DNA Origami Nanostructures Research Articles

Wireframe DNA origami nanostructure with the controlled opening of edges

Design and simulation of wireframe DNA origami nanostructures with fast and controlled opening capability using modified DAEDALUS code.

Quality Control of Mass-Encoded Nanodevices by Compartmented DNA Origami Frames for Precision Information Coding and Logic Mapping.

Encoded nanostructures afford an ideal platform carrying multi-channel signal components for multiplexed assay and information security. However, with the demand on exclusivity and reproducibility of coding signals, precise control on the structure and composition of nanomaterials featuring fully distinguishable signals remains challenging. By using the multiplexing capability of mass spectrometry (MS) and spatial addressability of DNA origami nanostructures, we herein propose a quality control methodology for constructing mass-encoded nanodevices (namely MNTs-TDOFs) in the scaffold of compartmented tetrahedral DNA origami frames (TDOFs), in which the arrangement and stoichiometry of four types of mass nanotags (MNTs) can be finely regulated and customized to generate characteristic MS patterns. The programmability of combinatorial MNTs and orthogonality of individual compartments allows further evolution of MNTs-TDOFs to static tagging agents and dynamic nanoprobes for labeling and sensing of multiple targets. More importantly, structure control at single TDOF level ensures the constancy of prescribed MS outputs, by which a high-capacity coding system was established for secure information encryption and decryption. In addition to the multiplexed outputs in parallel, the nanodevices could also map logic circuits with interconnected complexity and logic events of c-Met recognition and dimerization on cell surface for signaling regulation by MS interrogation.

Quality Control of Mass‐Encoded Nanodevices by Compartmented DNA Origami Frames for Precision Information Coding and Logic Mapping

AbstractEncoded nanostructures afford an ideal platform carrying multi‐channel signal components for multiplexed assay and information security. However, with the demand on exclusivity and reproducibility of coding signals, precise control on the structure and composition of nanomaterials featuring fully distinguishable signals remains challenging. By using the multiplexing capability of mass spectrometry (MS) and spatial addressability of DNA origami nanostructures, we herein propose a quality control methodology for constructing mass‐encoded nanodevices (namely MNTs‐TDOFs) in the scaffold of compartmented tetrahedral DNA origami frames (TDOFs), in which the arrangement and stoichiometry of four types of mass nanotags (MNTs) can be finely regulated and customized to generate characteristic MS patterns. The programmability of combinatorial MNTs and orthogonality of individual compartments allows further evolution of MNTs‐TDOFs to static tagging agents and dynamic nanoprobes for labeling and sensing of multiple targets. More importantly, structure control at single TDOF level ensures the constancy of prescribed MS outputs, by which a high‐capacity coding system was established for secure information encryption and decryption. In addition to the multiplexed outputs in parallel, the nanodevices could also map logic circuits with interconnected complexity and logic events of c‐Met recognition and dimerization on cell surface for signaling regulation by MS interrogation.

Analysis of DNA Origami Nanostructures Using Capillary Electrophoresis.

DNA origami nanostructures are engineered nanomaterials (ENMs) that possess significant customizability, biocompatibility, and tunable structural and functional properties, making them potentially useful materials in fields, such as medicine, biocomputing, biomedical engineering, and measurement science. Despite the potential of DNA origami as a functional nanomaterial, a major barrier to its applicability is the difficulty associated with obtaining pure, well-folded structures. Therefore, rapid methods of analysis to ensure purity are needed to support the rapid development of this class of nanomaterials. Here, we present the development of capillary electrophoresis (CE) as an analytical tool for DNA origami. CE was investigated under both capillary zone electrophoresis (CZE) and capillary transient isotachophoresis (ctITP) modes. Optimization of both systems yielded baseline resolved separations of folded DNA origami nanostructures from excess staple strands. The ctITP separation mode demonstrated superior performance in terms of peak resolution (Rs = 2.05 ± 0.3), peak efficiency (N = 12,200 ± 230), and peak symmetry (As = 1.29 ± 0.032). The SYBR family dyes (Gold, Green I, and Green II) were investigated as highly efficient, noncovalent fluorophores for on-column labeling of DNA origami and detection using laser-induced fluorescence. Finally, ctITP analysis conditions were also applied to DNA origami nanostructures with different shapes and for the differentiation of DNA origami aggregates.

