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Ribozymes with modular structures are attractive platforms for the construction of nanoscale RNA objects with biological functions. We designed group I ribozyme dimers as unit ribozyme dimers (Urds), which self-assembled to form their polymeric states and also oligomeric states with defined numbers of Urds. Assembly of Urds yielded catalytic ability of a pair of distinct ribozyme units to cleave two distinct substrates. The morphologies of the assembled ribozyme structures were observed directly by atomic force microscopy (AFM).

We report the use of DNA origami nanostructures, functionalized with aptamers, as a vehicle for delivering the antibacterial enzyme lysozyme in a specific and efficient manner. We test the system against Gram‐positive (Bacillus subtilis) and Gram‐negative (Escherichia coli) targets. We use direct stochastic optical reconstruction microscopy (dSTORM) and atomic force microscopy (AFM) to characterize the DNA origami nanostructures and structured illumination microscopy (SIM) to assess the binding of the origami to the bacteria. We show that treatment with lysozyme‐functionalized origami slows bacterial growth more effectively than treatment with free lysozyme. Our study introduces DNA origami as a tool in the fight against antibiotic resistance, and our results demonstrate the specificity and efficiency of the nanostructure as a drug delivery vehicle., Antibiotic resistance is a growing health issue that is now rendering humans vulnerable once again to infections that have been treatable for decades. Various approaches have been proposed to overcome this threat and effectively treat bacterial infections. DNA nanostructures, functionalized with aptamers, were used as a vehicle for delivering the antibacterial enzyme lysozyme in a specific and efficient manner, to destroy bacterial targets.

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

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

It is important to create new, specifically designed and controlled nanomaterials that can be used as molecular delivery systems for cells. Here, we describe a method for creating a nanosized DNA capsule (NC) using a photocaged unlocking system as a carrier for cell delivery. The photocaged NC (caged-NC) was designed and constructed to control the opening of the closed NC by photoirradiation. The opening of the NC was observed by atomic force microscopy, and the dynamic opening of the caged-NC was characterized by fluorescence quenching and recovery processes. The caged-NC was then introduced into the cytoplasm of a cell, where the photoinduced opening of the caged-NC was observed. The selective opening of the caged-NC in a single cell was successfully achieved by laser irradiation of individual cells. The caged-NC system could be used as a delivery system for relatively large nanomaterials in cells, similar to a native virus system.

Antibiotic resistance is a growing worldwide human health issue that is now rendering us vulnerable once again to infections that have been treatable for decades. Various approaches have been proposed in an effort to overcome this threat and effectively treat bacterial infections. We use a DNA origami nanostructure, functionalized with aptamers, as a vehicle for delivering the antibacterial peptide lysozyme in a specific and efficient manner, in order to destroy bacterial targets. We test the system against Gram-positive (Bacillus subtilis) and Gram - negative (Escherichia coli) targets. We use direct stochastic optical reconstruction microscopy (dSTORM) and atomic force microscopy (AFM) to characterize the DNA origami nanostructures and structured illumination microscopy (SIM) to assess the binding of 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.Abstract Figure

Ordered DNA origami arrays have the potential to compartmentalize space into distinct periodic domains that can incorporate a variety of nanoscale objects. Herein, we used the cavities of a preassembled 2D DNA origami framework to incorporate square-shaped DNA origami structures (SQ-origamis). The framework was self-assembled on a lipid bilayer membrane from cross-shaped DNA origami structures (CR-origamis) and subsequently exposed to the SQ-origamis. High-speed AFM revealed the dynamic adsorption/desorption behavior of the SQ-origamis, which resulted in continuous changing of their arrangements in the framework. These dynamic SQ-origamis were trapped in the cavities by increasing the Mg2+ concentration or by introducing sticky-ended cohesions between extended staples, both from the SQ- and CR-origamis, which enabled the directed docking of the SQ-origamis. Our study offers a platform to create supramolecular structures or systems consisting of multiple DNA origami components.

Understanding the mechanism responsible for the progression of amyloid deposition is important for developing methods to suppress this process in the treatment of Alzheimer's disease. The effects of physical damage during the transition phase of amyloid β fibril formation are unclear. In this study, we used high-speed atomic force microscopy to investigate the effects of damage to the intermediates of amyloid β in real time. Physical damage to intermediates did not suppress, but instead promoted fibrillization. This progression was accompanied by morphological changes from globular oligomers to protofibrils. These results suggest that the properties of the intermediates, such as structural fragility and stability, are highly related to the rate of fibrillization.

Bimolecular ribozymes derived by physical dissection of unimolecular ribozymes consisting of two structural modules are promising platforms for the design and construction of assembled RNA nanostructures. Unit RNAs to be assembled intermolecularly into one-dimensional (1D) oligomers are designed by reconnecting the two structural modules in a manner different from the parent ribozymes. This strategy was applied to the Tetrahymena group I ribozyme. We constructed 1D ribozyme oligomers the assembly of which was observed by atomic force microscopy (AFM) and also controlled rationally to design a heterooctamer by differentiating the interface between the two modules.

