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.     

Surface‐Assisted Large‐Scale Ordering of DNA Origami Tiles

The arrangement of DNA‐based nanostructures into extended higher order assemblies is an important step towards their utilization as functional molecular materials. We herein demonstrate that by electrostatically controlling the adhesion and mobility of DNA origami structures on mica surfaces by the simple addition of monovalent cations, large ordered 2D arrays of origami tiles can be generated. The lattices can be formed either by close‐packing of symmetric, non‐interacting DNA origami structures, or by utilizing blunt‐end stacking interactions between the origami units. The resulting crystalline lattices can be readily utilized as templates for the ordered arrangement of proteins.     

Multiscale Origami Structures as Interface for Cells

A DNA‐based platform was developed to address fundamental aspects of early stages of cell signaling in living cells. By site‐directed sorting of differently encoded, protein‐decorated DNA origami structures on DNA microarrays, we combine the advantages of the bottom‐up self‐assembly of protein–DNA nanostructures and top‐down micropatterning of solid surfaces to create multiscale origami structures as interface for cells (MOSAIC). In a proof‐of‐principle, we use this technology to analyze the activation of epidermal growth factor (EGF) receptors in living MCF7 cells using DNA origami structures decorated on their surface with distinctive nanoscale arrangements of EGF ligand entities. MOSAIC holds the potential to present to adhered cells well‐defined arrangements of ligands with full control over their number, stoichiometry, and precise nanoscale orientation. It therefore promises novel applications in the life sciences, which cannot be tackled by conventional technologies.     

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.     

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.     

Photocontrolled Dopamine Polymerization on DNA Origami with Nanometer Resolution

Temporal and spatial control over polydopamine formation on the nanoscale can be achieved by installing an irradiation‐sensitive polymerization system on DNA origami. Precisely distributed G‐quadruplex structures on the DNA template serve as anchors for embedding the photosensitizer protoporphyrin IX, which—upon irradiation with visible light—induces the multistep oxidation of dopamine to polydopamine, producing polymeric structures on designated areas within the origami framework. The photochemical polymerization process allows exclusive control over polydopamine layer formation through the simple on/off switching of the light source. The obtained polymer–DNA hybrid material shows significantly enhanced stability, paving the way for biomedical and chemical applications that are typically not possible owing to the sensitivity of DNA.     

Light-Activated Assembly of DNA Origami into Dissipative Fibrils

Hierarchical DNA nanostructures offer programmable functions at scale, but making these structures dynamic, while keeping individual components intact, is challenging. Here we show that the DNA A-motif—protonated, self-complementary poly(adenine) sequences—can propagate DNA origami into one-dimensional, micron-length fibrils. When coupled to a small molecule pH regulator, visible light can activate the hierarchical assembly of our DNA origami into dissipative fibrils. This system is recyclable and does not require DNA modification. By employing a modular and waste-free strategy to assemble and disassemble hierarchical structures built from DNA origami, we offer a facile and accessible route to developing well-defined, dynamic, and large DNA assemblies with temporal control. As a general tool, we envision that coupling the A-motif to cycles of dissipative protonation will allow the transient construction of diverse DNA nanostructures, finding broad applications in dynamic and non-equilibrium nanotechnology.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

3D DNA Origami Nanoparticles: From Basic Design Principles to Emerging Applications in Soft Matter and (Bio‐)Nanosciences

Scaffold‐based lattice‐engineered 3D DNA origami is a powerful and versatile technique for the rational design and build‐up of arbitrarily structured and monodisperse DNA‐based 3D nanoobjects. Relying on the unsurpassed molecular programmability of sequence‐specific DNA hybridization, a long DNA single strand (termed scaffold) is assembled with many short single‐stranded oligomers (termed staples), which organize the scaffold into a 3D lattice in a single step, thereby leading to 3D nanoparticulate structures of the highest precision in high yields. Applications of 3D DNA origami are increasingly wide‐spread and interface with numerous fields of sciences, for example, anisometric or anisotropically functionalized nanoparticles, fundamental investigations of superstructure formation, biomedicine, (bio)physics, sensors, and optical materials. This Minireview discusses the fundamentals and recent advances from structure formation to selected applications, with a mission to promote cross‐disciplinary exchange.     

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.     

Hydrophobic Actuation of a DNA Origami Bilayer Structure

Amphiphilic compounds have a strong tendency to form aggregates in aqueous solutions. It is shown that such aggregation can be utilized to fold cholesterol‐modified, single‐layered DNA origami structures into sandwich‐like bilayer structures, which hide the cholesterol modifications in their interior. The DNA bilayer structures unfold after addition of the surfactant Tween 80, and also in the presence of lipid bilayer membranes, with opening kinetics well described by stretched exponentials. It is also demonstrated that by combination with an appropriate lock and key mechanism, hydrophobic actuation of DNA sandwiches can be made conditional on the presence of an additional molecular input such as a specific DNA sequence.