning of various molecules and nanomaterials for expression of target functions.

Nanoscale Programmed Assembly and Control



We are developing programmed assembly systems to align DNA origami nanostructures into 1D, 2D, and 3D and further integration of the structures. We are also developing a photo-controlled assembly/disassembly system using photoresponsive DNA nanostructures.

Single-moleculeImaging of Biomolecules



We are studying on single-molecule observation of the enzymatic reactions and DNA structural changes by using target DNA strands attached to the DNA nanostructures. The imaging is carried out using a high-speed atomic force microscope which can capture molecular motions in real time.

Integrated Nanosystems



We are developing nanoscale molecular systems for control of the nanomachines by integrating molecules onto the DNA origami nanostructures. We are also developing a transcription regulation system to create genetic circuits on the DNA origami.

Mechanical Nanodevices



We are developing nanostructure-based molecular devices for physical and biological applications such as optical devices and molecular delivery. Their functions are expressed by the mechanical movement of the DNA nanostructure responsive to specific molecules and photoirradiation.

Single-molecule Sensing and Analysis System



We are developing a single-molecule detection system using DNA nanostructures and optical tweezers. We are also studying on physical property of biomolecules in a confined nanospace by mimicking the biological environments.


Cellular Applications



We are creating a delivery system to cellsUsing a capsule-shaped DNA structure that responds to light and performing selective optical manipulation inside the cells. We are also developing a photoresposive DNA materials to control the morphological changes of stem cells.


Current Research Topics

1. Construction & functionalization of novel DNA nanostructures

2.Programmed DNA nanosystem for multidimensional assembly and arrangements

3. Control of biomolecular reactions using a designed DNA nanospace

4. AFM-based single-molecule visualization of biomolecules in a designed DNA nanoscaffold

5. Single-molecule analysis of physical properties of biomolecules in a designed nanospace

6. Development of novel DNA crystals

7. Development of single-molecule switches and nanodevices

8. Development of optical nanomaterials

9. Applications for molecular nanomachines and molecular robotics

10. Development of novel delivery system for cellular applications

11. Diagnosis and medical applications




Non-invasive Regulation of Cellular Morphology Using a Photoswitchable Mechanical DNA Polymer

Angew. Chem Int. Ed. in press (2021)



The extracellular matrix, residing the cells provides a dynamic and reversible environment to the cells. These spatial and temporal cues are essential for the cells especially when they are undergoing morphogenesis, repair, and differentiation. Recapitulation of such an intricate system with reversible presentation of nanoscale cues can help us better understand important cellular processes and can allow us to precisely manipulate 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 the photoswitchable mechanical DNA polymer with photoirradiation. We found that the DNA polymer reversibly forms two different structures, a relaxed linear and shrinked compact form, which were 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-like shape, which thus provides us a tool to decipher the language of the extracellular matrix better. This study presents a general strategy to explore nanoscale interactions between stem cells and stimuli responsive mechanical DNA nanostructures.

Construction of optically controllable CRISPR-Cas9 system using a DNA origami nanostructure

Chem. Commun. 57, 5594-5596 (2021)



The Cas9 protein with a single guide RNA (sgRNA) has evolved into a powerful tool and attracts an attention for sequence-selective genome editing. We employed a DNA nanostructure and photoreaction to control the sequence-selective DNA cleavage using CRISPR-Cas9 system. In this study, we demonstrated the photo-controlled Cas9-mediated dsDNA cleavage using a DNA origami nanoring structure. Cas9 incorporated inside the nanoring did not show the cleavage activity. Cas9 was released from the nanoring by photoirradiation, and the released Cas9 expressed the activity for site-selective cleavage of dsDNA. We showed that the Cas9 activity can be suppressed by attaching the enzyme into the cavity of the DNA origami nanostructure, and the activity can be regenerated by the release of the enzyme using photoirradiation. This system can be applied for the gene expression control in cell.

Nanoscopic observation of a DNA crystal surface and its dynamic formation and degradation using atomic force microscopy

Chem. Commun. 57, 1651-1654 (2021)


Self-assembled DNA crystals have been attracting attention because the lattice of the crystals can be designed by changing the DNA sequence and functionalized by the incorporation of additional molecules. The DNA tensegrity triangle motif, which was created by Seeman’s group, was the first building block used for DNA crystallization. To understand the mechanism of crystal growth, we tried to observe the crystal surface at a molecular scale. In this study, we directly observed the formation and degradation of tensegrity triangle DNA crystals using AFM. We observed the crystal surface by AFM and characterized the lattice coordination of the assembled triangle units at a molecular level. We characterized the DNA crystal and assembly of the monomer units at nano-scale resolution, and visualized dynamics of the formation and degradation of the crystal at the layer level. We found that the assembly and disassembly can be controlled by adjusting the concentration of the monomer unit in the observation buffer, but the relationship between the formation speed and monomer concentration is still unclear. The method can be utilized for characterization of unknown crystals and their formation process.

Photocontrolled DNA Origami Assembly by Using Two Photoswitches

Chem. Eur. J. 27, 778−784 (2021)



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.

Direct observation of dynamic interactions between orientation-controlled nucleosomes in a DNA origami frame

Chem. Eur. J. 26, 15282−15289 (2020)



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 AFM. 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, and interactions were observed between them. 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, which means the DNA frame not only holds nucleosomes, but also retains their natural state. This method offers a promising platform for investigating nucleosome interactions and studying chromatin structure.

