The micrometer-scale assembly of various DNA nanostructures is one of the major challenges for further progress in DNA nanotechnology. Programmed patterns of 1D and 2D DNA origami assembly using ...specific DNA strands and micrometer-sized lattice assembly using cross-shaped DNA origami were performed on a lipid bilayer surface. During the diffusion of DNA origami on the membrane surface, the formation of lattices and their rearrangement in real-time were observed using high-speed atomic force microscopy (HS-AFM). The formed lattices were used to further assemble DNA origami tiles into their cavities. Various patterns of lattice–tile complexes were created by changing the interactions between the lattice and tiles. For the control of the nanostructure formation, the photo-controlled assembly and disassembly of DNA origami were performed reversibly, and dynamic assembly and disassembly were observed on a lipid bilayer surface using HS-AFM. Using a lipid bilayer for DNA origami assembly, it is possible to perform a hierarchical assembly of multiple DNA origami nanostructures, such as the integration of functional components into a frame architecture.
•Direct observation of target molecules using high-speed atomic force microscopy.•Use of custom-made DNA origami for visualizing DNA structural change and reaction.•Visualization of dynamic movement ...of target molecules at a molecular size resolution.•Analysis of the reaction mechanism by attaching substrates to the DNA origami.
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.
DNA can assemble various molecules and nanomaterials in a programmed fashion and is a powerful tool in the nanotechnology and biology research fields. DNA also allows the construction of desired ...nanoscale structures via the design of DNA sequences. Structural nanotechnology, especially DNA origami, is widely used to design and create functionalized nanostructures and devices. In addition, DNA molecular machines have been created and are operated by specific DNA strands and external stimuli to perform linear, rotational, and reciprocating movements. Furthermore, complicated molecular systems have been created on DNA nanostructures by arranging multiple molecules and molecular machines precisely to mimic biological systems. Currently, DNA nanomachines, such as molecular motors, are operated on DNA nanostructures. Dynamic DNA nanostructures that have a mechanically controllable system have also been developed. In this review, we describe recent research on new DNA nanomachines and nanosystems that were built on designed DNA nanostructures.
Conspectus Direct imaging of molecular motions is one of the most fundamental issues for elucidating the physical properties of individual molecules and their reaction mechanisms. Atomic force ...microscopy (AFM) enables direct molecular imaging, especially for biomolecules in the physiological environment. Because AFM can visualize the molecules at nanometer-scale spatial resolution, a versatile observation scaffold is needed for the precise imaging of molecule interactions in the reactions. The emergence of DNA origami technology allows the precise placement of desired molecules in the designed nanostructures and enables molecules to be detected at the single-molecule level. In our study, the DNA origami system was applied to visualize the detailed motions of target molecules in reactions using high-speed AFM (HS-AFM), which enables the analysis of dynamic motions of biomolecules in a subsecond time resolution. In this system, biochemical properties such as the placement of various double-stranded DNAs (dsDNAs) containing unrestricted DNA sequences, modified nucleosides, and chemical functions can be incorporated. From a physical point of view, the tension and rotation of dsDNAs can be controlled by placement into the DNA nanostructures. From a topological point of view, the orientations of dsDNAs and various shapes of dsDNAs including Holliday junctions can be incorporated for studies on reaction mechanisms. In this Account, we describe the combination of the DNA origami system and HS-AFM for imaging various biochemical reactions including enzymatic reactions and DNA structural changes. To observe the behaviors and reactions of DNA methyltransferase and DNA repair enzymes, the substrate dsDNAs were incorporated into the cavity of the DNA frame, and the enzymes that bound to the target dsDNA were observed using HS-AFM. DNA recombination was also observed using the recombination substrates and Holliday junction intermediates placed in the DNA frame, and the direction of the reactions was controlled by introducing structural stress to the substrates. In addition, the movement of RNA polymerase and its reaction were visualized using a template dsDNA attached to the origami structure. To observe DNA structural changes, G-quadruplex formation and disruption, the switching behaviors of photoresponsive oligonucleotides, and B–Z transition were visualized using the DNA frame observation system. For the formation and disruption of G-quadruplex and double-helix DNA, the two dsDNA chains incorporated into the DNA frame could amplify the small structural change to the global structural change, which enabled the visualization of their association and dissociation by HS-AFM. The dynamic motion of the helical rotation induced by the B–Z transition was also directly imaged in the DNA frame. Furthermore, the stepwise motions of mobile DNA along the DNA track were visualized on the DNA origami surface. These target-orientated observation systems should contribute to the detailed analysis of biomolecule motions in real time and at molecular resolution.
