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Ulrich F. Keyser - One of the best experts on this subject based on the ideXlab platform.
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Voltage-dependent properties of DNA Origami nanopores.
Nano Letters, 2014Co-Authors: Silvia Hernández-ainsa, Vivek V. Thacker, Karolis Misiunas, Elisa A. Hemmig, Ulrich F. KeyserAbstract:We show DNA Origami nanopores that respond to high voltages by a change in conformation on glass nanocapillaries. Our DNA Origami nanopores are voltage sensitive as two distinct states are found as a function of the applied voltage. We suggest that the origin of these states is a mechanical distortion of the DNA Origami. A simple model predicts the voltage dependence of the structural change. We show that our responsive DNA Origami nanopores can be used to lower the frequency of DNA translocation by 1 order of magnitude.
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Nanopores formed by DNA Origami: A review
FEBS Letters, 2014Co-Authors: Nicholas A.w. Bell, Ulrich F. KeyserAbstract:Nanopores have emerged over the past two decades to become an important technique in single molecule experimental physics and biomolecule sensing. Recently DNA nanotechnology, in particular DNA Origami, has been used for the formation of nanopores in insulating materials. DNA Origami is a very attractive technique for the formation of nanopores since it enables the construction of 3D shapes with precise control over geometry and surface functionality. DNA Origami has been applied to nanopore research by forming hybrid architectures with solid state nanopores and by direct insertion into lipid bilayers. This review discusses recent experimental work in this area and provides an outlook for future avenues and challenges.
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DNA Origami nanopores for controlling DNA translocation
ACS Nano, 2013Co-Authors: Silvia Hernández-ainsa, Nicholas A.w. Bell, Vivek V. Thacker, Kerstin Göpfrich, Karolis Misiunas, Maria Eugenia Fuentes-perez, Fernando Moreno-herrero, Ulrich F. KeyserAbstract:We combine DNA Origami structures with glass nanocapillaries to reversibly form hybrid DNA Origami nanopores. Trapping of the DNA Origami onto the nanocapillary is proven by imaging fluorescently labeled DNA Origami structures and simultaneous ionic current measurements of the trapping events. We then show two applications highlighting the versatility of these DNA Origami nanopores. First, by tuning the pore size we can control the folding of dsDNA molecules ("physical control"). Second, we show that the specific introduction of binding sites in the DNA Origami nanopore allows selective detection of ssDNA as a function of the DNA sequence ("chemical control").
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Ionic Current Detection of DNA Origami Nanostructures with Nanocapillaries
Nanopores for Bioanalytical Applications, 2012Co-Authors: Nicholas A.w. Bell, Christian R. Engst, Silvia Hernández-ainsa, Tim Liedl, Ulrich F. KeyserAbstract:DNA Origami is a powerful technique for the construction of designer nanopores. Nanocapillaries can be used both to detect the translocation of DNA Origami structures and to trap the Origami to form a hybrid pore by tuning the diameter of the nanopore. The detection of translocations is a potential new tool for assessing the folding quality and aggregation of DNA Origami in solution. The formation of a hybrid Origami-nanocapillary nanopore represents a novel method for creating hybrid nanopores with high throughput and low cost.
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DNA Origami nanopores
Nano Letters, 2012Co-Authors: Nicholas A.w. Bell, Christian R. Engst, Marc Ablay, Tim Liedl, Caterina Ducati, Giorgio Divitini, Ulrich F. KeyserAbstract:We demonstrate the assembly of functional hybrid nanopores for single molecule sensing by inserting DNA Origami structures into solid-state nanopores. In our experiments, single artificial nanopores based on DNA Origami are repeatedly inserted in and ejected from solid-state nanopores with diameters around 15 nm. We show that these hybrid nanopores can be employed for the detection of λ-DNA molecules. Our approach paves the way for future development of adaptable single-molecule nanopore sensors based on the combination of solid-state nanopores and DNA self-assembly.
Philip Tinnefeld - One of the best experts on this subject based on the ideXlab platform.
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Toward quantitative fluorescence microscopy with DNA Origami nanorulers.