CytoDirect: A Nucleic Acid Nanodevice for Specific and Efficient Delivery of Functional Payloads to the Cytoplasm.

Direct and efficient delivery of functional payloads such as chemotherapy drugs, siRNA, or small-molecule inhibitors into the cytoplasm, bypassing the endo/lysosomal trapping, is a challenging task for intracellular medicine. Here, we take advantage of the programmability of DNA nanotechnology to develop a DNA nanodevice called CytoDirect, which incorporates disulfide units and human epidermal growth factor receptor 2 (HER2) affibodies into a DNA origami nanostructure, enabling rapid cytosolic uptake into targeted cancer cells and deep tissue penetration. We further demonstrated that therapeutic oligonucleotides and small-molecule chemotherapy drugs can be easily delivered by CytoDirect and showed notable effects on gene knockdown and cell apoptosis, respectively. This study demonstrates the synergistic effect of disulfide and HER2 affibody modifications on the rapid cytosolic delivery of DNA origami and its payloads to targeted cells and deep tissues, thereby expanding the delivery capabilities of DNA nanostructures in a new direction for disease treatment.

ZIMFLUX: Single molecule localization microscopy with patterned illumination in 3D

Three dimensional modulation-enhanced single-molecule localization techniques, such as ModLoc, offer advancements in axial localization precision across the entire field of view and axial capture range, by applying phase shifting to the illumination pattern. However, this improvement is limited by the pitch of the illumination pattern that can be used and requires registration between separate regions of the camera. To overcome these limitations, we present ZIMFLUX, a method that combines astigmatic point-spread-function (PSF) engineering with a structured illumination pattern in all three spatial dimensions. In order to achieve this we address challenges such as optical aberrations, refractive index mismatch, supercritical angle fluorescence (SAF), and imaging at varying depths within a sample, by implementing a vectorial PSF model. In scenarios involving refractive index mismatch between the sample and immersion medium, the astigmatic PSF loses its ellipticity at greater imaging depths, leading to a deterioration in axial localization precision. In contrast, our simulations demonstrate that ZIMFLUX maintains high axial localization precision even when imaging deeper into the sample. Experimental results show unbiased localization of 3D 80 nm DNA-origami nanostructures in SAF conditions with a 1.5-fold improvement in axial localization precision when comparing ZIMFLUX to conventional SMLM methods that rely solely on astigmatic PSF engineering.

Wireframe DNA Origami for the Cellular Delivery of Platinum(II)-Based Drugs

The DNA origami method has revolutionized the field of DNA nanotechnology since its introduction. These nanostructures, with their customizable shape and size, addressability, nontoxicity, and capacity to carry bioactive molecules, are promising vehicles for therapeutic delivery. Different approaches have been developed for manipulating and folding DNA origami, resulting in compact lattice-based and wireframe designs. Platinum-based complexes, such as cisplatin and phenanthriplatin, have gained attention for their potential in cancer and antiviral treatments. Phenanthriplatin, in particular, has shown significant antitumor properties by binding to DNA at a single site and inhibiting transcription. The present work aims to study wireframe DNA origami nanostructures as possible carriers for platinum compounds in cancer therapy, employing both cisplatin and phenanthriplatin as model compounds. This research explores the assembly, platinum loading capacity, stability, and modulation of cytotoxicity in cancer cell lines. The findings indicate that nanomolar quantities of the ball-like origami nanostructure, obtained in the presence of phenanthriplatin and therefore loaded with that specific drug, reduced cell viability in MCF-7 (cisplatin-resistant breast adenocarcinoma cell line) to 33%, while being ineffective on the other tested cancer cell lines. The overall results provide valuable insights into using wireframe DNA origami as a highly stable possible carrier of Pt species for very long time-release purposes.

Spatiotemporal Control over Polynucleotide Brush Growth on DNA Origami Nanostructures

AbstractDNA nanotechnology provides an approach to create precise, tunable, and biocompatible nanostructures for biomedical applications. However, the stability of these structures is severely compromised in biological milieu due to their fast degradation by nucleases. Recently, we showed how enzymatic polymerization could be harnessed to grow polynucleotide brushes of tunable length and location on the surface of DNA origami nanostructures, which greatly enhances their nuclease stability. Here, we report on strategies that allow for both spatial and temporal control over polymerization through activatable initiation, cleavage, and regeneration of polynucleotide brushes using restriction enzymes. The ability to site‐specifically decorate DNA origami nanostructures with polynucleotide brushes in a spatiotemporally controlled way provides access to “smart” functionalized DNA architectures with potential applications in drug delivery and supramolecular assembly.

