Dielectrophoretic trapping of multilayer DNA origami nanostructures and DNA origami‐induced local destruction of silicon dioxide

DNA origami is a widely used method for fabrication of custom‐shaped nanostructures. However, to utilize such structures, one needs to controllably position them on nanoscale. Here we demonstrate how different types of 3D scaffolded multilayer origamis can be accurately anchored to lithographically fabricated nanoelectrodes on a silicon dioxide substrate by DEP. Straight brick‐like origami structures, constructed both in square (SQL) and honeycomb lattices, as well as curved “C”‐shaped and angular “L”‐shaped origamis were trapped with nanoscale precision and single‐structure accuracy. We show that the positioning and immobilization of all these structures can be realized with or without thiol‐linkers. In general, structural deformations of the origami during the DEP trapping are highly dependent on the shape and the construction of the structure. The SQL brick turned out to be the most robust structure under the high DEP forces, and accordingly, its single‐structure trapping yield was also highest. In addition, the electrical conductivity of single immobilized plain brick‐like structures was characterized. The electrical measurements revealed that the conductivity is negligible (insulating behavior). However, we observed that the trapping process of the SQL brick equipped with thiol‐linkers tended to induce an etched “nanocanyon” in the silicon dioxide substrate. The nanocanyon was formed exactly between the electrodes, that is, at the location of the DEP‐trapped origami. The results show that the demonstrated DEP‐trapping technique can be readily exploited in assembling and arranging complex multilayered origami geometries. In addition, DNA origamis could be utilized in DEP‐assisted deformation of the substrates onto which they are attached.     

DNA Origami Post‐Processing by CRISPR‐Cas12a

Customizable nanostructures built through the DNA‐origami technique hold tremendous promise in nanomaterial fabrication and biotechnology. Despite the cutting‐edge tools for DNA‐origami design and preparation, it remains challenging to separate structural components of an architecture built from—thus held together by—a continuous scaffold strand, which in turn limits the modularity and function of the DNA‐origami devices. To address this challenge, here we present an enzymatic method to clean up and reconfigure DNA‐origami structures. We target single‐stranded (ss) regions of DNA‐origami structures and remove them with CRISPR‐Cas12a, a hyper‐active ssDNA endonuclease without sequence specificity. We demonstrate the utility of this facile, selective post‐processing method on DNA structures with various geometrical and mechanical properties, realizing intricate structures and structural transformations that were previously difficult to engineer. Given the biocompatibility of Cas12a‐like enzymes, this versatile tool may be programmed in the future to operate functional nanodevices in cells.     

Reconfigurable T‐junction DNA Origami

DNA self‐assembly allows the construction of nanometre‐scale structures and devices. Structures with thousands of unique components are routinely assembled in good yield. Experimental progress has been rapid, based largely on empirical design rules. Herein, we demonstrate a DNA origami technique designed as a model system with which to explore the mechanism of assembly. The origami fold is controlled through single‐stranded loops embedded in a double‐stranded DNA template and is programmed by a set of double‐stranded linkers that specify pairwise interactions between loop sequences. Assembly is via T‐junctions formed by hybridization of single‐stranded overhangs on the linkers with the loops. The sequence of loops on the template and the set of interaction rules embodied in the linkers can be reconfigured with ease. We show that a set of just two interaction rules can be used to assemble simple T‐junction origami motifs and that assembly can be performed at room temperature.     

Amphipathic DNA Origami Nanoparticles to Scaffold and Deform Lipid Membrane Vesicles

We report a synthetic biology‐inspired approach for the engineering of amphipathic DNA origami structures as membrane‐scaffolding tools. The structures have a flat membrane‐binding interface decorated with cholesterol‐derived anchors. Sticky oligonucleotide overhangs on their side facets enable lateral interactions leading to the formation of ordered arrays on the membrane. Such a tight and regular arrangement makes our DNA origami capable of deforming free‐standing lipid membranes, mimicking the biological activity of coat‐forming proteins, for example, from the I‐/F‐BAR family.     