Methods in Cell Biology, 2020Co-Authors: Susanne Beater, Mario Raab, Philip TinnefeldAbstract:Abstract The dynamic development of fluorescence microscopy has created a large number of new techniques, many of which are able to overcome the diffraction limit. This chapter describes the use of DNA Origami nanostructures as scaffold for quantifying microscope properties such as sensitivity and resolution. The DNA Origami technique enables placing of a defined number of fluorescent dyes in programmed geometries. We present a variety of DNA Origami nanorulers that include nanorulers with defined labeling density and defined distances between marks. The chapter summarizes the advantages such as practically free choice of dyes and labeling density and presents examples of nanorulers in use. New triangular DNA Origami nanorulers that do not require photoinduced switching by imaging transient binding to DNA nanostructures are also reported. Finally, we simulate fluorescence images of DNA Origami nanorulers and reveal that the optimal DNA nanoruler for a specific application has an intermark distance that is roughly 1.3-fold the expected optical resolution.
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DNA Origami-based standards for quantitative fluorescence microscopy
Nature Protocols, 2014Co-Authors: Jürgen J. Schmied, Enrico Pibiri, Bettina Wünsch, Mario Raab, Carsten Forthmann, Thorben Dammeyer, Philip TinnefeldAbstract:Validating and testing a fluorescence microscope or a microscopy method requires defined samples that can be used as standards. DNA Origami is a new tool that provides a framework to place defined numbers of small molecules such as fluorescent dyes or proteins in a programmed geometry with nanometer precision. The flexibility and versatility in the design of DNA Origami microscopy standards makes them ideally suited for the broad variety of emerging super-resolution microscopy methods. As DNA Origami structures are durable and portable, they can become a universally available specimen to check the everyday functionality of a microscope. The standards are immobilized on a glass slide, and they can be imaged without further preparation and can be stored for up to 6 months. We describe a detailed protocol for the design, production and use of DNA Origami microscopy standards, and we introduce a DNA Origami rectangle, bundles and a nanopillar as fluorescent nanoscopic rulers. The protocol provides procedures for the design and realization of fluorescent marks on DNA Origami structures, their production and purification, quality control, handling, immobilization, measurement and data analysis. The procedure can be completed in 1-2 d. © 2014 Nature America, Inc.
Hendrik Dietz - One of the best experts on this subject based on the ideXlab platform.
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The sequence of events during folding of a DNA Origami
Science Advances, 2019Co-Authors: Fabian Schneider, Natalie Möritz, Hendrik DietzAbstract:We provide a comprehensive reference dataset of the kinetics of a multilayer DNA Origami folding. To this end, we measured the folding kinetics of every staple strand and its two terminal segments during constant-temperature assembly of a multilayer DNA Origami object. Our data illuminate the processes occurring during folding of the DNA Origami in fine detail, starting with the first nucleating double-helical domains and ending with the fully folded DNA Origami object. We found a complex sequence of folding events that cannot be explained with simplistic local design analysis. Our real-time data, although derived from one specific DNA Origami object, through its sheer massive detail, could provide the crucial input needed to construct and test a quantitatively predictive, general model of DNA Origami assembly.
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biotechnological mass production of DNA Origami
Nature, 2017Co-Authors: Florian Praetorius, Benjamin Kick, Karl L. Behler, Maximilian N. Honemann, Dirk Weusterbotz, Hendrik DietzAbstract:All necessary strands for DNA Origami can be created in a single scalable process by using bacteriophages to generate single-stranded precursor DNA containing the target sequences interleaved with self-excising DNA enzymes. DNA Origami can readily be used to create micrometre-scale objects with nanometre-precise features using a very long single-stranded DNA 'scaffold' that is held in place by many short single-stranded DNA 'staples'. These objects could find many uses, but the cost of manufacturing them could be prohibitive for conducting research into their potential applications. Hendrik Dietz and colleagues now show that single strands of DNA of random length and sequence can be mass-produced at low cost. They used a litre-scale bioreactor to generate single-stranded precursor