Patterning protein complexes on DNA nanostructures using a GFP nanobody

DNA nanostructures have become an important and powerful tool for studying protein function over the last 5 years. One of the challenges, though, has been the development of universal methods for patterning protein complexes on DNA nanostructures. Herein, we present a new approach for labeling DNA nanostructures by functionalizing them with a GFP nanobody. We demonstrate the ability to precisely control protein attachment via our nanobody linker using two enzymatic model systems, namely adenylyl cyclase activity and myosin motility. Finally, we test the power of this attachment method by patterning unpurified, endogenously expressed Arp2/3 protein complex from cell lysate. By bridging DNA nanostructures with a fluorescent protein ubiquitous throughout cell and developmental biology and protein biochemistry, this approach significantly streamlines the application of DNA nanostructures as a programmable scaffold in biological studies.     

Nanopore fingerprinting of supramolecular DNA nanostructures

DNA nanotechnology has paved the way for new generations of programmable nanomaterials. Utilizing the DNA origami technique, various DNA constructs can be designed, ranging from single tiles to the self-assembly of large-scale, complex, multi-tile arrays. This technique relies on the binding of hundreds of short DNA staple strands to a long single-stranded DNA scaffold that drives the folding of well-defined nanostructures. Such DNA nanostructures have enabled new applications in biosensing, drug delivery, and other multifunctional materials. In this study, we take advantage of the enhanced sensitivity of a solid-state nanopore that employs a poly-ethylene glycol enriched electrolyte to deliver real-time, non-destructive, and label-free fingerprinting of higher-order assemblies of DNA origami nanostructures with single-entity resolution. This approach enables the quantification of the assembly yields for complex DNA origami nanostructures using the nanostructure-induced equivalent charge surplus as a discriminant. We compare the assembly yield of four supramolecular DNA nanostructures obtained with the nanopore with agarose gel electrophoresis and atomic force microscopy imaging. We demonstrate that the nanopore system can provide analytical quantification of the complex supramolecular nanostructures within minutes, without any need for labeling and with single-molecule resolution. We envision that the nanopore detection platform can be applied to a range of nanomaterial designs and enable the analysis and manipulation of large DNA assemblies in real time.     

Switchable DNA-origami nanostructures that respond to their environment and their applications

Structural DNA nanotechnology, in which Watson-Crick base pairing drives the formation of self-assembling nanostructures, has rapidly expanded in complexity and functionality since its inception in 1981. DNA nanostructures can now be made in arbitrary three-dimensional shapes and used to scaffold many other functional molecules such as proteins, metallic nanoparticles, polymers, fluorescent dyes and small molecules. In parallel, the field of dynamic DNA nanotechnology has built DNA circuits, motors and switches. More recently, these two areas have begun to merge—to produce switchable DNA nanostructures, which change state in response to their environment. In this review, we summarise switchable DNA nanostructures into two major classes based on response type: molecular actuation triggered by local chemical changes such as pH or concentration and external actuation driven by light, electric or magnetic fields. While molecular actuation has been well explored, external actuation of DNA nanostructures is a relatively new area that allows for the remote control of nanoscale devices. We discuss recent applications for DNA nanostructures where switching is used to perform specific functions—such as opening a capsule to deliver a molecular payload to a target cell. We then discuss challenges and future directions towards achieving synthetic nanomachines with complexity on the level of the protein machinery in living cells.     

CRISPR–Cas9-mediated nuclear transport and genomic integration of nanostructured genes in human primary cells

DNA nanostructures are a promising tool to deliver molecular payloads to cells. DNA origami structures, where long single-stranded DNA is folded into a compact nanostructure, present an attractive approach to package genes; however, effective delivery of genetic material into cell nuclei has remained a critical challenge. Here, we describe the use of DNA nanostructures encoding an intact human gene and a fluorescent protein encoding gene as compact templates for gene integration by CRISPR-mediated homology-directed repair (HDR). Our design includes CRISPR–Cas9 ribonucleoprotein binding sites on DNA nanostructures to increase shuttling into the nucleus. We demonstrate efficient shuttling and genomic integration of DNA nanostructures using transfection and electroporation. These nanostructured templates display lower toxicity and higher insertion efficiency compared to unstructured double-stranded DNA templates in human primary cells. Furthermore, our study validates virus-like particles as an efficient method of DNA nanostructure delivery, opening the possibility of delivering nanostructures in vivo to specific cell types. Together, these results provide new approaches to gene delivery with DNA nanostructures and establish their use as HDR templates, exploiting both their design features and their ability to encode genetic information. This work also opens a door to translate other DNA nanodevice functions, such as biosensing, into cell nuclei.     