Programmed‐Assembly System Using DNA Jigsaw Pieces

A novel method for assembling multiple DNA origami structures has been developed by using designed 2D DNA origami rectangles, so‐called “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 π‐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‐Nanotechnologie

For the past two decades the extraordinary molecular recognition properties of DNA molecules have been used for the creation of artificial molecular structures. Following the initial production of simple molecular objects and lattices, with the recent invention of the DNA origami technique the complexity of these structures has considerably increased. Now the construction of almost arbitrary molecular nanostructures from DNA in two and even three dimensions is feasible – and first concrete applications in biomedicine and nanotechnology are in reach. In addition to static molecular structures, also dynamical systems such as molecular machines, molecular motors, and molecular computers can be realized. The combination of these functions within integrated systems currently leads to the development of first molecular “robots” and assembly lines for nanotechnology.     

On the Stability of DNA Origami Nanostructures in Low‐Magnesium Buffers

DNA origami structures have great potential as functional platforms in various biomedical applications. Many applications, however, are incompatible with the high Mg²⁺ concentrations commonly believed to be a prerequisite for maintaining DNA origami integrity. Herein, we investigate DNA origami stability in low‐Mg²⁺ buffers. DNA origami stability is found to crucially depend on the availability of residual Mg²⁺ ions for screening electrostatic repulsion. The presence of EDTA and phosphate ions may thus facilitate DNA origami denaturation by displacing Mg²⁺ ions from the DNA backbone and reducing the strength of the Mg²⁺–DNA interaction, respectively. Most remarkably, these buffer dependencies are affected by DNA origami superstructure. However, by rationally selecting buffer components and considering superstructure‐dependent effects, the structural integrity of a given DNA origami nanostructure can be maintained in conventional buffers even at Mg²⁺ concentrations in the low‐micromolar range.     

Fabrication of Defined Polydopamine Nanostructures by DNA Origami‐Templated Polymerization

A versatile, bottom‐up approach allows the controlled fabrication of polydopamine (PD) nanostructures on DNA origami. PD is a biosynthetic polymer that has been investigated as an adhesive and promising surface coating material. However, the control of dopamine polymerization is challenged by the multistage‐mediated reaction mechanism and diverse chemical structures in PD. DNA origami decorated with multiple horseradish peroxidase‐mimicking DNAzyme motifs was used to control the shape and size of PD formation with nanometer resolution. These fabricated PD nanostructures can serve as “supramolecular glue” for controlling DNA origami conformations. Facile liberation of the PD nanostructures from the DNA origami templates has been achieved in acidic medium. This presented DNA origami‐controlled polymerization of a highly crosslinked polymer provides a unique access towards anisotropic PD architectures with distinct shapes that were retained even in the absence of the DNA origami template.     

Complexing DNA Origami Frameworks through Sequential Self‐Assembly Based on Directed Docking

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.     

Vesicle Tubulation with Self‐Assembling DNA Nanosprings

A major goal of nanotechnology and bioengineering is to build artificial nanomachines capable of generating specific membrane curvatures on demand. Inspired by natural membrane‐deforming proteins, we designed DNA‐origami curls that polymerize into nanosprings and show their efficacy in vesicle deformation. DNA‐coated membrane tubules emerge from spherical vesicles when DNA‐origami polymerization or high membrane‐surface coverage occurs. Unlike many previous methods, the DNA self‐assembly‐mediated membrane tubulation eliminates the need for detergents or top‐down manipulation. The DNA‐origami design and deformation conditions have substantial influence on the tubulation efficiency and tube morphology, underscoring the intricate interplay between lipid bilayers and vesicle‐deforming DNA structures.     