DNA strands that contain target strand sequences interspersed with self-excising DNA enzyme cassettes. This makes it possible to efficiently generate all of the single strands of DNA needed to assemble different target Origami objects in one process, which should expand the scope of DNA nanotechnology in many areas of science and technology. Three related papers is this issue report further advances in DNA Origami, and all four are summarized in a News & Views. DNA nanotechnology, in particular DNA Origami, enables the bottom-up self-assembly of micrometre-scale, three-dimensional structures with nanometre-precise features1,2,3,4,5,6,7,8,9,10,11,12. These structures are customizable in that they can be site-specifically functionalized13 or constructed to exhibit machine-like14,15 or logic-gating behaviour16. Their use has been limited to applications that require only small amounts of material (of the order of micrograms), owing to the limitations of current production methods. But many proposed applications, for example as therapeutic agents or in complex materials3,16,17,18,19,20,21,22, could be realized if more material could be used. In DNA Origami, a nanostructure is assembled from a very long single-stranded scaffold molecule held in place by many short single-stranded staple oligonucleotides. Only the bacteriophage-derived scaffold molecules are amenable to scalable and efficient mass production23; the shorter staple strands are obtained through costly solid-phase synthesis24 or enzymatic processes25. Here we show that single strands of DNA of virtually arbitrary length and with virtually arbitrary sequences can be produced in a scalable and cost-efficient manner by using bacteriophages to generate single-stranded precursor DNA that contains target strand sequences interleaved with self-excising ‘cassettes’, with each cassette comprising two Zn2+-dependent DNA-cleaving DNA enzymes. We produce all of the necessary single strands of DNA for several DNA Origami using shaker-flask cultures, and demonstrate end-to-end production of macroscopic amounts of a DNA Origami nanorod in a litre-scale stirred-tank bioreactor. Our method is compatible with existing DNA Origami design frameworks and retains the modularity and addressability of DNA Origami objects that are necessary for implementing custom modifications using functional groups. With all of the production and purification steps amenable to scaling, we expect that our method will expand the scope of DNA nanotechnology in many areas of science and technology.
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Biotechnological mass production of DNA Origami
Nature, 2017Co-Authors: Florian Praetorius, Benjamin Kick, Karl L. Behler, Maximilian N. Honemann, Dirk Weuster-botz, Hendrik DietzAbstract:DNA nanotechnology, in particular DNA Origami, enables the bottom-up self-assembly of micrometre-scale, three-dimensional structures with nanometre-precise features1–12. These structures are customizable in that they can be site-specifically functionalized13 or constructed to exhibit machine-like14,15 or logic-gating behaviour16. Their use has been limited to applications that require only small amounts of material (of the order of micrograms), owing to the limitations of current production methods. But many proposed applications, for example as therapeutic agents or in complex materials3,16–22, could be realized if more material could be used. In DNA Origami, a nanostructure is assembled from a very long single-stranded scaffold molecule held in place by many short single-stranded staple oligonucleotides. Only the bacteriophage- derived scaffold molecules are amenable to scalable and efficient mass production23; the shorter staple strands are obtained through costly solid-phase synthesis24 or enzymatic processes25. Here we show that single strands of DNA of virtually arbitrary length and with virtually arbitrary sequences can be produced in a scalable and cost-efficient manner by using bacteriophages to generate single-stranded precursor DNA that contains target strand sequences interleaved with self-excising ‘cassettes’, with each cassette comprising two Zn2+-dependent DNA-cleaving DNA enzymes. We produce all of the necessary single strands of DNA for several DNA Origami using shaker-flask cultures, and demonstrate end-to-end production of macroscopic amounts of a DNA Origami nanorod in a litre-scale stirred-tank bioreactor. Our method is compatible with existing DNA Origami design frameworks and retains the modularity and addressability of DNA Origami objects that are necessary for implementing custom modifications using functional groups. With all of the production and purification steps amenable to scaling, we expect that our method will expand the scope of DNA nanotechnology in many areas of science and technology.