X-ray crystal structure of a cyclic-PIP–DNA complex in the reverse-binding orientation

J. Am. Chem. Soc. 142, 10544−10549 (2020)



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 designing the sequence of pyrrole, imidazole, and other synthetic units, PIP binds 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 Å resolution and reveal that cPIP binds in the reverse orientation in the DNA minor groove. Analysis of the crystal structure revealed that the position 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 g-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 towards 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.


DNA Nanostructures for Targeted Antimicrobial Delivery

Angew. Chem. Int. Ed. 59, 12698–12702 (2020)



Antibiotic resistance is a growing worldwide human health issue, and alternative antimicrobial strategies are needed urgently. 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 (d STORM) 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.     


Duplex DNA is Weakened in Nanoconfinement

J. Am. Chem. Soc. 142, 10042−10049 (2020)



For proteins and DNA secondary structures such as G-quadruplexes and i-motifs, nanoconfinement can facilitate their folding and increase structural stabilities. However, properties of physiologically prevalent B-DNA duplex have not been elucidated inside nanocavity. By 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 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 nanocage. All these indicate that nanoconfinement weakens the stability of 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, the 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 non-canonical DNA over B-DNA in cellular environment where nanocavity is abundant.


Regulation of the TET-mediated oxidation process in a DNA origami Nanochip

Nucleic Acids Res. 48, 4041–4051 (2020)



TET proteins play a central role in epigenetic regulation to control the gene expression by continuous oxidation of 5-methylcytosine (5mC) at CpG site. In this study, we regulated TET-mediated oxidation of 5mC in a DNA origami nanochip and observed the behavior of single-molecule TET protein in the DNA nanochip by atomic force microscopy (AFM). We used a reformative frame-like DNA origami in which 5mC-containing substrate double-stranded DNAs (dsDNA) were incorporated in parallel orientations. First we controlled tense and relaxed states of dsDNAs in the DNA nanochip to examine the effect on TET oxidation and TDG excision. From the enzyme binding and detection of the oxidation reactions in the DNA nanochip, TET prefer a relaxed substrate no matter what kinds of 5-oxidated-mthylated Cytosine modification. Then we used multi-5mCG sites model to obtain the substrate preference of TET and found that TET preferred fully-methylated site than hemi-methylated site. We also observed the dynamic movement of TET by high-speed AFM (HS-AFM) and observed the TET motion trail directly in a physical processive manner such as sliding and interstrand jumping. In addition, we characterize the TDG reactions in the DNA nanochip. Thus, our system could physically regulate the enzyme reactions, and be applied in observation and characterization of DNA demethylation process in a nanoscale level.


Direct Observation and Analysis of the Dynamics of the Photoresponsive Transcription Factor GAL4

Angew. Chem. Int. Ed. 58, 7626-7630 (2019)



We report the direct visualization of the dynamic behavior of transcription factor GAL4 with photo-switching function (GAL4-VVD) in the DNA origami structure. Using high-speed atomic force microscopy (HS-AFM), we observed photo-induced complex formation of GAL4-VVD and substrate DNAs. Dynamic behaviors of GAL4-VVD such as binding, sliding, stalling, and dissociation with two substrate DNA strands, containing specific GAL4 binding sites, were observed. We also observed inter-strand hopping on two double-stranded (ds) DNAs. On a long substrate DNA strand that contained five binding sites, a series of GAL4-VVD/DNA interactions including binding, sliding, stalling, and dissociation could be identified while interacting with the surface. We also found the clear difference in the movement of GAL4-VVD between sliding and stalling in the AFM images. Detailed analysis revealed that GAL4-VVD randomly moved on the dsDNA using sliding and hopping for rapidly searching specific binding sites, and then stalled to the specific sites for the stable complex formation. The results suggest the existence of the different conformational mode of the protein for sliding and stalling. This single-molecule imaging system at the nanoscale resolution provides the insight of the searching mechanism of the DNA binding proteins.


A Photocaged DNA Nanocapsule for Controlled Unlocking and Opening inside the Cell

Bioconjugate Chem. 30, 1860-1863 (2019)



We report a nano-sized DNA capsule with a photo-inducible mechanical unlocking system for creation of a carrier for delivery system to the cells. Photocage system was introduced into the nanocapsule (NC) for control of opening of the NC with photoirradiation. The open 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 localized to the cytoplasm, and the photo-induced open 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.


Translation-dependent unwinding of stem-loops by UPF1 licenses Regnase-1 to degrade inflammatory mRNAs

Nucleic Acids Res. 47, 8838–8859 (2019)



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.


Direct observation of the double-stranded DNA formation through metal ion-mediated base pairing in the nanoscale structure

Chem. Eur. J. 25, 1446-1450 (2019)



We demonstrate 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, we observed the dynamic formation of unique dsDNA and its dissociation. The formation of an unusual shape of dsDNA with consecutive T-Hg-T base pairs was visualized in the designed nanoscale structure.


Decreased water activity in nanoconfinement contributes to the folding of G-quadruplex and i-motif structures

Proc. Natl. Acad. Sci. USA, 115, 9539-9544 (2018)



Due to the small size of a nanoconfinement, 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 X 5 to 7 X 7 helix bundles (cross sections: 9 X 9 nm to 15 X 15 nm). We found water activities decrease with reducing cage size. In the 9X9 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.


