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.     

Light‐Directing Omnidirectional Circularly Polarized Reflection from Liquid‐Crystal Droplets

Constructing and tuning self‐organized three‐dimensional (3D) superstructures with tailored functionality is crucial in the nanofabrication of smart molecular devices. Herein we fabricate a self‐organized, phototunable 3D photonic superstructure from monodisperse droplets of one‐dimensional cholesteric liquid crystal (CLC) containing a photosensitive chiral molecular switch with high helical twisting power. The droplets are obtained by a glass capillary microfluidic technique by dispersing into PVA solution that facilitates planar anchoring of the liquid‐crystal molecules at the droplet surface, as confirmed by the observation of normal incidence selective circular polarized reflection in all directions from the core of individual droplet. Photoirradiation of the droplets furnishes dynamic reflection colors without thermal relaxation, whose wavelength can be tuned reversibly by variation of the irradiation time. The results provided clear evidence on the phototunable reflection in all directions.     

Peptide Conjugates for Directing the Morphology and Assembly of 1D Nanoparticle Superstructures

Designed peptide conjugates molecules are used to direct the synthesis and assembly of gold nanoparticles into complex 1D nanoparticle superstructures with various morphologies. Four peptide conjugates, each based on the gold‐binding peptide (AYSSGAPPMPPF; PEP_Au), are prepared: C₁₂H₂₃O‐AYSSGAPPMPP ( 1 ), C₁₂H₂₃O‐AYSSGAPPMPPF ( 2 ), C₁₂H₂₃O‐AYSSGAPPMPPFF ( 3 ), and C₁₂H₂₃O‐AYSSGAPPMPPFFF ( 4 ). The affect that C‐terminal hydrophobic F residues have on both the soft‐assembly of the peptide conjugates and the resulting assembly of gold nanoparticle superstructures is examined. It is shown that the addition of two C‐terminal F residues ( 3 ) leads to thick, branched 1D gold nanoparticle superstructures, whereas the addition of three C‐terminal F residues ( 4 ) leads to bundling of thin 1D nanoparticle superstructures.     

Biosensing by Tandem Reactions of Structure Switching,Nucleolytic Digestion,and DNA Amplification of a DNA Assembly

ϕ29 DNA polymerase (ϕ29DP) is able to carry out repetitive rounds of DNA synthesis using a circular DNA template by rolling circle amplification (RCA). It also has the ability to execute 3′–5′ digestion of single‐stranded but not double‐stranded DNA. A biosensor engineering strategy is presented that takes advantage of these two properties of ϕ29DP coupled with structure‐switching DNA aptamers. The design employs a DNA assembly made of a circular DNA template, a DNA aptamer, and a pre‐primer. The DNA assembly is unable to undergo RCA in the absence of cognate target owing to the formation of duplex structures. The presence of the target, however, triggers a structure‐switching event that causes nucleolytic conversion of the pre‐primer by ϕ29DP into a mature primer to facilitate RCA. This method relays target detection by the aptamer to the production of massive DNA amplicons, giving rise to dramatically enhanced detection sensitivity.     

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.     

Biosensing by Tandem Reactions of Structure Switching,Nucleolytic Digestion,and DNA Amplification of a DNA Assembly

?29 DNA polymerase (?29DP) is able to carry out repetitive rounds of DNA synthesis using a circular DNA template by rolling circle amplification (RCA). It also has the ability to execute 3′–5′ digestion of single‐stranded but not double‐stranded DNA. A biosensor engineering strategy is presented that takes advantage of these two properties of ?29DP coupled with structure‐switching DNA aptamers. The design employs a DNA assembly made of a circular DNA template, a DNA aptamer, and a pre‐primer. The DNA assembly is unable to undergo RCA in the absence of cognate target owing to the formation of duplex structures. The presence of the target, however, triggers a structure‐switching event that causes nucleolytic conversion of the pre‐primer by ?29DP into a mature primer to facilitate RCA. This method relays target detection by the aptamer to the production of massive DNA amplicons, giving rise to dramatically enhanced detection sensitivity.     

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.     

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.     

A Spatially Programmable DNA Nanorobot Arm to Modulate Anisotropic Gold Nanoparticle Assembly by Enzymatic Excision

Programmable assembly of gold nanoparticle superstructures with precise spatial arrangement has drawn much attention for their unique characteristics in plasmonics and biomedicine. Bio-inspired methods have already provided programmable, molecular approaches to direct AuNP assemblies using biopolymers. The existing methods, however, predominantly use DNA as scaffolds to directly guide the AuNP interactions to produce intended superstructures. New paradigms for regulating AuNP assembly will greatly enrich the toolbox for DNA-directed AuNP manipulation and fabrication. Here, we developed a strategy of using a spatially programmable enzymatic nanorobot arm to modulate anisotropic DNA surface modifications and assembly of AuNPs. Through spatial controls of the proximity of the reactants, the locations of the modifications were precisely regulated. We demonstrated the control of the modifications on a single 15 nm AuNP, as well as on a rectangular DNA origami platform, to direct unique anisotropic AuNP assemblies. This method adds an alternative enzymatic manipulation to DNA-directed AuNP superstructure assembly.     

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.     

Ultrasensitive Fluorescence Polarization Aptasensors Based on Exonuclease Signal Amplification and Polystyrene Nanoparticle Amplification

Here, we combine T7 exonuclease (T7 Exo) signal amplification and polystyrene nanoparticle (PS NP) amplification to develop novel fluorescence polarization (FP) aptasensors. The binding of a target/open aptamer hairpin complex or a target/single‐stranded aptamer complex to dye‐labeled DNA bound to PS NPs, or the self‐assembly of two aptamer subunits (one of them labeled with a dye) into a target/aptamer complex on PS NPs leads to the cyclic T7 Exo‐catalyzed digestion of the dye‐labeled DNA or the dye‐labeled aptamer subunit. This results in a substantial decrease in the FP value for the amplified sensing process. Our newly developed aptasensors exhibit a sensitivity five orders of magnitude higher than that of traditional homogeneous aptasensors and a high specificity for the target molecules. These distinct advantages of our proposed assay protocol make it a generic platform for the design of amplified aptasensors for ultrasensitive detection of various target molecules.     