Preeclampsia is a systemic vascular disorder characterized by new-onset hypertension and proteinuria after 20 weeks of gestation. This condition targets several organs, including the kidneys, liver ...and brain, and is the leading cause of maternal and perinatal morbidity and mortality. Furthermore, recent evidence has revealed preeclampsia as a significant risk factor for future cardiovascular diseases in these women. Over the past decade, increasing evidence has indicated that maternal angiogenic imbalances caused by placental antiangiogenic factors play a central role in the systemic vascular dysfunction underling preeclampsia. The severity of the maternal antiangiogenic state correlates closely with maternal and perinatal outcomes. Assessing angiogenic imbalance and several vascular function tests have also emerged as a way of detecting systemic vascular dysfunction during pregnancy. This review summarizes the current understanding of the pathophysiology of preeclampsia, its clinical applications and clinical evidence for future cardiovascular risks.
Preeclampsia, a systemic vascular disorder characterized by new-onset hypertension and proteinuria after 20 weeks of gestation, is the leading cause of maternal and perinatal morbidity and mortality. ...Maternal endothelial dysfunction caused by placental factors has long been accepted with respect to the pathophysiology of preeclampsia. Over the past decade, increased production of placental antiangiogenic factors has been identified as a placental factor leading to maternal endothelial dysfunction and systemic vascular dysfunction. This review summarizes the recent advances in understanding the molecular mechanisms of endothelial dysfunction caused by placental antiangiogenic factors, and the novel clinical strategies based on these discoveries.
Single-Molecule Analysis Using DNA Origami Rajendran, Arivazhagan; Endo, Masayuki; Sugiyama, Hiroshi
Angewandte Chemie (International ed.),
January 23, 2012, Letnik:
51, Številka:
4
Journal Article
Recenzirano
During the last two decades, scientists have developed various methods that allow the detection and manipulation of single molecules, which have also been called “in singulo” approaches. Fundamental ...understanding of biochemical reactions, folding of biomolecules, and the screening of drugs were achieved by using these methods. Single‐molecule analysis was also performed in the field of DNA nanotechnology, mainly by using atomic force microscopy. However, until recently, the approaches used commonly in nanotechnology adopted structures with a dimension of 10–20 nm, which is not suitable for many applications. The recent development of scaffolded DNA origami by Rothemund made it possible for the construction of larger defined assemblies. One of the most salient features of the origami method is the precise addressability of the structures formed: Each staple can serve as an attachment point for different kinds of nanoobjects. Thus, the method is suitable for the precise positioning of various functionalities and for the single‐molecule analysis of many chemical and biochemical processes. Here we summarize recent progress in the area of single‐molecule analysis using DNA origami and discuss the future directions of this research.
Origami for singles: Concurrent to the development of single‐molecule analytical techniques has been rapid progress in nanobiotechnology and efforts at building lab‐on‐a‐chip systems for high‐throughput analytical biochemistry. The scaffolded DNA origami method is suitable for the construction of defined larger assemblies that can act as a platform for the positioning of various functionalities and their single‐molecule analysis (see picture).
Here, we report the direct visualization of the assembly/disassembly processes of photoresponsive DNA origami nanostructures which can be placed on a lipid bilayer surface. The observation relies on ...controlled interactions between the bilayer components and cholesterol moieties introduced to the hexagonal origami structures, one of whose outer edges carries Azo-ODNs. The bilayer-placed hexagonal dimer was disassembled into monomer units by UV irradiation, and reversibly assembled again during visible light irradiation. These dynamic processes were directly monitored with high-speed atomic force microscopy. The successful application of our approach should facilitate studies of interactive and functional behaviors of various DNA nanostructures.
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.
Back to the fold: Using a DNA origami frame as a nanoscaffold for the structural control, the unprecedented solution‐state structures of a tetramolecular antiparallel and (3+1)‐type G‐quadruplex intermediates in the G‐quadruplex folding, such as G‐hairpin and G‐triplex, can be visualized directly at the single‐molecule level with nanometer resolution by AFM.
Nature has developed striking light-powered proteins such as bacteriorhodopsin, which can convert light energy into conformational changes for biological functions. Such natural machines are a great ...source of inspiration for creation of their synthetic analogues. However, synthetic molecular machines typically operate at the nanometre scale or below. Translating controlled operation of individual molecular machines to a larger dimension, for example, to 10-100 nm, which features many practical applications, is highly important but remains challenging. Here we demonstrate a light-driven plasmonic nanosystem that can amplify the molecular motion of azobenzene through the host nanostructure and consequently translate it into reversible chiroptical function with large amplitude modulation. Light is exploited as both energy source and information probe. Our plasmonic nanosystem bears unique features of optical addressability, reversibility and modulability, which are crucial for developing all-optical molecular devices with desired functionalities.
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