Construction of integrated gene logic-chip

Nature Nanotechnology, 13, 933-940 (2018)



In synthetic biology, the control of gene expression requires a multi-step computation of biological signals. However, the complexity of genetic circuits remains low because it is difficult to completely avoid crosstalk between genetic circuits. Here, we made an orthogonal self-contained device by integrating an actuator and sensors onto a DNA origami-based nano-chip that contains an enzyme, T7 RNA polymerase, and multiple target gene substrates. This gene nano-chip orthogonally transcribes its own genes, and the nano-layout ability of DNA origami allows us to rationally design the gene expression levels by controlling the intermolecular distances between the enzyme and the substrate genes. We further integrated reprogrammable logic gates so that the nano-chip responds to water-in-oil droplets and computes their small RNA (miRNA) profiles, which demonstrates that the nano-chip can function as a gene logic-chip. Our approach to component integration on a nano-chip may provide a basis for large-scale, integrated genetic circuits.


Complexing DNA origami frameworks through sequential self-assembly based on directed docking

Angew. Chem. Int. Ed. 57, 7061-7065 (2018)



Ordered DNA origami arrays have the potential to compartmentalize space into distinct periodic domains that can incorporate a variety of nanoscale objects. Here, we aimed to use the internal cavities of a preassembled two-dimensional (2D) DNA origami framework to incorporate supplemental square-shaped DNA origamis (SQ-origamis) with shapes that were complementary to those of the cavities. In our approach, the framework was first self-assembled on a mica-supported lipid bilayer membrane from cross-shaped DNA origamis (CR-origamis) and subsequently exposed to the SQ-origamis. Using high-speed atomic force microscopy, we revealed that our SQ-origamis exhibited a dynamic adsorption/desorption behavior, which resulted in continuous changing of their arrangements in the framework. These dynamic SQ-origamis were trapped in the cavities by increasing Mg2+ concentration or by introducing sticky-ended cohesions between extended staples, both from the SQ- and CR-origamis, which enabled the directed placement of the SQ-origamis. Based on these features of our system, the framework that was self-assembled from two different CR-origami components was complexed into a checkerboard-like pattern through sequential self-assembly. Our study offers a platform to create supramolecular structures or systems consisting of multiple DNA origami components.  


DNA Origami Scaffolds as Templates for Functional Tetrameric Kir3 K+ Channels

Angew. Chem. Int. Ed. 57, 2586-2591 (2018)



In native systems, scaffolding proteins play important roles in assembling proteins into complexes to elicit signal transduction. This concept is yet to be incorporated in assembling functional transmembrane protein complexes in artificial systems. To address this issue, DNA origami can be used to construct ideal scaffolds that serve to arrange proteins at specific positions. Here, we report that Kir3 K+ channel proteins are assembled through zinc-finger protein (ZFP)-adaptors at specific locations on DNA origami scaffolds. Specific binding of the ZFP-fused Kir3 channels and ZFP-based adaptors on DNA origami were confirmed by atomic force microscopy and gel electrophoretic analyses. Furthermore, we observed that the DNA origami with ZFP binding sites nearly tripled the K+ channel current of heterotetrameric Kir3 channels compared with cells without the DNA origami. Thus, our method provides a useful template to artificially control the oligomerization states of membrane proteins.


Direct Single-Molecule Observation of Mode and Geometry of RecA-Mediated Homology Search

ACS Nano, 12, 272-278 (2018)



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.



  

Single-Molecule Observation of The Photo Regulated Conformational Dynamics of DNA Origami Nanoscissors

Angew. Chem. Int. Ed. 56, 15324-15328 (2017)



A key goal of nanotechnology is to construct molecular robots, mechanical devices, and nanomachines. To create such objects, stimuli-responsive molecular actuators are required. We demonstrate direct observation of the dynamic opening and closing behavior of photo-controllable DNA origami nanoscissors using high-speed atomic force microscopy (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 irradiation. Using photo-switchable 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, via gel electrophoresis and AFM imaging. Additionally, these reversible changes in shape during photo-irradiation were directly visualized using high-speed AFM. Moreover, four photo-switchable nanoscissors were assembled into a scissor-actuator-like higher-order object whose configuration could be controlled by the open and close switching as induced by UV and Vis light irradiation.     



Confined Space Facilitates G-quadruplex Formation

Nature Nanotechnology, 12, 582–588 (2017)



Molecular simulations have suggested that confined space increases the stability of a folded structure due to entropic effects. Owing to the potential interaction between the wall of a constrained space and the confined structure, clear-cut evidence for this prediction is lacking. Using DNA origami nanocages, here we investigated the pure effect of confined space on the property of individual human telomeric DNA G-quadruplexes. By targeted mechanical unfolding of the G-quadruplex while leaving the nanocage intact, we found that mechanical and thermodynamic stabilities of the G-quadruplex inside the nanocage increase significantly with decreasing cage size. The increased stabilities are more pronounced than in diluted or molecularly crowded buffer. Compared to the latter two solutions, folding rate of a G-quadruplex in confined space is 100 times faster, suggesting the possibility of co-replicational or co-transcriptional folding of G-quadruplex inside polymerase machinery.