Gold‐Nanoparticle‐Mediated Jigsaw‐Puzzle‐like Assembly of Supersized Plasmonic DNA Origami

DNA origami has rapidly emerged as a powerful and programmable method to construct functional nanostructures. However, the size limitation of approximately 100 nm in classic DNA origami hampers its plasmonic applications. Herein, we report a jigsaw‐puzzle‐like assembly strategy mediated by gold nanoparticles (AuNPs) to break the size limitation of DNA origami. We demonstrated that oligonucleotide‐functionalized AuNPs function as universal joint units for the one‐pot assembly of parent DNA origami of triangular shape to form sub‐microscale super‐origami nanostructures. AuNPs anchored at predefined positions of the super‐origami exhibited strong interparticle plasmonic coupling. This AuNP‐mediated strategy offers new opportunities to drive macroscopic self‐assembly and to fabricate well‐defined nanophotonic materials and devices.     

Ion‐Selective Formation of a Guanine Quadruplex on DNA Origami Structures

DNA origami nanostructures are a versatile tool that can be used to arrange functionalities with high local control to study molecular processes at a single‐molecule level. Here, we demonstrate that DNA origami substrates can be used to suppress the formation of specific guanine (G) quadruplex structures from telomeric DNA. The folding of telomeres into G‐quadruplex structures in the presence of monovalent cations (e.g. Na⁺ and K⁺) is currently used for the detection of K⁺ ions, however, with insufficient selectivity towards Na⁺. By means of FRET between two suitable dyes attached to the 3′‐ and 5′‐ends of telomeric DNA we demonstrate that the formation of G‐quadruplexes on DNA origami templates in the presence of sodium ions is suppressed due to steric hindrance. Hence, telomeric DNA attached to DNA origami structures represents a highly sensitive and selective detection tool for potassium ions even in the presence of high concentrations of sodium ions.     

Block Copolymer Micellization as a Protection Strategy for DNA Origami

DNA nanotechnology enables the synthesis of nanometer‐sized objects that can be site‐specifically functionalized with a large variety of materials. For these reasons, DNA‐based devices such as DNA origami are being considered for applications in molecular biology and nanomedicine. However, many DNA structures need a higher ionic strength than that of common cell culture buffers or bodily fluids to maintain their integrity and can be degraded quickly by nucleases. To overcome these deficiencies, we coated several different DNA origami structures with a cationic poly(ethylene glycol)–polylysine block copolymer, which electrostatically covered the DNA nanostructures to form DNA origami polyplex micelles (DOPMs). This straightforward, cost‐effective, and robust route to protect DNA‐based structures could therefore enable applications in biology and nanomedicine where unprotected DNA origami would be degraded.     

Self‐assembly of DNA Origami Using Rolling Circle Amplification Based DNA Nanoribbons

During the development of structural DNA nanotechnology, the emerging of scaffolded DNA origami is marvelous. It utilizes DNA double helix inherent specificity of Watson‐Crick base pairing and structural features to create self‐assembling structures at the nanometer scale exhibiting the addressable character. However, the assembly of DNA origami is disorderly and unpredictable. Herein, we present a novel strategy to assemble the DNA origami using rolling circle amplification based DNA nanoribbons as the linkers. Firstly, long single‐stranded DNA from Rolling Circle Amplification is annealed with several staples to form kinds of DNA nanoribbons with overhangs. Subsequently, the rectangle origami is formed with overhanged staple strands at any edge that would hybridize with the DNA nanoribbons. By mixing them up, we illustrate the one‐dimensional even two‐dimensional assembly of DNA origami with good orientation.     

Folding and Imaging of DNA Nanostructures in Anhydrous and Hydrated Deep‐Eutectic Solvents

There is great interest in DNA nanotechnology, but its use has been limited to aqueous or substantially hydrated media. The first assembly of a DNA nanostructure in a water‐free solvent, namely a low‐volatility biocompatible deep‐eutectic solvent composed of a 4:1 mixture of glycerol and choline chloride (glycholine), is now described. Glycholine allows for the folding of a two‐dimensional DNA origami at 20 °C in six days, whereas in hydrated glycholine, folding is accelerated (≤3 h). Moreover, a three‐dimensional DNA origami and a DNA tail system can be folded in hydrated glycholine under isothermal conditions. Glycholine apparently reduces the kinetic traps encountered during folding in aqueous solvent. Furthermore, folded structures can be transferred between aqueous solvent and glycholine. It is anticipated that glycholine and similar solvents will allow for the creation of functional DNA structures of greater complexity by providing a milieu with tunable properties that can be optimized for a range of applications and nanostructures.     

