DNA Origami Technology Research Articles

Development of an indirect ELISA to detect antigens using DNA origami nanoantenna labeled specific monoclonal antibodies

This research examines the development of an indirect ELISA to detect antigens using DNA origami nanoantenna-labeled specific monoclonal antibodies. Hepatocellular carcinoma (HCC) is one of the five most common cancers and the second leading cause of cancer-related death worldwide. More than 60% of cases are detected in the late stage, with the absence of specific symptoms in the early stage of the disease. Adding measurements of AFP (Alpha-Fetoprotein), TXN (Thioredoxin), and DCP (Des-γ-Carboxy Prothrombin) has the potential to improve the accuracy of diagnosing hepatocellular carcinoma. DNA origami technology is revolutionizing the forefront of cancer diagnostics, particularly for HCC. In this study, an indirect enzyme-linked immunosorbent assay (ELISA) method targeting the assessment of the effectiveness of specific monoclonal antibody-conjugated DNA origami particles was developed. The results of DNA origami folding into complex nanostructures and labeled biomarkers like AFP, DCP, and TXN have explored the high specificity and sensitivity for targeting HCC. We found the cut-off values for AFP, DCP, and TXN to be 0.132, 0.156, and 0.150, respectively. The coefficients of variation for samples remained consistently under 10%, indicating the method's high degree of stability and reproducibility. This innovation, combined with biomarkers, promised in early HCC detection, holds the potential to significantly enhance patient outcomes in the fight against liver cancer. The platform also has enormous potential for advancing personalized medicine.

Controllable self-cleaning FET self-assembled RNA-cleaving DNAzyme based DNA nanotree for culture-free Staphylococcus aureus detection

Staphylococcus aureus (SA) poses a serious risk to human and animal health, necessitating a low-cost and high-performance analytical platform for point-of-care diagnostics. Cellulose paper-based field-effect transistors (FETs) with RNA-cleaving DNAzymes (RCDs) can fulfill the low-cost requirements, however, its high hydrophilicity and lipophilicity hinder biochemical modification and result in low sensitivity, poor mechanical stability and poor fouling performance. Herein, we proposed a controllable self-cleaning FET to simplify biochemical modification and improve mechanical stability and antifouling performance. Then, we constructed an RCD-based DNA nanotree to significantly enhance the sensitivity for SA detection. For controllable self-cleaning FET, 1 H,1 H,2 H,2 H-perfluorodecyltrimethoxysilane based-polymeric nanoparticles were synthesized to decorate cellulose paper and whole carbon nanofilm wires. O2 plasma was applied to regulate to reduce fluorocarbon chain density, and then control the hydrophobic-oleophobic property in sensitive areas. Because negatively charged DNA affected the sensitivity of semiconducting FETs, three Y-shaped branches with low-cost were designed and applied to synthesize an RCD-based DNA-Nanotree based on similar DNA-origami technology, which further improved the sensitivity. The trunk of DNA-Nanotree was composed of RCD, and the canopy was self-assembled using multiple Y-shaped branches. The controllable self-cleaning FET biosensor was applied for SA detection without cultivation, which had a wide linear range from 1 to 105 CFU/mL and could detect a low value of 1 CFU/mL.

Layer‐Controllable “2.5D” DNA Origami Crystals Synthesized by a Hierarchical Assembly Strategy

AbstractThe finite periodic arrangement of functional nanomaterials on the two‐dimensional scale enables the integration and enhancement of individual properties, making them an important research topic in the field of tuneable nanodevices. Although layer‐controllable lattices such as graphene have been successfully synthesized, achieving similar control over colloidal nanoparticles remains a challenge. DNA origami technology has achieved remarkable breakthroughs in programmed nanoparticle assembly. Based on this technology, we proposed a hierarchical assembly strategy to construct a universal DNA origami platform with customized layer properties, which we called 2.5‐dimensional (2.5D) DNA origami crystals. Methodologically, this strategy divides the assembly procedure into two steps: 1) array synthesis, and 2) lattice synthesis, which means that the layer properties, including layer number, interlayer distance, and surface morphology, can be flexibly customized based on the independent designs in each step. In practice, these synthesized 2.5D crystals not only pioneer the expansion of the DNA origami crystal library to a wider range of dimensions, but also highlight the technological potential for templating 2.5D colloidal nanomaterial lattices.