Holliday junction resolvases mediate chloroplast nucleoid segregation

Science, 356, 631-634 (2017)



Holliday junctions, four-stranded DNA structures formed during homologous recombination, are disentangled by resolvases that have been found in prokaryotes and eukaryotes but not in plant organelles. Here, we identify monokaryotic chloroplast 1 (MOC1) as a Holliday junction resolvase in chloroplasts by analyzing a green alga Chlamydomonas reinhardtii mutant defective in chloroplast nucleoid (DNA-protein complex) segregation. MOC1 is structurally similar to a bacterial Holliday junction resolvase, resistance to ultraviolet (Ruv) C, and genetically conserved among green plants. Reduced or no expression of MOC1 in Arabidopsis thaliana leads to growth defects and aberrant chloroplast nucleoid segregation. In vitro biochemical analysis and high-speed atomic force microscopic analysis revealed that A. thaliana MOC 1 (AtMOC1) binds and cleaves the core of Holliday junctions symmetrically. MOC1 may mediate chloroplast nucleoid segregation in green plants by resolving Holliday junctions.      


Torsional Constraints of DNA Substrates Impact Cas9 Cleavage.

J. Am. Chem. Soc. 138, 13842-13845 (2016)



To examine the effect of the torsional constraints imposed on DNA substrates on Cas9 cleavage used for genome editing, we prepared constrained DNA substrates using a DNA origami frame. By fixing the dsDNA at the connectors of the DNA frame, we created torsionally constrained or relaxed substrates. We quantified the cleavage of constrained and relaxed substrates by Cas9 with qPCR. Moreover, we observed the Cas9/sgRNA complex bound to the DNA substrates and characterized the dissociation of the complex with high-speed AFM. The results revealed that the constrained non-target strand reduced the cleavage efficiency of Cas9 drastically, whereas torsional constraints on the target strand had little effect on the cleavage. The present study suggests that highly ordered and constrained DNA structures could be obstacles for Cas9 and additionally provides insights in Cas9 dissociation at a single molecule level.


A photoregulated DNA-based rotary nanosystem and direct visualization of its rotational movements.

Chem. Eur. J. 23, 3979-3985 (2017)



Light has been used for DNA-based nanomachines to perform tasks in nanometer scale. The programed controllability and the energy inputs are still major challenges for DNA mechanical devices. Here a rotary DNA nanomachine is constructed on a nanostructure, in which the rotations are driven and also can be reversibly regulated by light inputs in different wavelengths. The DNA rotor in a bar-shaped stiff double-crossover molecule supported by a rigid molecular wire is placed on the top of a rectangular DNA tile. By employing two pair of photoresponsive oligonucleotides as switching motifs, the rotor can be regulated to rotate specified degree to be locked in the two reconfigurable states. Furthermore, the real-time rotational motions of the rotor are directly visualized by high-speed atomic force microscopy. Two types of photoresponsive DNA switches are combined together to trigger the rotary motions in a DNA nanostructure. Our rotary nanostructure represents a unique prototype for DNA-based nanodevices.


Triple Helix Formation in a Topologically Controlled DNA Nanosystem.

Chem. Eur. J. 22, 5494-5498 (2016)



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 the two different types of triplet base pairs. The target DNA strand and the third strand were incorporated into the DNA frame, and third-strand binding was controlled by the formation of Watson–Crick base pairing. The triple-helix formation was monitored by observing the changes in the incorporated DNA structure. The triple-helix formation was also examined using a photocaged third strand, and the third-strand binding was directly observed using high-speed atomic force microscopy with photoirradiation. The binding of the third strand could be controlled by controlling the duplex formation and photocaged strands in the designed nanospace.


A light-driven three-dimensional plasmonic nanosystem that translates molecular motion into reversible chiroptical function.

Nature Communications, 7, 10591 (2016)



We demonstrate an all-optically controlled plasmonic nanosystem constructed on a DNA designed DNA nanostructure. Our system can amplify the sub-nanometre conformation changes of azobenzene through the active host nanostructure and consequently translate the light-induced molecular motion of azobenzene into reversible plasmonic chiroptical response, which can be in situ read out by optical spectroscopy. The plasmonic nanostructure comprises two gold nanorods (AuNRs) assembled on a reconfigurable DNA origami template. A photoresponsive active site is introduced on the template with an photoresponsive DNA segment. Light can cyclically ‘write’ and ‘erase’ the conformation states of the nanostructure through photoisomerization at a localized region. Different conformation states are read by probe light.


Emergent Mechanical Properties of DNA Origami Nanoassemblies are Determined by Holliday Junction Mechanophores.

Nucleic Acids Res. 44, 6574-6582 (2016)



DNA origami structure can be shaped into a desired geometry by using short DNA staples that bring together distal regions of a single-stranded DNA template through Holliday junctions. Using laser tweezers, we have dissected two mechanical properties of individual DNA origami assembly. First, the mechanical stability of DNA nanotubes (42-50 pN) is anisotropic.  Comparison between different DNA tubes and 2-D DNA tiles has revealed that mechanical stability is determined by the density of Holliday junctions along a particular stress direction. Second, in a lower force range (26-30 pN), DNA tubes undergo mechanical isomerization between short and long tubular forms, which has been attributed to collective actions of individual Holliday junctions. Our results indicate that Holliday junctions serve as mechanophores to govern the mechanical behaviors of DNA origami structures. The emergent 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.


Direct visualization of walking motions of a photo-controlled DNA nanomachine on the DNA nanostructure.