Spatiotemporal Control over Polynucleotide Brush Growth on DNA Origami Nanostructures.

DNA nanotechnology provides an approach to create precise, tunable, and biocompatible nanostructures for biomedical applications. However, the stability of these structures is severely compromised in biological milieu due to their fast degradation by nucleases. Recently, we showed how enzymatic polymerization could be harnessed to grow polynucleotide brushes of tunable length and location on the surface of DNA origami nanostructures, which greatly enhances their nuclease stability. Here, we report on strategies that allow for both spatial and temporal control over polymerization through activatable initiation, cleavage, and regeneration of polynucleotide brushes using restriction enzymes. The ability to site-specifically decorate DNA origami nanostructures with polynucleotide brushes in a spatiotemporally controlled way provides access to "smart" functionalized DNA architectures with potential applications in drug delivery and supramolecular assembly.

High-Affinity Host-Guest Recognition for Efficient Assembly and Enzymatic Responsiveness of DNA Nanostructures.

The combination of multiple orthogonal interactions enables hierarchical complexity in self-assembled nanoscale materials. Here, efficient supramolecular polymerization of DNA origami nanostructures is demonstrated using a multivalent display of small molecule host-guest interactions. Modification of DNA strands with cucurbit[7]uril (CB[7]) and its adamantane guest, yielding a supramolecularcomplex with an affinity of order 1010 m-1 , directs hierarchical assembly of origami monomers into 1D nanofibers. This affinity regime enables efficient polymerization; a lower-affinity β-cyclodextrin-adamantane complex does not promote extended structures at a similar valency. Finally, the utility of the high-affinity CB[7]-adamantane interactions is exploited to enable responsive enzymatic actuation of origami nanofibers assembled using peptide linkers. This work demonstrates the power of high-affinity CB[7]-guest recognition as an orthogonal axis to drive self-assembly in DNA nanotechnology.

Nano- and Microscale Confinements in DNA-Scaffolded Enzyme Cascade Reactions.

Artificial reconstruction of naturally evolved principles, such as compartmentalization and cascading of multienzyme complexes, offers enormous potential for the development of biocatalytic materials and processes. Due to their unique addressability at the nanoscale, DNA origami nanostructures (DON) have proven to be an exceptionally powerful tool for studying the fundamental processes in biocatalytic cascades. To systematically investigate the diffusion-reaction network of (co)substrate transfer in enzyme cascades, a model system of stereoselective ketoreductase (KRED) with cofactor regenerating enzyme is assembled in different spatial arrangements on DNA nanostructures and is located in the sphere of microbeads (MB) as a spatially confining nano- and microenvironment, respectively. The results, obtained through the use of highly sensitive analytical methods, Western blot-based quantification of the enzymes, and mass spectrometric (MS) product detection, along with theoretical modeling, provide strong evidence for the presence of two interacting compartments, the diffusion layers around the microbead and the DNA scaffold, which influence the catalytic efficiency of the cascade. It is shown that the microscale compartment exerts a strong influence on the productivity of the cascade, whereas the nanoscale arrangement of enzymes has no influence but can be modulated by the insertion of a diffusion barrier.

Light-induced damage to DNA origami nanostructures in the 193 nm–310 nm range

DNA origami nanostructures provide precisely addressable substrates for in singulo experiments as well as for applications in nanotechnology. We report on experiments evaluating the stability of DNA origami upon irradiation with light at different wavelengths and buffer solutions. DNA is irradiated with nanosecond pulsed lasers and the damage is evaluated using UV–Vis spectroscopy and atomic force microscopy imaging. We show that the wavelength dependence of the damage follows the UV absorption spectrum of DNA. Electronic excitation of DNA is primarily responsible for DNA origami damage at present wavelengths. We also demonstrate UV–Vis absorption of tris reaction products, influencing the UV–Vis absorption evaluation in experiments studying DNA damage.

Metallic Nanowires Self-Assembled in Quasi-Circular Nanomolds Templated by DNA Origami.