Chiral plasmonic metasurface assembled by DNA origami

Chiral materials are essential to perceive photonic devices that control the helicity of light. However, the chirality of natural materials is rather weak, and relatively thick films are needed for noticeable effects. To overcome this limitation, artificial photonic materials were suggested to affect the chiral response in a much more substantial manner. Ideally, a single layer of such a material, a metasurface, should already be sufficient. While various structures fabricated with top-down nanofabrication technologies have already been reported, here we propose to utilize scaffolded DNA origami technology, a scalable bottom-up approach for metamolecule production, to fabricate a chiral metasurface. We introduce a chiral plasmonic metamolecule in the shape of a tripod and simulate its optical properties. By fixing the metamolecule to a rectangular planar origami, the tripods can be assembled into a 2D DNA origami crystal that forms a chiral metasurface. We simulate the optical properties but also fabricate selected devices to assess the experimental feasibility of the suggested approach critically.

Particle Deformability Enables Control of Interactions between Membrane-Anchored Nanoparticles.

Nanoparticles adsorbed on a membrane can induce deformations of the membrane that give rise to effective interactions between the particles. Previous studies have focused primarily on rigid nanoparticles with fixed shapes. However, DNA origami technology has enabled the creation of deformable nanostructures with controllable shapes and mechanical properties, presenting new opportunities to modulate interactions between particles adsorbed on deformable surfaces. Here we use coarse-grained molecular dynamics simulations to investigate deformable, hinge-like nanostructures anchored to lipid membranes via cholesterol anchors. We characterize deformations of the particles and membrane as a function of the hinge stiffness. Flexible particles adopt open configurations to conform to a flat membrane, whereas stiffer particles induce deformations of the membrane. We further show that particles spontaneously aggregate and that cooperative effects lead to changes in their shape when they are close together. Using umbrella sampling methods, we quantify the effective interaction between two particles and show that stiffer hinge-like particles experience stronger and longer-ranged attraction. Our results demonstrate that interactions between deformable, membrane-anchored nanoparticles can be controlled by modifying mechanical properties of the particles, suggesting new ways to modulate the self-assembly of particles on deformable surfaces.

DNA origami-mediated plasmonic dimer nanoantenna-based SERS biosensor for ultrasensitive determination of trace diethylstilbestrol

Diethylstilbestrol (DES) is a threatening factor to the human endocrine system. Here, we reported a DNA origami-assembled plasmonic dimer nanoantenna-based surface-enhanced Raman scattering (SERS) biosensor for measuring trace DES in foods. A critical factor influencing the SERS effect is interparticle gap modulation of SERS hotspots with nanometer-scale accuracy. DNA origami technology aims to generate naturally perfect structures with nano-scale precision. Exploiting the specificity of base-pairing and spatial addressability of DNA origami to form plasmonic dimer nanoantenna, the designed SERS biosensor generated electromagnetic-enhancement and uniform-enhancement hotspots to improve sensitivity and uniformity. Owing to their high target-binding affinity, aptamer-functionalized DNA origami biosensors transduced the target recognition into dynamic structural transformations of plasmonic nanoantennas, which were further converted to amplified Raman outputs. A broad linear range from 10−10 to 10−5 M was obtained with the detection limit of 0.217 nM. Our findings demonstrate the utility of aptamer-integrated DNA origami-based biosensors as a promising approach for trace analysis of environmental hazards.

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.

Highly selective preparation of N-terminus Horseradish peroxidase-DNA conjugate with fully retained enzymatic activity: HRP-DNA structure – activity relation

Within the last decade, the field of bio-nanoengineering has achieved significant advances allowing us to generate, e.g., nanoscaled molecular machineries with arbitrary shapes. To unleash the full potential of novel methods such as DNA origami technology, it is important to functionalise complex molecules and nanostructures precisely. Thus, considerable attention has been given to site-selective modifications of proteins allowing further incorporation of various functionalities. Here, we describe a method for the covalent attachment of oligonucleotides to the glycosylated horseradish peroxidase protein (HRP) with high N-terminus selectivity and significant yield while conserving the enzymatic activity. This two-step process includes a pH-controlled metal-free diazotransfer reaction using imidazole-1-sulfonyl azide hydrogen sulfate, which at pH 8.5 results in an N-terminal azide-functionalized protein, followed by the Cu-free click SPAAC reaction to dibenzocyclooctyne- (DBCO) modified oligonucleotides. The reaction conditions were optimised to achieve maximum yield and the best performance. The resulting protein–oligonucleotide conjugates (HRP-DNA) were characterised by electrophoresis and mass spectrometry (MS). Native-PAGE experiments demonstrated different migration patterns for HRP-DNA and the azido-modified protein allowing zymogram experiments. Structure–activity relationships of novel HRP-DNA conjugates were assessed using molecular dynamics simulations, characterising the molecular interactions that define the structural and dynamical properties of the obtained protein-oligonucleotide conjugates (POC).