Nano Lett. 15, 6672-6676 (2015)



A light-driven artificial molecular nanomachine was demonstrated based on DNA scaffolding. A single stranded DNA carrying two pyrene molecules employed as the walker was assembled with a 2D DNA tile carrying four stator strands as the linear track. The walker-stator duplex was located on the surface of DNA tile. The excited pyrene molecules (λex = 350 nm) of the walker can induce the cleavage of disulfide bond in stator strands, initiating the DNA walker migrating from one cleaved stator to the next intact one on the DNA tile continuously until moving to the final stator. The entire walking process of the walker was determined by characterizing the distribution ratios of walker-stator duplex at four anchorage sites on the tile separately under different irradiation times. Importantly, the light-fuelled movements of the nanomachine were directly observed in real-time during UV irradiation using high-speed atomic force microscopy (AFM). Our photocontrolled nanomachine shows promise for applications such as cargo transport and manual configuration change in mesoscopic systems.


Single-Molecule Manipulation of the Duplex Formation and Dissociation at the G-quadruplex/i-motif Site in the DNA Nanostructure.

ACS Nano, 9, 9922-9929 (2015)



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


Lipid bilayer-assisted two-dimensional self-assembly of DNA origami nanostructures.

Nature Communications, 6, 8052 (2015)



Self-assembly is a ubiquitous approach for design and fabrication of novel supermolecular architectures. We describe a strategy referred as ‘lipid-bilayer-assisted self-assembly’ to assemble DNA origami nanostructures into two-dimensional lattices. DNA origami structures are electrostatically adsorbed onto a mica-supported twitterionic lipid bilayer in the presence of divalent cations. We demonstrate that the bilayer-adsorbed origami units are mobile on the surface and can self-assemble into large lattices with micrometer size in their lateral dimensions. By using high-speed atomic force microscopy (HS-AFM) imaging technique, a variety of dynamic processes such as growth and reorganization of lattices were successfully visualized. The surface modifiability of the assembled lattice was also proven by in situ decoration with streptavidin molecules. Our approach provides a new strategy for preparing versatile scaffold for nanofabrication and paves the way for organizing functional nanodevices in a micrometer space.


Single-Molecule Visualization of the Activity of Zn2+-Dependent DNAzyme.

Angew. Chem. Int. Ed. 54, 10550-10554 (2015)



We demonstrate the single-molecule imaging of the catalytic reaction of the Zn2+-dependent DNAzyme in a DNA origami nanostructure. The single-molecule catalytic activity of a Zn2+-dependent DNAzyme was examined in the designed nanostructure called DNA frame. The DNAzyme and a substrate strands attached to two dsDNAs were incorporated into the DNA frame in two different configurations. The reaction progress was monitored by observing the change of the incorporated dsDNAs structures, and the configuration arrangement of the DNA strands in the DNA frame was clearly observed after the reaction. The separation of the dsDNAs, induced by the cleavage by the DNAzyme, was directly visualized by high-speed atomic force microscopy (AFM). This AFM-based imaging system is a practical tool for monitoring chemical and biochemical catalytic reactions.


Engineering RNA-Protein Complexes with Different Shapes for Imaging and Therapeutic Applications

ACS Nano, 8, 8130–8140 (2014)



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


Preparation of Chemically Modified RNA Origami Nanostructures.

Chem. Eur. J. 20, 15330-15333 (2014)



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


Photoresponsive DNA Nanocapsule Having an Open/Close System for Capture and Release of Nanomaterials.

Chem. Eur. J. 20, 14951-14954 (2014)



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 into the NC was carried out via hybridization of a specific DNA (capture strand), and the release of the AuNP was examined by addition of toehold-containing complementary DNA (release strand). The release of the AuNP from the NC was successfully achieved by the opening of the NC and subsequent strand displacement.


Single-Molecule Mechanochemical Sensing Using DNA Origami Nanostructures.

Angew. Chem. Int. Ed. 53, 8137-8141 (2014)



While single-molecular biosensing offers ultimate detection limit, its throughput is often compromised due to restricted platforms, in which sensing events are carried out one at a time in most cases. To address this problem, we introduced DNA origami nanostructures as expanded platforms in a new sensing strategy that exploits mechanochemical principles. As a proof-of-concept, we incorporated six sensing probes at different locations of a 7-tile DNA origami template. Binding of a target molecule to any of these probes induces rearrangement of the 2D or 3D origami nanostructure, which is monitored in real time by optical tweezers. This platform is able to detect 10 pM Platelet Derived Growth Factor (PDGF) within 10 minutes while differentiating the PDGF and a DNA target in a multiplexing fashion. By tapping into the rapid development of the versatile DNA origami nanoassembly, we anticipate this mechanochemical platform offers the long-sought solution for high-throughput sensing at the single molecular level.


Helical DNA origami tubular structures in various sizes and arrangements.

Angew. Chem. Int. Ed. 53, 7484-7490 (2014)



We developed a novel method to design various helical tubular structures using DNA origami method. The size-controlled tubular structures 6-tube, 8-tube, and 10-tube, which have 192, 256, and 320 base-pairs for one-round were prepared, respectively. By detailed analysis of the tube surface patterns, expected short tubes and unexpected long tubes were formed. The short tube has mainly a left-handed helical structure. We found that the unexpected long tube was extended to the axial directions of the double-helices and formed mainly a right-handed structure. By changing the annealing conditions, the folding process was estimated from the proportions of short and long tubes. Depending on the numbers of base-pairs involved in one-round of tubes, the population of the left-/right-handed and short/long tubes changed. The helical stress caused by stiffness of double-helices and non-natural helical pitch in DNA origami design determines the variable formation of tubular structures.