Organizing protein-DNA hybrids as nanostructures with programmed functionalities

The structural and functional information encoded in the base sequence of nucleic acids provides a means to organize hybrid protein-DNA nanostructures with pre-designed, programmed functionality. This review discusses the activation of enzyme cascades in supramolecular DNA-protein hybrid structures, the bioelectrocatalytic activation of redox enzymes on DNA scaffolds, and the programmed positioning of enzymes on 1D, 2D and 3D DNA nanostructures. These systems provide starting points towards the design of interconnected enzyme networks. Substantial progress in the tailoring of functional protein-DNA nanostructures has been accomplished in recent years, and advances in this field warrant a comprehensive discussion. The application of these systems for the control of biocatalytic transformations, for amplified biosensing, and for the synthesis of metallic nanostructures are addressed, and future prospects for these systems are highlighted.     

Modulating the chemo-mechanical response of structured DNA assemblies through binding molecules

Recent advances in DNA nanotechnology led the fabrication and utilization of various DNA assemblies, but the development of a method to control their global shapes and mechanical flexibilities with high efficiency and repeatability is one of the remaining challenges for the realization of the molecular machines with on-demand functionalities. DNA-binding molecules with intercalation and groove binding modes are known to induce the perturbation on the geometrical and mechanical characteristics of DNA at the strand level, which might be effective in structured DNA assemblies as well. Here, we demonstrate that the chemo-mechanical response of DNA strands with binding ligands can change the global shape and stiffness of DNA origami nanostructures, thereby enabling the systematic modulation of them by selecting a proper ligand and its concentration. Multiple DNA-binding drugs and fluorophores were applied to straight and curved DNA origami bundles, which demonstrated a fast, recoverable, and controllable alteration of the bending persistence length and the radius of curvature of DNA nanostructures. This chemo-mechanical modulation of DNA nanostructures would provide a powerful tool for reconfigurable and dynamic actuation of DNA machineries.     

DNA四面体纳米结构及其在生物技术领域的应用进展

DNA四面体纳米结构是一种通过精确巧妙的DNA序列设计,应用碱基互补配对的原则,由4条单链自动杂交结合而成的具有四面体形状的DNA三维纳米结构。其具有良好的生物相容性和优异的细胞膜通透性,同时制备较为简单且产率高、尺寸以及动态性均可调节,因而在微生物鉴定、医学诊断和生物传感器等领域得到了广泛的研究与应用。基于此,介绍了DNA四面体纳米结构及其功能化修饰,综述了DNA四面体纳米结构在生物技术领域的应用进展,以期为推动DNA四面体纳米结构的研究、拓宽其应用领域提供参考。     

DNA Tetrominoes: The Construction of DNA Nanostructures Using Self-Organised Heterogeneous Deoxyribonucleic Acids Shapes

The unique programmability of nucleic acids offers alternative in constructing excitable and functional nanostructures. This work introduces an autonomous protocol to construct DNA Tetris shapes (L-Shape, B-Shape, T-Shape and I-Shape) using modular DNA blocks. The protocol exploits the rich number of sequence combinations available from the nucleic acid alphabets, thus allowing for diversity to be applied in designing various DNA nanostructures. Instead of a deterministic set of sequences corresponding to a particular design, the protocol promotes a large pool of DNA shapes that can assemble to conform to any desired structures. By utilising evolutionary programming in the design stage, DNA blocks are subjected to processes such as sequence insertion, deletion and base shifting in order to enrich the diversity of the resulting shapes based on a set of cascading filters. The optimisation algorithm allows mutation to be exerted indefinitely on the candidate sequences until these sequences complied with all the four fitness criteria. Generated candidates from the protocol are in agreement with the filter cascades and thermodynamic simulation. Further validation using gel electrophoresis indicated the formation of the designed shapes. Thus, supporting the plausibility of constructing DNA nanostructures in a more hierarchical, modular, and interchangeable manner.     