Ultrathin Hexagonal Hybrid Nanosheets Synthesized by Graphene Oxide‐Assisted Exfoliation of β‐Co(OH)2 Mesocrystals

In the present study, we report the synthesis of a high‐quality, single‐crystal hexagonal β‐Co(OH)₂ nanosheet, exhibiting a thickness down to ten atomic layers and an aspect ratio exceeding 900, by using graphene oxide (GO) as an exfoliant of β‐Co(OH)₂ nanoflowers. Unlike conventional approaches using ionic precursors in which morphological control is realized by structure‐directing molecules, the β‐Co(OH)₂ flower‐like superstructures were first grown by a nanoparticle‐mediated crystallization process, which results in large 3D superstructure consisting of ultrathin nanosheets interspaced by polydimethoxyaniline (PDMA). Thereafter, β‐Co(OH)₂ nanoflowers were chemically exfoliated by surface‐active GO under hydrothermal conditions into unilamellar single‐crystal nanosheets. In this reaction, GO acts as a two‐dimensional (2D) amphiphile to facilitate the exfoliation process through tailored interactions between organic and inorganic molecules. Meanwhile, the on‐site conjugation of GO and Co(OH)₂ promotes the thermodynamic stability of freestanding ultrathin nanosheets and restrains further growth through Oswald ripening. The unique 2D structure combined with functionalities of the hybrid ultrathin Co(OH)₂ nanosheets on rGO resulted in a remarkably enhanced lithium‐ion storage performance as anode materials, maintaining a reversible capacity of 860 mA h g^?1 for as many as 30 cycles. Since mesocrystals are ubiquitous and rich in morphological diversity, the strategy of the GO‐assisted exfoliation of mesocrystals developed here provides an opportunity for the synthesis of new functional nanostructures that could bear importance in clean renewable energy, catalysis, photoelectronics, and photonics.     

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.     

Isothermal assembly of DNA origami structures using denaturing agents

DNA origami is one of the most promising recent developments in DNA self-assembly. It allows for the construction of arbitrary nanoscale patterns and objects by folding a long viral scaffold strand using a large number of short "staple" strands. Assembly is usually accomplished by thermal annealing of the DNA molecules in buffer solution. We here demonstrate that both 2D and 3D origami structures can be assembled isothermally by annealing the DNA strands in denaturing buffer, followed by a controlled reduction of denaturant concentration. This opens up origami assembly for the integration of temperature-sensitive components.     

Arrest of Rolling Circle Amplification by Protein‐Binding DNA Aptamers

Certain DNA polymerases, such as ?29 DNA polymerase, can isothermally copy the sequence of a circular template round by round in a process known as rolling circle amplification (RCA), which results in super‐long single‐stranded (ss) DNA molecules made of tandem repeats. The power of RCA reflects the high processivity and the strand‐displacement ability of these polymerases. In this work, the ability of ?29DNAP to carry out RCA over circular templates containing a protein‐binding DNA aptamer sequence was investigated. It was found that protein–aptamer interactions can prevent this DNA polymerase from reading through the aptameric domain. This finding indicates that protein‐binding DNA aptamers can form highly stable complexes with their targets in solution. This novel observation was exploited by translating RCA arrest into a simple and convenient colorimetric assay for the detection of specific protein targets, which continues to showcase the versatility of aptamers as molecular recognition elements for biosensing applications.     

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.     

Unusually Stable Triazine‐based Organic Superstructures

Solid‐state reactions have been rapidly gaining popularity in organic chemistry owing to their simplicity, efficiency, and selectivity compared to liquid‐phase reactions. Herein, we describe the formation of superstructures through the solid‐state reaction of an organic single‐crystal. The superstructure of 5,5′,5′′‐(1,3,5‐triazine‐2,4,6‐triyl)triisophthalonitrile (TIPN) can be formed by cyclotrimerization of 1,3,5‐tricyanobenzene (TCB) single crystals. The TIPN superstructure was confirmed by single crystal X‐ray diffraction and visualized by transmission electron microscopy. The superstructure has hexagonally packed 1‐dimensional (1D) channels along the crystal axis. Furthermore, the superstructure arises from interdigitated nitrile interactions in the crystal lattice, and thus has electron‐beam tolerance and very high thermal stability.     

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.     

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.     

A Three‐Dimensional Origami Paper‐Based Device for Potentiometric Biosensing

Current paper‐based potentiometric ion‐sensing platforms are planar devices used for clinically relevant ions. These devices, however, have not been designed for the potentiometric biosensing of proteins or small molecule analytes. A three‐dimensional origami paper‐based device, in which a solid‐contact ion‐selective electrode is integrated with an all‐solid‐state reference electrode, is described for the first time. The device is made by impregnation of paper with appropriate bioreceptors and reporting reagents on different zones. By folding and unfolding the paper structures, versatile potentiometric bioassays can be performed. A USB‐controlled miniaturized electrochemical detector can be used for simple and in situ measurements. Using butyrylcholinesterase as a model enzyme, the device has been successfully applied to the detection of enzyme activities and organophosphate pesticides involved in the enzymatic system as inhibitors. The proposed 3D origami paper device allows the potentiometric biosensing of proteins and small molecules in a simple, portable, and cost‐effective way.