Dynamic Assembly/Disassembly Processes of Photoresponsive DNA Origami Nanostructures Directly Visualized on a Lipid Membrane Surface.

J. Am. Chem. Soc. 136, 1714-1717 (2014)



Direct visualization of the assembly/disassembly processes of photoresponsive DNA origami nanostructures was achieved on a lipid bilayer surface. The observation relies on controlled interactions between the bilayer components and cholesterol moieties introduced to the hexagonal origami structures with azobenzene-modified oligonucleotides on one outer edge. The bilayer-placed hexagonal dimer was disassembled into monomer units by ultraviolet irradiation,and reversibly assembled again through irradiation with visible light. These dynamic processes were monitored directly by high-speed atomic force microscopy. Successful application of our approach should facilitate the study of interactive and functional behaviors of various DNA nanostructures.


DNA origami based visualization system for studying site-specific recombination events.

J. Am. Chem. Soc. 136, 211-218 (2014)



Cre recombinase recognizes specific loxP sequence and forms a synaptic complex with two-loxP dsDNAs as a tetramer. Cre performs two-step cleavage/strand exchange reactions including formation of Holiday junction (HJ) and recombinant product. For the direct imaging of Cre-mediated recombination events, we introduced a pair of substrate dsDNAs in the DNA frames by controlling orientations. In the antiparallel arrangement, he Cre-mediated recombination proceeded even in the nanospace. DNA recombination was directly observed by high-speed AFM. During the observation of the Cre-loxP complex, the Cre tetramer forming synaptic complex dissociated into four monomers, and the recombinant product appeared In addition, control of the angles of Holiday junction intermediate in the DNA frames changed the direction of DNA recombination. The topological control of the substrate dsDNA in the DNA frames is important to study the recombination events by controlling the orientation of the substrate dsDNAs and angle of HJ intermediates.


Direct analysis of Holliday Junction resolving enzyme in a DNA origami nanostructure.

Nucleic Acids Res. 42, 7421-7428 (2014)



Holliday Junction (HJ) resolution is a fundamental step for completion of homologous recombination. HJ resolving enzymes (resolvases) generally distort the junction structure upon binding, raising the possibility that the reactivity of the enzyme can be affected by a particular geometric and topology at the junction. Here, we employed a DNA origami nano-scaffold in which each arm of a HJ can be tethered through the base-pair hybridization, allowing us to make the junction core flexible/inflexible by adjusting the length of the DNA arms. Both flexible and inflexible junctions bound to Bacillus subtilis RecU HJ resolvase, while 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.


Single molecule visualization and characterization of Sox2-Pax6 complex formation on a regulatory DNA element using a DNA origami frame.

Nano Lett. 14, 2286-2292 (2014)



We report here the use of AFM to study Sox2-Pax6 complex formation on its DNA element, DC5, which is a key event in the development of the central nervous system. We used an origami DNA scaffold containing two DNA strands of the DC5 element with different levels of tensile force. By observing the Sox2 binding to DNA using AFM, we confirmed that DNA bending is necessary for Sox2 binding to DNA. Further, we showed that the formation of the Sox2/DC5 complex is a prerequisite for the binding of Pax6 to DNA by observing the enhancement of the binding capability of Pax6 to DNA. AFM imaging also showed that occupancy of Sox2-Pax6 on the DNA element is much higher than Sox2 alone, giving an indication of the cooperative interaction that is a key characteristic of paired transcription systems.


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

Angew. Che. Int. Ed. 53, 4107-4012 (2014)



Using DNA origami frame as a nanoscaffold for the structural control, we present the unprecedented solution-state structures of a tetramolecular antiparallel and (3+1)-type G-quadruplex intermediates such as G-hairpin and G-triplex visualized directly at single-molecule level with nanometer resolution. These intermediates are stable and formed with relatively good yield. Finally, we proposed a possible model of G-quadruplex folding.


HIV-1 Nucleocapsid Proteins as Molecular Chaperones for Tetramolecular Antiparallel G‑Quadruplex Formation.

J. Am. Chem. Soc. 135, 18575−18585 (2013)



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.


Controlling the stoichiometry and strand polarity of a tetramolecular G-quadruplex structure by using a DNA origami frame.

Nucleic Acids Res. 41, 8738–8747 (2013)



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.


Regulation of B-Z conformational transition and Zαβ protein binding by introduction of constraint to double-stranded DNA using a DNA nanoscaffold.

Chem. Eur. J. 19, 16887-16890 (2013)



The B–Z DNA conformational transition requires the rotation of a double helix from a right-handed B-form to a left-handed Z-form structure. We herein designed a constrained and rotatable double-stranded DNA in which the rotational freedom was controlled by its placement into a DNA nanoscaffold. The Zab protein specifically binds to CG repeat sequences in Z-form double helices. In this system, the Zab protein bound preferentially to the rotatable double helix, even when using the same sequence. This result indicates that the Zab protein binds to the rotatable CG repeat sequence to form a left-handed Z-form structure. In addition, a rotatable 5-methyl-CG repeat sequence facilitated the binding of the Zab protein, indicating that Zab protein binding can be clearly controlled on the DNA nanostructure.