Purification of DNA-origami nanostructures by rate-zonal centrifugation

Most previously reported methods for purifying DNA-origami nanostructures rely on agarose-gel electrophoresis (AGE) for separation. Although AGE is routinely used to yield 0.1–1 µg purified DNA nanostructures, obtaining >100 µg of purified DNA-origami structure through AGE is typically laborious because of the post-electrophoresis extraction, desalting and concentration steps. Here, we present a readily scalable purification approach utilizing rate-zonal centrifugation, which provides comparable separation resolution as AGE. The DNA nanostructures remain in aqueous solution throughout the purification process. Therefore, the desired products are easily recovered with consistently high yield (40–80%) and without contaminants such as residual agarose gel or DNA intercalating dyes. Seven distinct three-dimensional DNA-origami constructs were purified at the scale of 0.1–100 µg (final yield) per centrifuge tube, showing the versatility of this method. Given the commercially available equipment for gradient mixing and fraction collection, this method should be amenable to automation and further scale up for preparation of larger amounts (e.g. milligram quantities) of DNA nanostructures.     

Reconfiguration of DNA nanostructures induced by enzymatic ligation treatment

Enzymatic ligation is a popular method in DNA nanotechnology for structural enforcement. When employed as stability switch for chosen components, ligation can be applied to induce DNA nanostructure reconfiguration. In this study, we investigate the reinforcement effect of ligation on addressable DNA nanostructures assembled entirely from short synthetic strands as the basis of structural reconfiguration. A careful calibration of ligation efficiency is performed on structures with programmable nicks. Systematic investigation using comparative agarose gel electrophoresis enables quantitative assessment of enhanced survivability with ligation treatment on a number of unique structures. The solid ligation performance sets up the foundation for the ligation-based structural reconfiguration. With the capability of switching base pairing status between permanent and transient (ON and OFF) by a simple round of enzymatic treatment, ligation induced reconfiguration can be engineered for DNA nanostructures accordingly.     

Size-controllable quartz nanostructure for signal enhancement of DNA chip

A mask-free, cost-effective dry-etching method for the fabrication of height- and spacing-controlled, pillar-like nanostructures was established in order to detect DNA molecules. The height and spacing of the quartz nanostructure were regulated by successive O(2) and CF(4) reactive ion etching times. The height and spacing of the nanostructures were tuned between 118 and 269 nm and between 107 and 161 nm, respectively. Probe DNA was immobilized on the structure and hybridized with fluorescently-labeled target DNA. Increases in the height and spacing of the nanopillar structure positively correlated with the fluorescence intensity of bound DNA. Usage of the nanostructure increased the DNA detection limit by up to 100-fold.     

DNA microarrays on silicon nanostructures: optimization of the multilayer stack for fluorescence detection

To improve the sensitivity of fluorescence detection in DNA microarrays, the use of silicon nanostructures based on chemical vapor deposition (CVD) processes adopted for the growth of rough polycrystalline silicon was investigated. These substrates present advantages of two main properties which could lead to an enhancement of the fluorescence detection, i.e. (i) the increase of the available surface area in order to achieve a high loading capacity of biomolecules and (ii) the optimization of the stack of silicon nanostructures support. Indeed, the structures were elaborated on an initial thermal oxide layer and then covered with a silicon oxide layer, obtained by oxidation and allowing the functionalization for the subsequent grafting of DNA probes. Moreover, these oxide layers play a part in the fluorescence detection. The influence of the silicon oxide layer thickness above and below the silicon grains in close relation with the density of nanostructures on the emitted fluorescence was emphasized. This paper presents an experimental characterization of the fluorescence intensity and the optimization of the different layers that composed the substrate used for DNA microarrays. The performances of the microarrays were investigated by means of hybridization experiments using complementary fluorescent labeled-oligonucleotides targets. Our results indicate that an optimized substrate can be designed and that the use of oxidized silicon nanostructures for support of biochip could be a strategy for improving the sensitivity of fluorescence detection.     

DNA Nano-Gears

DNA is a nanoscale material for programmable self-assembly, using the selective affinity of pairs of DNA strands to form DNA nanostructures. Self-assembly is the spontaneous self-ordering of substructures into superstructures which driven by the selective affinity of the substructures. DNA self-assembly is the most advanced and versatile system that has been experimentally demonstrated for programmable construction of patterned systems on the molecular scale. This programmability renders the scaffolding have the patterning required for fabricating complex devices made of these components. We present various strategies to assemble DNA based gears for application in nano-machines, nano-motors and nano-devices. In this paper, some fundamental parts of mechanical nano-machines with DNA blocks are designed. These kinds of nanostructures, nano-gears, are fundamental for the development of future useful molecular-level devices.     