The self-assembly of conducting nanostructures is currently being investigated intensively in order to evaluate the feasibility of creating novel nanoelectronic devices and circuits using such pathways. In particular, methods based on so-called DNA Origami nanostructures have shown great potential in the formation of metallic nanowires. The main challenge of this method is the reproducible generation of very well-connected metallic nanostructures, which may be used as interconnects in future devices. Here, we use a novel design of nanowires with a quasi-circular cross-section as opposed to rectangular or uncontrolled cross-sections in earlier studies. We find indications that the reliability of the fabrication scheme is enhanced and the overall resistance of the wires is comparable to metallic nanostructures generated by electrochemistry or top-down methods. In addition, we observe that some of the nanowires are annealed when passing a current through them, which leads to a clear enhancement for the conductance. We envision that these nanowires provide further steps towards the successful generation of nanoelectronics using self-assembly.

A Ratiometric Fluorescent Probe for pH Measurement over a Wide Range Composed of Three Types of Fluorophores Assembled on a DNA Scaffold

The desirable properties of the sophisticated fluorescent pH probe are ratiometric detection properties and a wide detection range. In this study, three types of fluorophores with different fluorescence properties were assembled on a DNA origami nanostructure. DNA nanostructure has the advantage of being a scaffold that can assemble different types of fluorophores with control over their number and position. The defined number of three different fluorophores, i.e., pH-sensitive fluorescein (CF) and Oregon Green (OG), and pH-insensitive tetramethylrhodamine (CR), assembled on the DNA scaffold provided a ratiometric fluorescent pH probe with a wide pH detection range that could cover the variation of intracellular pH.

Advanced applications of DNA nanostructures dominated by DNA origami in antitumor drug delivery.

DNA origami is a cutting-edge DNA self-assembly technique that neatly folds DNA strands and creates specific structures based on the complementary base pairing principle. These innovative DNA origami nanostructures provide numerous benefits, including lower biotoxicity, increased stability, and superior adaptability, making them an excellent choice for transporting anti-tumor agents. Furthermore, they can considerably reduce side effects and improve therapy success by offering precise, targeted, and multifunctional drug delivery system. This comprehensive review looks into the principles and design strategies of DNA origami, providing valuable insights into this technology's latest research achievements and development trends in the field of anti-tumor drug delivery. Additionally, we review the key function and major benefits of DNA origami in cancer treatment, some of these approaches also involve aspects related to DNA tetrahedra, aiming to provide novel ideas and effective solutions to address drug delivery challenges in cancer therapy.

Gradient-mixing LEGO robots for purifying DNA origami nanostructures of multiple components by rate-zonal centrifugation.

DNA origami purification is essential for many fields, including biophysics, molecular engineering, and therapeutics. The increasing interest in DNA origami has led to the development of rate-zonal centrifugation (RZC) as a scalable, high yield, and contamination-free method for purifying DNA origami nanostructures. RZC purification uses a linear density gradient of viscous media, such as glycerol or sucrose, to separate molecules according to their mass and shape. However, many methods for creating density gradients are time-consuming because they rely on slow passive diffusion. To expedite the preparation time, we used a LEGO gradient mixer to generate rotational motion and rapidly create a quasi-continuous density gradient with a minimal layering of the viscous media. Rotating two layers of differing concentrations at an angle decreases the time needed to form the density gradient from a few hours to minutes. In this study, the density gradients created by the LEGO gradient mixer were used to purify 3 DNA origami shapes that have different aspect ratios and numbers of components, with an aspect ratio ranging from 1:1 to 1:100 and the number of components up to 2. The gradient created by our LEGO gradient mixer is sufficient to purify folded DNA origami nanostructures from excess staple strands, regardless of their aspect ratios. Moreover, the gradient was able to separate DNA origami dimers from DNA origami monomers. In light of recent advances in large-scale DNA origami production, our method provides an alternative for purifying DNA origami nanostructures in large (gram) quantities in resource-limited settings.

DNA-origami-directed virus capsid polymorphism.

Viral capsids can adopt various geometries, most iconically characterized by icosahedral or helical symmetries. Importantly, precise control over the size and shape of virus capsids would have advantages in the development of new vaccines and delivery systems. However, current tools to direct the assembly process in a programmable manner are exceedingly elusive. Here we introduce a modular approach by demonstrating DNA-origami-directed polymorphism of single-protein subunit capsids. We achieve control over the capsid shape, size and topology by employing user-defined DNA origami nanostructures as binding and assembly platforms, which are efficiently encapsulated within the capsid. Furthermore, the obtained viral capsid coatings can shield the encapsulated DNA origami from degradation. Our approach is, moreover, not limited to a single type of capsomers and can also be applied to RNA-DNA origami structures to pave way for next-generation cargo protection and targeting strategies.