Direct and real-time observation of rotary movement of a DNA nanomechanical device.

J. Am. Chem. Soc. 135, 1117-1123 (2013)



A right-handed B-form double-stranded containing CG repeat sequence is known to transit to the left-handed helical structure by increasing the salt concentration. To visualize the B-Z transition, a dsDNA with a 5-methyl-CG six repeat sequence and a flag marker having three dsDNAs was introduced to the DNA frame structure. A dsDNA having a CG sequence was introduced to the top as a B-Z transition system, and a dsDNA having a random sequence was introduced to the bottom as a control. To allow the rotation during B-Z transition, four connectors were designed to lift up the dsDNAs from the surface of DNA frame. A ratio of flag marker rotated to the upper side increased according to the increase of the concentration of Mg ion. Further, by controlling the concentration of Mg ions, the rotation of the flag was directly observe by high-speed AFM under the equilibrium condition for the B-Z state .


Direct observation of the dual-switching behaviors inducing state transition in a single nanoframe.

Chem. Commun. 50, 4211-4213 (2014)



A DNA nanoframe was designed and adopted as a scaffold to observe the communication behaviors between three finite states by high-speed atomic force microscopy in real time. Here, two DNA multistrand motifs—pseudo-complementary photoresponsive oligonucleotides and G-telomeric repeat strands for G-quadruplex formation—were integrated together into three parallel double-stranded DNAs in the nanoframe system. Different photoirradiation wavelengths and K+ were employed as stimuli to regulate the “kissing” and “unkissing” behaviors of three double-stranded DNAs in a logical manner. Cascade reactions from photoinduced dissociation to G-quadruplex formation were implemented successfully. Furthermore, the single logical configuration conversions corresponding to conformational changes were sequentially visualized by combining high-speed atomic force microscopy and DNA origami methodology.


Photocontrollable DNA Origami Nanostructures Assembling into Predesigned Multiorientational Patterns.

J. Am. Chem. Soc. 134, 20645-20653 (2012)



We demonstrate a novel strategy for constructing multidirectional programmed two-dimensional (2D) DNA nanostructures in various unique patterns by introducing photoresponsive DNAs into hexagonal DNA origami structures. Hexagonal DNA units were designed and constructed that were then employed as self-assembly units for building up nanosized architectures in regulated arrangements. The regulation of repeated assembly and disassembly of the hexagonal structures having photoresponsive DNAs was examined by irradiation of different wavelength in a programmed manner. By adjusting the numbers and the positions of photoresponsive DNAs in the hexagonal units, specific face-controlling nanostructures can be achieved, which permitted the construction of curved and ring-shaped nanostructures.


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

Angew. Chem. Int. Ed. 51, 10518-10522 (2012)



The hybridization and dissociation of photoresponsive oligonucleotides and their mechanical switching behavior was directly imaged using high-speed atomic force microscopy (AFM). A pair of photoresponsive oligonucleotides containing azobenzene moieties (X. Liang, T. Mochizuki, H. Asanuma, Small, 2009, 5, 1761) was introduced to double-stranded DNA (dsDNA) and then placed in the cavity of the DNA nanostructure (DNA frame). Two dsDNAs in contact at the center were dissociated during ultraviolet irradiation, and this was directly observed using this observation system and high-speed AFM. After visible-light irradiation, the two separated dsDNAs contacted again in the center. Reversible switching of the hybridization and dissociation of the photoresponsive domains was visualized at the single-molecule level by observing the global change of the two dsDNAs in the DNA frame


RNA-templated DNA origami structures.

Chem. Commun. 49, 2879-2881 (2013)



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 modified RNA/DNA hybrid origami structures were prepared by using RNA templates containing chemically modified uracils.


Direct visualization of the movement of a single T7 RNA polymerase and transcription on a DNA nanostructure.

Angew. Chem. Int. Ed. 51, 8778-8782 (2012)



Movement of single T7 RNA polymerase (RNAP) and its transcription were visualized directly at a molecular resolution using a double-stranded DNA (dsDNA) template attached to a DNA origami scaffold. High-speed atomic force microscopy (AFM) was employed for the observation. A template dsDNA (1.0 kbp) containing the T7 promoter was attached to a designed nanoscale observation scaffold (ca. 400 x 15 nm) at two positions. Flexible movement of the template dsDNA on the scaffold was observed directly using high-speed AFM. Using the template dsDNA-attached scaffold system, transcription occurred both in solution and on mica surface. RNAP sliding on the template dsDNA was observed as unidirectional movement of RNAP. Finally, we observed transcription on the template-attached scaffold using AFM. In the presence of NTP, RNAP moved in one direction from the promoter, and the RNA synthesis occurred on this scaffold. Using this new observation system, we successfully visualized the sliding and transcription of single RNAP on a template-attached DNA nanoscaffold.


Transcription Regulation System Mediated by Mechanical Operation of DNA Nanostructure.

J. Am. Chem. Soc. 134, 2852-2855 (2012)


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 six-helix bundled 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.


Sequence-Selective Single-Molecule Alkylation with a Pyrrole-Imidazole Polyamide Visualized in a DNA Nanoscaffold.