Thiol-mediated anchoring of silver cations to DNA for construction of nanofibers on DNA scaffold

The formation of metal-containing Ag-mercaptoethanol (-Ag-S(R)-)(n) complexes on DNA chain scaffold was studied by UV spectroscopy, zeta potential measurement, and fluorescence and transmission electron microscopies. Experimental results made clear the mechanism of DNA mineralization and compaction, according to which intercalation of silver cations into DNA scaffold and further formation of (-Ag-S(R)-)(n) oligomeric complexes on DNA induce efficient DNA chain compaction by terminal Ag(+) cations. By transmission electron microscopy the formation of fiber-like DNA-templated nanostructures was observed. DNA-Ag-thiol complexes are promising for DNA-templated engineering of hybrid 1D nanostructures with adjustable chemical functionalities by choosing appropriate thiol ligand.     

Binding of DNA origami to lipids: maximizing yield and switching via strand displacement

Liposomes are widely used as synthetic analogues of cell membranes and for drug delivery. Lipid-binding DNA nanostructures can modify the shape, porosity and reactivity of liposomes, mediated by cholesterol modifications. DNA nanostructures can also be designed to switch conformations by DNA strand displacement. However, the optimal conditions to facilitate stable, high-yield DNA–lipid binding while allowing controlled switching by strand displacement are not known. Here, we characterized the effect of cholesterol arrangement, DNA structure, buffer and lipid composition on DNA–lipid binding and strand displacement. We observed that binding was inhibited below pH 4, and above 200 mM NaCl or 40 mM MgCl₂, was independent of lipid type, and increased with membrane cholesterol content. For simple motifs, binding yield was slightly higher for double-stranded DNA than single-stranded DNA. For larger DNA origami tiles, four to eight cholesterol modifications were optimal, while edge positions and longer spacers increased yield of lipid binding. Strand displacement achieved controlled removal of DNA tiles from membranes, but was inhibited by overhang domains, which are used to prevent cholesterol aggregation. These findings provide design guidelines for integrating strand displacement switching with lipid-binding DNA nanostructures. This paves the way for achieving dynamic control of membrane morphology, enabling broader applications in nanomedicine and biophysics.     

Formation of non-base-pairing DNA microgels using directed phase transition of amphiphilic monomers

Programmability of DNA sequences enables the formation of synthetic DNA nanostructures and their macromolecular assemblies such as DNA hydrogels. The base pair-level interaction of DNA is a foundational and powerful mechanism to build DNA structures at the nanoscale; however, its temperature sensitivity and weak interaction force remain a barrier for the facile and scalable assembly of DNA structures toward higher-order structures. We conducted this study to provide an alternative, non-base-pairing approach to connect nanoscale DNA units to yield micrometer-sized gels based on the sequential phase transition of amphiphilic unit structures. Strong electrostatic interactions between DNA nanostructures and polyelectrolyte spermines led to the formation of giant phase-separated aggregates of monomer units. Gelation could be initiated by the addition of NaCl, which weakened the electrostatic DNA-spermine interaction while attractive interactions between cholesterols created stable networks by crosslinking DNA monomers. In contrast to the conventional DNA gelation techniques, our system used solid aggregates as a precursor for DNA microgels. Therefore, in situ gelation could be achieved by depositing aggregates on the desired substrate and subsequently initiating a phase transition. Our approach can expand the utility and functionality of DNA hydrogels by using more complex nucleic acid assemblies as unit structures and combining the technique with top-down microfabrication methods.     

Cooperativity-based modeling of heterotypic DNA nanostructure assembly

DNA origami is a robust method for the fabrication of nanoscale 2D and 3D objects with complex features and geometries. The process of DNA origami folding has been recently studied, however quantitative understanding of it is still elusive. Here, we describe a systematic quantification of the assembly process of DNA nanostructures, focusing on the heterotypic DNA junction—in which arms are unequal—as their basic building block. Using bulk fluorescence studies we tracked this process and identified multiple levels of cooperativity from the arms in a single junction to neighboring junctions in a large DNA origami object, demonstrating that cooperativity is a central underlying mechanism in the process of DNA nanostructure assembly. We show that the assembly of junctions in which the arms are consecutively ordered is more efficient than junctions with randomly-ordered components, with the latter showing assembly through several alternative trajectories as a potential mechanism explaining the lower efficiency. This highlights consecutiveness as a new design consideration that could be implemented in DNA nanotechnology CAD tools to produce more efficient and high-yield designs. Altogether, our experimental findings allowed us to devise a quantitative, cooperativity-based heuristic model for the assembly of DNA nanostructures, which is highly consistent with experimental observations.     