DNA Origami Tessellations.

Molecular tessellation research aims to elucidate the underlying principles that govern intricate patterns in nature and to leverage these principles to create precise and ordered structures across multiple scales, thereby facilitating the emergence of novel functionalities. DNA origami nanostructures are excellent building blocks for constructing tessellation patterns. However, the size and complexity of DNA origami tessellation systems are currently limited by several unexplored factors relevant to the accuracy of essential design parameters, the applicability of design strategies, and the compatibility between different tiles. Here, we present a general method for creating DNA origami tiles that grow into tessellation patterns with micrometer-scale order and nanometer-scale precision. Interhelical distance (D) was identified as a critical design parameter determining tile conformation and tessellation outcome. Finely tuned D facilitated the accurate geometric design of monomer tiles with minimized curvature and improved tessellation capability, enabling the formation of single-crystalline lattices ranging from tens to hundreds of square micrometers. The general applicability of the design method was demonstrated by 9 tile geometries, 15 unique tile designs, and 12 tessellation patterns covering Platonic, Laves, and Archimedean tilings. Particularly, we took two strategies to increase the complexity of DNA origami tessellation, including reducing the symmetry of monomer tiles and coassembling tiles of different geometries. Both yielded various tiling patterns that rivaled Platonic tilings in size and quality, indicating the robustness of the optimized tessellation system. This study will promote DNA-templated, programmable molecular and material patterning and open up new opportunities for applications in metamaterial engineering, nanoelectronics, and nanolithography.

The Creation of DNA Origami-Based Supramolecular Nanostructures for Cancer Therapy.

DNA origami technology, a unique type of DNA nanotechnology, has attracted much attention from researchers and is applied in various fields. Through exquisite design and precise self-assembly of four kinds of deoxyribonucleotides, DNA origami nanostructures are endowed with excellent programmability and addressability and show outstanding biocompatibility in bio-related applications, especially in cancer treatment. In this review, nanomaterials based on DNA origami for cancer therapy are concluded, whereby chemotherapy and photo-assisted therapy are the main focus. Furthermore, the working mechanisms of the functional materials attached to the rigid DNA structures to enable targeted delivery and circumvent drug resistance are also discussed. DNA origami nanostructures are valuable carriers for delivering multifunctional therapeutic agents and demonstrate great potential in cancer treatment both in vitro and in vivo. It is undoubted that DNA origami technology is a promising strategy for constructing versatile nanodevices in biological fields and will excel in human healthcare.

A Comprehensive Review of DNA Origami Stabilization Techniques

In recent years, DNA has emerged as a powerful tool in the field of nanotechnology. The DNA origami technique is largely responsible for this, revolutionizing nanofabrication due to its controllability, precision, and ability to leverage DNA’s unique properties. The technique consists of folding a long, single-stranded DNA (called a scaffold strand) by binding it with shorter staple strands to create almost any shape desired. With a desired structure in mind, researchers can design and assemble scaffold and staple strands using computer software like Cadnano or Tiamat. This is possible because of the Watson-Crick base pairing of DNA strands, which allows for programmable self-assembly of DNA nanostructures and therefore, the synthesis of arbitrary 2D and 3D shapes. Because DNA is a biomolecule,the nanostructures are also biocompatible and can be employed in biological applications including drug delivery. DNA origami nanostructures are not only limited to biological applications; they have also found uses in nanophotonics, plasmonics, and electronics. However, DNA origami still faces many challenges before it can be widely adopted. One such challenge is ensuring stability, and thus guaranteeing the performance of the DNA origami, in the presence of heat, nuclease in organic bodies, and chaotropic agents. This warrants the question: what methodologies can be employed to best stabilize DNA origami structures? This paper further focuses on two methods: covalently binding various molecules by cross-linking and non-binding encapsulation. Detailed analysis and comparison between various molecules used to bind and coat DNA nanostructures is used to evaluate performance and applicability of each method. In the end an oligolysines coating cross-linked with glutaraldehyde was found to have the strongest biological stability, thymine cross-linking had the strongest thermal stability, a silica coating had the best stability against the largest number of factors, and both graphene and Al3O2 coatings had the best mechanical stability.