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.     

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.     

Designed Intercalators for Modification of DNA Origami Surface Properties

The modification of the backbone properties of DNA origami nanostructures through noncovalent interactions with designed intercalators, based on acridine derivatized with side chains containing esterified fatty acids or oligo(ethylene glycol) residues is reported. Spectroscopic analyses indicate that these intercalators bind to DNA origami structures. Atomic force microscopy studies reveal that intercalator binding does not affect the structural intactness but leads to altered surface properties of the highly negatively charged nanostructures, as demonstrated by their interaction with solid mica or graphite supports. Moreover, the noncovalent interaction between the intercalators and the origami structures leads to alteration in cellular uptake, as shown by confocal microscopy studies using two different eukaryotic cell lines. Hence, the intercalator approach offers a potential means for tailoring the surface properties of DNA nanostructures.     

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.     

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.     

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.     

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.     

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.     

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.     

Preparation of Chemically Modified RNA Origami Nanostructures

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.     

DNA Nanostructures for Targeted Antimicrobial Delivery

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.     

DNA Origami Scaffolds as Templates for Functional Tetrameric Kir3 K+ Channels

In native systems, scaffolding proteins play important roles in assembling proteins into complexes to transduce signals. This concept is yet to be applied to the assembly of functional transmembrane protein complexes in artificial systems. To address this issue, DNA origami has the potential to serve as scaffolds that arrange proteins at specific positions in complexes. Herein, we report that Kir3 K⁺ channel proteins are assembled through zinc‐finger protein (ZFP)‐adaptors at specific locations on DNA origami scaffolds. Specific binding of the ZFP‐fused Kir3 channels and ZFP‐based adaptors on DNA origami were confirmed by atomic force microscopy and gel electrophoresis. Furthermore, the DNA origami with ZFP binding sites nearly tripled the K⁺ channel current activity elicited by heterotetrameric Kir3 channels in HEK293T cells. Thus, our method provides a useful template to control the oligomerization states of membrane protein complexes in vitro and in living cells.     

Platinum-DNA Origami Hybrid Structures in Concentrated Hydrogen Peroxide

The DNA origami technique allows fast and large-scale production of DNA nanostructures that stand out with an accurate addressability of their anchor points. This enables the precise organization of guest molecules on the surfaces and results in diverse functionalities. However, the compatibility of DNA origami structures with catalytically active matter, a promising pathway to realize autonomous DNA machines, has so far been tested only in the context of bio-enzymatic activity, but not in chemically harsh reaction conditions. The latter are often required for catalytic processes involving high-energy fuels. Here, we provide proof-of-concept data showing that DNA origami structures are stable in 5 % hydrogen peroxide solutions over the course of at least three days. We report a protocol to couple these to platinum nanoparticles and show catalytic activity of the hybrid structures. We suggest that the presented hybrid structures are suitable to realize catalytic nanomachines combined with precisely engineered DNA nanostructures.     

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.     

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.     

Assembly and Purification of Enzyme‐Functionalized DNA Origami Structures

The positioning of enzymes on DNA nanostructures for the study of spatial effects in interacting biomolecular assemblies requires chemically mild immobilization procedures as well as efficient means for separating unbound proteins from the assembled constructs. We herein report the exploitation of free‐flow electrophoresis (FFE) for the purification of DNA origami structures decorated with biotechnologically relevant recombinant enzymes: the S‐selective NADP⁺/NADPH‐dependent oxidoreductase Gre2 from S. Cerevisiae and the reductase domain of the monooxygenase P450 BM3 from B. megaterium. The enzymes were fused with orthogonal tags to facilitate site‐selective immobilization. FFE purification yielded enzyme–origami constructs whose specific activity was quantitatively analyzed. All origami‐tethered enzymes were significantly more active than the free enzymes, thereby suggesting a protective influence of the large, highly charged DNA nanostructure on the stability of the proteins.     

Enhancing Biocompatible Stability of DNA Nanostructures Using Dendritic Oligonucleotides and Brick Motifs

The use of DNA‐based nanomaterials in biomedical applications is continuing to grow, yet more emphasis is being put on the need for guaranteed structural stability of DNA nanostructures in physiological conditions. Various methods have been developed to stabilize DNA origami against low concentrations of divalent cations and the presence of nucleases. However, existing strategies typically require the complete encapsulation of nanostructures, which makes accessing the encased DNA strands difficult, or chemical modification, such as covalent crosslinking of DNA strands. We present a stabilization method involving the synthesis of DNA brick nanostructures with dendritic oligonucleotides attached to the outer surface. We find that nanostructures assembled from DNA brick motifs remain stable against denaturation without any chemical modifications. Furthermore, densely coating the outer surface of DNA brick nanostructures with dendritic oligonucleotides prevents nuclease digestion.     

Triple Helix Formation in a Topologically Controlled DNA Nanosystem

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.     

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.     

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.