DNA complexes, formed on aqueous phase surfaces: new planar polymeric and composite nanostructures

The formation of DNA complexes with Langmuir monolayers of the cationic lipid octadecylamine (ODA) and the new amphiphilic polycation poly-4-vinylpyridine with 16% of cetylpyridinium groups (PVP-16) on the surface of an aqueous solution of native DNA of low ionic strength was studied. Topographic images of Langmuir-Blodgett films of DNA/ODA and DNA/PVP-16 complexes applied to micaceous substrates were investigated by the method of atomic force microscopy. It was found that films of the amphiphilic polycation have an ordered planar polycrystalline structure. The morphology of planar DNA complexes with the amphiphilic cation substantially depended on the incubation time and the phase state of the monolayer on the surface of the aqueous DNA solution. Complex structures and individual DNA molecules were observed on the surface of the amphiphilic monolayer. Along with quasi-linear individual bound DNA molecules, characteristic extended net-like structures and quasi-circular toroidal condensed conformations of planar DNA complexes were detected. Mono- and multilayer films of DNA/PVP-16 complexes were used as templates and nanoreactors for the synthesis of inorganic nanostructures via the binding of metal cations from the solution and subsequent generation of the inorganic phase. As a result, ultrathin polymeric composite films with integrated DNA building blocks and quasi-linear arrays of inorganic semiconductor (CdS) and iron oxide nanoparticles and nanowires were obtained. The nanostructures obtained were characterized by scanning probe microscopy and transmission electron microscopy techniques. The methods developed are promising for investigating the mechanisms of structural organization and transformation in DNA and polyelectrolyte complexes at the gas-liquid interface and for the design of new extremely thin highly ordered planar polymeric and composite materials, films, and coatings with controlled ultrastructure for applications in nanoelectronics and nanobiotechnology.     

Self-assembled DNA-based giant thrombin nanoparticles for controlled release

Protein-aptamer interactions have been used in a wide range of fields, including medical diagnosis and protein delivery. Herein, we report a method for thrombin delivery with thrombin-binding aptamer (TBA), which is one of the well-known aptamers for thrombin, by generating giant thrombin nanoparticles (GTNPs). GTNPs can be synthesized by crosslinking thrombin with DNA nanostructures that possess several TBA molecules. To generate GTNPs, two different DNA nanostructures were used. Y-shaped DNA with TBA and X-shaped DNA with TBA were used for 250 and 650 nm GTNPs, respectively. Controlled release of thrombin from GTNPs was performed by adding complementary DNA (cDNA) to TBA. To investigate thrombin release from GTNPs, the sizes of the GTNPs were measured using dynamic light scattering, atomic force microscopy (AFM), and scanning electron microscopy (SEM). We confirmed a decrease in the size of GTNPs with various concentrations of cDNA, suggesting the release of thrombin. Based on these results, we expect that our method can be used to control the amount of thrombin released effectively. Our method is also widely applicable for effective protein delivery.     

Heat-resistant DNA tile arrays constructed by template-directed photoligation through 5-carboxyvinyl-2'-deoxyuridine

Template-directed DNA photoligation has been applied to a method to construct heat-resistant two-dimensional (2D) DNA arrays that can work as scaffolds in bottom-up assembly of functional biomolecules and nano-electronic components. DNA double-crossover AB-staggered (DXAB) tiles were covalently connected by enzyme-free template-directed photoligation, which enables a specific ligation reaction in an extremely tight space and under buffer conditions where no enzymes work efficiently. DNA nanostructures created by self-assembly of the DXAB tiles before and after photoligation have been visualized by high-resolution, tapping mode atomic force microscopy in buffer. The improvement of the heat tolerance of 2D DNA arrays was confirmed by heating and visualizing the DNA nanostructures. The heat-resistant DNA arrays may expand the potential of DNA as functional materials in biotechnology and nanotechnology.