J. Am. Chem. Soc. 134, 4654-4660 (2012)


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 the alkylating agent and biotin for sequence-selective alkylation at the target sequence and subsequent streptavidin labeling, respectively. Selective alkylation of the target site of the substrate DNA was observed by analysis using sequencing gel electrophoresis. For the single-molecule observation of the alkylation, 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 fully matched sequence was alkylated as 88% selectivity over other mismatched sequences. 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 DNA-based molecular motor that can navigate a network of tracks.

Nature Nanotechnology, 7, 169-173 (2012)



We designed the branched motor-track on the DNA origami scaffold and controlled the movement of a DNA motor using the instructions for navigation of the DNA motor. To control the programmable movement of the motor strand, a branched track was constructed on the DNA origami scaffold, and three branching points and four final destinations were created. The block strands were introduced at both sides of the branching points to control the direction of the DNA motor. When one side of the block strand was removed by the corresponding release strand, the motor strand could pass the branching point in the predefined direction. The DNA passed the two branching points; therefore, the two releasing strands can determine the pathway and destinations in a programmed fashion. Reaching the final destination was observed using AFM and the fluorescence quenching method. The DNA motor was found at the predefined destinations by following the programmed instructions.


Direct observation of stepwise movement of a synthetic molecular transporter.

Nature Nanotechnology, 6, 166-169 (2011)



A DNA transportation system was constructed with a mobile DNA nanomachine moving along a designed track on the DNA origami surface. We have designed and constructed a controlled DNA nanomachine system in which a DNA motor moves along the track on the DNA origami scaffold. The motor track on the DNA scaffold was constructed for observation of the multi-step movement of a DNA motor strand. We introduced the 17 stators on the DNA origami scaffold as a motor track to observe movement of DNA motor. We examined the DNA motor movement by incubation with nicking enzyme. The movement of the DNA motor was visualized as single spot of duplex on the motor track by AFM. The distribution of the DNA motor position revealed one-directional and time-dependent movement of the motor strand on the track. During scanning of AFM, the DNA motor spot moved forward along the track. The stepwise movement of theDNA motor was directly observed using this origami scaffold and high-speed AFM.


Visualization of Dynamic Conformational Switching of the G-Quadruplex in a DNA Nanostructure.

J. Am. Chem. Soc. 132, 16311–16313 (2010)


The real-time observation of G-quadruplex formation was investigated 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 realtime with fast-scanning AFM.


A versatile DNA nanochip for direct analysis of DNA base-excision repair.

Angew. Chem. Int. Ed. 49, 9412–9416 (2010)


The single DNA-repair enzymes 8-oxoguanine glycosylase and T4 pyrimidine dimer glycosylase were analyzed by a nanoscale DNA chip containing two substrate double-stranded DNAs. To examine the structural effect on glycosylase/AP lyase activity including cleavage of the DNA strand and the trapping of reaction intermediates, two different lengths of substrate dsDNAs, tense 64 bp and relaxed 74 bp dsDNAs, were placed into the DNA frame. The enzymes more favorably cleaved the relaxed dsDNA and were covalently trapped after NaBH4 reduction compared with the tense dsDNA.Dynamic movement of the enzymes and the single DNA-repair reaction on the DNA nanochip was visualized by high-speed AFM.


Regulation of DNA Methylation Using Different Tensions of Double Strands Constructed in a Defined DNA Nanostructure.

J. Am. Chem. Soc. 132, 1592–1597 (2010)


A novel strategy for regulation of an enzymatic DNA modification reaction has been developed by employing a designed nanoscale DNA scaffold “DNA frame”. Two different tensions of double-stranded DNAs (dsDNAs), tense and relaxed dsDNAs were incorporated into the DNA frame, and EcoRI methyltransferase (M.EcoRI) was employed for the single molecule analysis of the reactions caused by dsDNA tensions. High-speed atomic force microscope (AFM) imaging revealed the different dynamic movement of M.EcoRI complexes on tense and relaxed dsDNAs. AFM analysis and biochemical analysis revealed that the methylation preferentially occurred in the relaxed dsDNA. The 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.


Photo-cross-linking-assisted thermal stability of DNA origami structures and its application for higher-temperature self-assembly.

J. Am. Chem. Soc. 133, 14488-14491 (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.


Programmed Two-Dimensional Self-Assembly of Multiple DNA Origami Jigsaw Pieces.

ACS Nano, 5, 665-671 (2011)



A novel strategy of self-assembly to scale up origami structures in two dimensional (2D) space was developed 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 -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 x 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.


Two-dimensional DNA origami assemblies using a four-way connector.

Chem. Commun. 47, 3213-3215 (2011)



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.


Programmed-Assembly System Using DNA Jigsaw Pieces.

Chem. Eur. J. 16, 5362–5368 (2010)



A novel method for assembling multiple DNA origami structures has been developed by using designed 2D DNA origami rectangles,“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 p-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.


DNA Prism Structures Constructed by Folding of Multiple Rectangular Arms.

J. Am. Chem. Soc. 131, 15570–15571 (2009)



Novel multi-arm DNA structures were designed using 2D-DNA origami scaffolds, and these structures were folded into hollow 3D-structures by introduction of connection strands into the arms. The opening of the prism structures were examined by high-speed AFM imaging, which showed the dissociation events of the connecting arms in the 3D-structures.



バナースペース

DNA Nanotechnology Group
Graduate School of Science
Institute for Integrated Cell-Material Sciences
Kyoto University

Yoshida-ushinomiyacho, Sakyo-ku, Kyoto 606-8501