Transfer of Two‐Dimensional Oligonucleotide Patterns onto Stereocontrolled Plasmonic Nanostructures through DNA‐Origami‐Based Nanoimprinting Lithography

The precise functionalization of self‐assembled nanostructures with spatial and stereocontrol is a major objective of nanotechnology and holds great promise for many applications. Herein, the nanoscale addressability of DNA origami was exploited to develop a precise copy‐machine‐like platform that can transfer two‐dimensional oligonucleotide patterns onto the surface of gold nanoparticles (AuNPs) through a deliberately designed toehold‐initiated DNA displacement reaction. This strategy of DNA‐origami‐based nanoimprinting lithography (DONIL) demonstrates high precision in controlling the valence and valence angles of AuNPs. These DNA‐decorated AuNPs act as precursors in the construction of discrete AuNP clusters with desired chirality.     

Bottom‐Up Fabrication of Nanopatterned Polymers on DNA Origami by In Situ Atom‐Transfer Radical Polymerization

Bottom‐up strategies to fabricate patterned polymers at the nanoscale represent an emerging field in the development of advanced nanodevices, such as biosensors, nanofluidics, and nanophotonics. DNA origami techniques provide access to distinct architectures of various sizes and shapes and present manifold opportunities for functionalization at the nanoscale with the highest precision. Herein, we conduct in situ atom‐transfer radical polymerization (ATRP) on DNA origami, yielding differently nanopatterned polymers of various heights. After cross‐linking, the grafted polymeric nanostructures can even stably exist in solution without the DNA origami template. This straightforward approach allows for the fabrication of patterned polymers with low nanometer resolution, which provides access to unique DNA‐based functional hybrid materials.     

Computer‐Aided Production of Scaffolded DNA Nanostructures from Flat Sheet Meshes

The use of DNA as a nanoscale construction material has been a rapidly developing field since the 1980s, in particular since the introduction of scaffolded DNA origami in 2006. Although software is available for DNA origami design, the user is generally limited to architectures where finding the scaffold path through the object is trivial. Herein, we demonstrate the automated conversion of arbitrary two‐dimensional sheets in the form of digital meshes into scaffolded DNA nanostructures. We investigate the properties of DNA meshes based on three different internal frameworks in standard folding buffer and physiological salt buffers. We then employ the triangulated internal framework and produce four 2D structures with complex outlines and internal features. We demonstrate that this highly automated technique is capable of producing complex DNA nanostructures that fold with high yield to their programmed configurations, covering around 70 % more surface area than classic origami flat sheets.     

Single‐Molecule Mechanochemical Sensing Using DNA Origami Nanostructures

While single‐molecule sensing offers the ultimate detection limit, its throughput is often restricted as sensing events are carried out one at a time in most cases. 2D and 3D DNA origami nanostructures are used as expanded single‐molecule platforms in a new mechanochemical sensing strategy. As a proof of concept, six sensing probes are incorporated in a 7‐tile DNA origami nanoassembly, wherein binding of a target molecule to any of these probes leads to mechanochemical rearrangement of the origami nanostructure, which is monitored in real time by optical tweezers. Using these platforms, 10 pM platelet‐derived growth factor (PDGF) are detected within 10 minutes, while demonstrating multiplex sensing of the PDGF and a target DNA in the same solution. By tapping into the rapid development of versatile DNA origami nanostructures, this mechanochemical platform is anticipated to offer a long sought solution for single‐molecule sensing with improved throughput.