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How We Make DNA Origami
ChemBioChem, 2017Co-Authors: Klaus F. Wagenbauer, Floris A.s. Engelhardt, Evi Stahl, Vera K. Hechtl, Pierre Stömmer, Letizia Meregalli, Thomas Gerling, Philip Ketterer, Fabian Seebacher, Hendrik DietzAbstract:DNA Origami has attracted substantial attention since its inven- tion ten years ago, due to the seemingly infinite possibilities that it affords for creating customized nanoscale objects. Al- though the basic concept of DNA Origami is easy to under- stand, using custom DNA Origami in practical applications re- quires detailed know-how for designing and producing the particles with sufficient quality and for preparing them at ap- propriate concentrations with the necessary degree of purity in custom environments. Such know-how is not readily avail- able for newcomers to the field, thus slowing down the rate at which new applications outside the field of DNA nanotechnol- ogy may emerge. To foster faster progress, we share in this ar- ticle the experience in making and preparing DNA Origami that we have accumulated over recent years. We discuss design solutions for creating advanced structural motifs including cor- ners and various types of hinges that expand the design space for the more rigid multilayer DNA Origami and provide guide- lines for preventing undesired aggregation and on how to induce specific oligomerization of multiple DNA Origami build- ing blocks. In addition, we provide detailed protocols and discuss the expected results for five key methods that allow efficient and damage-free preparation of DNA Origami. These methods are agarose-gel purification, filtration through molec- ular cut-off membranes, PEG precipitation, size-exclusion chro- matography, and ultracentrifugation-based sedimentation. The guide for creating advanced design motifs and the detailed protocols with their experimental characterization that we de- scribe here should lower the barrier for researchers to accom- plish the full DNA Origami production workflow.
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a primer to scaffolded DNA Origami
Nature Methods, 2011Co-Authors: Carlos E. Castro, Enrique Lin Shiao, Tobias Wauer, Philipp Wortmann, Fabian Kilchherr, Mark Bathe, Hendrik DietzAbstract:Molecular self-assembly with scaffolded DNA Origami enables building custom-shaped nanometer-scale objects with molecular weights in the megadalton regime. Here we provide a practical guide for design and assembly of scaffolded DNA Origami objects. We also introduce a computational tool for predicting the structure of DNA Origami objects and provide information on the conditions under which DNA Origami objects can be expected to maintain their structure.
Hao Yan - One of the best experts on this subject based on the ideXlab platform.
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DNA Origami: Scaffolds for Creating Higher Order Structures
Chemical Reviews, 2017Co-Authors: Fan Hong-yi, Yan Liu, Fei Zhang, Hao YanAbstract:DNA has become one of the most extensively used molecular building blocks for engineering self-assembling materials. DNA Origami is a technique that uses hundreds of short DNA oligonucleotides, called staple strands, to fold a long single-stranded DNA, which is called a scaffold strand, into various designer nanoscale architectures. DNA Origami has dramatically improved the complexity and scalability of DNA nanostructures. Due to its high degree of customization and spatial addressability, DNA Origami provides a versatile platform with which to engineer nanoscale structures and devices that can sense, compute, and actuate. These capabilities open up opportunities for a broad range of applications in chemistry, biology, physics, material science, and computer science that have often required programmed spatial control of molecules and atoms in three-dimensional (3D) space. This review provides a comprehensive survey of recent developments in DNA Origami structure, design, assembly, and directed self-assembly, as well as its broad applications.
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Stability of DNA Origami nanoarrays in cell lysate
Nano Letters, 2011Co-Authors: Qian Mei, Xixi Wei, Roger Johnson, Yan Liu, Fengyu Su, Hao Yan, Stuart Lindsay, Cody Youngbull, Deirdre MeldrumAbstract:Scaffolded DNA Origami, a method to create self-assembled nanostructures with spatially addressable features, has recently been used to develop water-soluble molecular chips for label-free RNA detection, platforms for deterministic protein positioning, and single molecule reaction observatories. These applications highlight the possibility of exploiting the unique properties and biocompatibility of DNA nanostructures in live, cellular systems. Herein, we assembled several DNA Origami nanostructures of differing shape, size and probes, and investigated their interaction with lysate obtained from various normal and cancerous cell lines. We separated and analyzed the Origami-lysate mixtures using agarose gel electrophoresis and recovered the DNA structures for functional assay and subsequent microscopic examination. Our results demonstrate that DNA Origami nanostructures are stable in cell lysate and can be easily separated from lysate mixtures, in contrast to natural, single- and double-stranded DNA. Atomic force microscope (AFM) and transmission electron microscope (TEM) images show that the DNA Origami structures are fully intact after separation from cell lysates and hybridize to their targets, verifying the superior structural integrity and functionality of self-assembled DNA Origami nanostructures relative to conventional oligonucleotides. The stability and functionality of DNA Origami structures in cell lysate validate their use for biological applications, for example, as programmable molecular rafts or disease detection platforms. © 2011 American Chemical Society.