We demonstrate the single-molecule operation and observation of the formation and resolution of double-stranded DNA (dsDNA) containing G-quadruplex (GQ)- and counterpart i-motif-forming sequences in the DNA nanostructure. Sequential manipulation of DNA strands in the DNA frame was performed to prepare a topologically controlled GQ/i-motif dsDNA. Using the strand displacement and the addition and removal of K+, the topologically controlled GQ/i-motif dsDNA in the DNA frame was obtained in high yield. The dsDNA was resolved into the single-stranded DNA, GQ, and i-motif by the addition of K+ and operation under acidic conditions. The dissociation of the dsDNA under the GQ and i-motif formation conditions was monitored by high-speed atomic force microscopy. The results indicate that the dsDNA containing the GQ- and i-motif sequence is effectively dissolved when the duplex is helically loosened in the DNA nanoscaffold.

In the present study, we demonstrate single-molecule imaging of triple helix formation in DNA nanostructures. The binding of the single-molecule third strand to double-stranded DNA in a DNA origami frame was examined using two different types of triplet base pairs. The target DNA strand and the third strand were incorporated into the DNA frame, and the binding of the third strand was controlled by the formation of Watson-Crick base pairing. Triple helix formation was monitored by observing the structural changes in the incorporated DNA strands. It was also examined using a photocaged third strand wherein the binding of the third strand was directly observed using high-speed atomic force microscopy during photoirradiation. We found that the binding of the third strand could be controlled by regulating duplex formation and the uncaging of the photocaged strands in the designed nanospace.

Direct visualization of the biomolecules of interest is a straightforward way to elucidate the physical properties of individual molecules and their reaction processes. Atomic force microscopy (AFM) enables direct imaging of biomolecules in suitable solution conditions. As AFM visualizes the molecules at a nanometer-scale spatial resolution, a versatile observation platform is required for precise imaging of the molecules in action. The DNA origami technology allows precise placement of target molecules in a designed nanostructure, enabling their detection at the single-molecule level. We used DNA origami technology for visualizing the detailed movement of target molecules in reactions using high-speed AFM (HS-AFM), which enables the analysis of dynamic movement of biomolecules with a subsecond time resolution. By combining the DNA origami system and HS-AFM, DNA conformational changes, including G-quadruplex formation and disruption and B-Z transition, were visualized. In addition, enzyme-based reactions such as DNA recombination were also visualized at the single-molecule level using this combined observation system. Moreover, the enzyme-based reaction could be directly regulated in the DNA origami frame by imposing structural stress on the substrate DNAs to elucidate the reaction mechanism. These target-orientated observation systems should contribute to a detailed analysis of biomolecular motions in real time at molecular resolution.

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

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

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

In vitro gliding assay of microtubules (MTs) on kinesins has provided us with valuable biophysical and chemo-mechanical insights of this biomolecular motor system. Visualization of MTs in an in vitro gliding assay has been mainly dependent on optical microscopes, limited resolution of which often render them insufficient sources of desired information. In this work, using high speed atomic force microscopy (HS-AFM), which allows imaging with higher resolution, we monitored MTs and protofilaments (PFs) of tubulins while gliding on kinesins. Moreover, under the HS-AFM, we also observed splitting of gliding MTs into single PFs at their leading ends. The split single PFs interacted with kinesins and exhibited translational motion, but with a slower velocity than the MTs. Our investigation at the molecular level, using the HS-AFM, would provide new insights to the mechanics of MTs in dynamic systems and their interaction with motor proteins.

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.

To examine the effect of the torsional constraints imposed on DNA substrates on Cas9 cleavage, 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 atomic force microscopy. The results revealed that the constrained nontarget 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.

RNA is a biopolymer that is attractive for constructing nano-scale objects with complex structures. Three-dimensional (3D) structures of naturally occurring RNAs often have modular architectures. The 3D structure of a group I (GI) ribozyme from Tetrahymena has a typical modular architecture, which can be separated into two structural modules (ΔP5 and P5abc). The fully active ribozyme can be reconstructed by assembling the two separately prepared modules through highly specific and strong assembly between ΔP5 ribozyme and P5abc RNA. Such non-covalent assembly of the two modules allows the design of polygonal RNA nano-structures. Through rational redesign of the parent GI ribozyme, we constructed variant GI ribozymes as unit RNAs for polygonal-shaped (closed) oligomers with catalytic activity. Programmed trimerization and tetramerization of the unit RNAs afforded catalytically active nano-sized RNA triangles and squares, the structures of which were directly observed by atomic force microscopy (AFM).

Visualization of biomolecules is one of the straightforward ways to elucidate the physical properties of individual molecules and their reaction processes. Atomic force microscopy (AFM) enables direct imaging of biomolecules in the physiological environment. Because AFM visualizes the molecules at nanometer-scale spatial resolution, a versatile observation scaffold should be required for the precise imaging of molecules in the reactions. The emergence of DNA origami technology allows the precise placement of target molecules in the designed nanostructures and enables molecules to be detected at the single-molecule level. The DNA origami is applied for visualizing the detailed motions of target molecules in the reaction using high-speed AFM (HS-AFM), which enables the analysis of dynamic motions of biomolecules in a subsecond time resolution.