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Interconnecting gold islands with DNA Origami nanotubes
Nano Letters, 2010Co-Authors: Baoquan Ding, Yan Liu, Hongbin Yu, Zhao Zhao, Wei Xu, Hao Wu, Hao YanAbstract:Scaffolded DNA Origami has recently emerged as a versatile, programmable method to fold DNA into arbitrarily shaped nanostructures that are spatially addressable, with sub-10-nm resolution. Toward functional DNA nanotechnology, one of the key challenges is to integrate the bottom-up self-assembly of DNA Origami with the top-down lithographic methods used to generate surface patterning. In this report we demonstrate that fixed length DNA Origami nanotubes, modified with multiple thiol groups near both ends, can be used to connect surface patterned gold islands (tens of nanometers in diameter) fabricated by electron beam lithography (EBL). Atomic force microscopic imaging verified that the DNA Origami nanotubes can be efficiently aligned between gold islands with various interisland distances and relative locations. This development represents progress toward the goal of bridging bottom-up and top-down assembly approaches.
Nicholas A.w. Bell - One of the best experts on this subject based on the ideXlab platform.
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Nanopores formed by DNA Origami: A review
FEBS Letters, 2014Co-Authors: Nicholas A.w. Bell, Ulrich F. KeyserAbstract:Nanopores have emerged over the past two decades to become an important technique in single molecule experimental physics and biomolecule sensing. Recently DNA nanotechnology, in particular DNA Origami, has been used for the formation of nanopores in insulating materials. DNA Origami is a very attractive technique for the formation of nanopores since it enables the construction of 3D shapes with precise control over geometry and surface functionality. DNA Origami has been applied to nanopore research by forming hybrid architectures with solid state nanopores and by direct insertion into lipid bilayers. This review discusses recent experimental work in this area and provides an outlook for future avenues and challenges.
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DNA Origami nanopores for controlling DNA translocation
ACS Nano, 2013Co-Authors: Silvia Hernández-ainsa, Nicholas A.w. Bell, Vivek V. Thacker, Kerstin Göpfrich, Karolis Misiunas, Maria Eugenia Fuentes-perez, Fernando Moreno-herrero, Ulrich F. KeyserAbstract:We combine DNA Origami structures with glass nanocapillaries to reversibly form hybrid DNA Origami nanopores. Trapping of the DNA Origami onto the nanocapillary is proven by imaging fluorescently labeled DNA Origami structures and simultaneous ionic current measurements of the trapping events. We then show two applications highlighting the versatility of these DNA Origami nanopores. First, by tuning the pore size we can control the folding of dsDNA molecules ("physical control"). Second, we show that the specific introduction of binding sites in the DNA Origami nanopore allows selective detection of ssDNA as a function of the DNA sequence ("chemical control").
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Ionic Current Detection of DNA Origami Nanostructures with Nanocapillaries
Nanopores for Bioanalytical Applications, 2012Co-Authors: Nicholas A.w. Bell, Christian R. Engst, Silvia Hernández-ainsa, Tim Liedl, Ulrich F. KeyserAbstract:DNA Origami is a powerful technique for the construction of designer nanopores. Nanocapillaries can be used both to detect the translocation of DNA Origami structures and to trap the Origami to form a hybrid pore by tuning the diameter of the nanopore. The detection of translocations is a potential new tool for assessing the folding quality and aggregation of DNA Origami in solution. The formation of a hybrid Origami-nanocapillary nanopore represents a novel method for creating hybrid nanopores with high throughput and low cost.
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DNA Origami nanopores
Nano Letters, 2012Co-Authors: Nicholas A.w. Bell, Christian R. Engst, Marc Ablay, Tim Liedl, Caterina Ducati, Giorgio Divitini, Ulrich F. KeyserAbstract:We demonstrate the assembly of functional hybrid nanopores for single molecule sensing by inserting DNA Origami structures into solid-state nanopores. In our experiments, single artificial nanopores based on DNA Origami are repeatedly inserted in and ejected from solid-state nanopores with diameters around 15 nm. We show that these hybrid nanopores can be employed for the detection of λ-DNA molecules. Our approach paves the way for future development of adaptable single-molecule nanopore sensors based on the combination of solid-state nanopores and DNA self-assembly.