DNA origami technology for biomedical applications: Challenges and opportunities

AbstractDNA origami, a promising branch of structural DNA technology, refers to the technique of folding a single‐stranded DNA scaffold into well‐defined nanostructures. In recent years, DNA origami nanostructures have shown considerable promise in a variety of biomedical applications, owing to their biodegradability, unique programmability, and addressability. Despite their popularity, the biomedical application of DNA origami techniques, which exploits their unique programmability and addressability, is rare in previous studies. Most recently, mounting evidence has demonstrated the robustness of DNA origami nanostructures in the spatial organization of functional components at the nanoscale in the biomedical field. These examples provide typical paradigms to fully realize the potential of DNA origami techniques by taking advantage of their unique programmability and addressability. This minireview summarizes the recent advancements of DNA origami techniques in biosensing, biocatalysis, and drug delivery, and the representative examples using DNA origami nanostructures for the spatial organization of functional molecules with nanometric precision are highlighted. We further discuss the possible limitations and challenges for in vivo applications, including stability issues and potential immunogenicity, and finally, propose some strategies to overcome these obstacles to fully realize the potential of DNA origami techniques in biomedical applications.

Multi-Reconfigurable DNA Origami Nanolattice Driven by the Combination of Orthogonal Signals.

The progress of the scaffolded DNA origami technology has enabled the construction of various dynamic nanodevices imitating the shapes and motions of mechanical elements. To further expand the achievable configurational changes, the incorporation of multiple movable joints into a single DNA origami structure and their precise control are desired. Here, we propose a multi-reconfigurable 3 × 3 lattice structure consisting of nine frames with rigid four-helix struts connected with flexible 10-nucleotide joints. The configuration of each frame is determined by the arbitrarily selected orthogonal pair of signal DNAs, resulting in the transformation of the lattice into various shapes. We also demonstrated sequential reconfiguration of the nanolattice and its assemblies from one into another via an isothermal strand displacement reaction at physiological temperatures. Our modular and scalable design approach could serve as a versatile platform for a variety of applications that require reversible and continuous shape control with nanoscale precision.

Quantum Yield of DNA Strand Breaks under Photoexcitation of a Molecular Ruby.

Photodynamic therapy (PDT) used for treating cancer relies on the generation of highly reactive oxygen species, for example, singlet oxygen 1 O2 , by light-induced excitation of a photosensitizer (PS) in the presence of molecular oxygen, inducing DNA damage in close proximity of the PS. Although many precious metal complexes have been explored as PS for PDT and received clinical approval, only recently, the potential of photoactive complexes of non-noble metals as PS has been discovered. Using the DNA origami technology that can absolutely quantify DNA strand break cross sections, we assessed the potential of the luminescent transition metal complex [Cr(ddpd)2 ]3+ (ddpd=N,N'-dimethyl-N,N'-dipyridine-2-ylpyridine-2,6-diamine) to damage DNA in an air-saturated aqueous environment upon UV/Vis illumination. The quantum yield for strand breakage, that is, the ratio of DNA strand breaks to the number of absorbed photons, was determined to 1-4 %, indicating efficient transformation of photons into DNA strand breaks by [Cr(ddpd)2 ]3+ .

Recent Advances of DNA Origami Technology and Its Application in Nanomaterial Preparation

In recent years, the unrivalled self‐assembly properties of DNA molecules have driven the rapid development of a new class of DNA self‐assembly technology—DNA origami technology. Over the past decade, this technology has enabled the construction of DNA nanostructures with different shapes, such as 2D and 3D structures. Meanwhile, the application of DNA origami are also developed rapidly. DNA origami structures are widely used in nanomaterial preparation and drug delivery due to their characteristics of accurate addressability, excellent programmability, and good biocompatibility, especially in the field of nanomaterial preparation. Such structures provide a new platform to construct nanomaterials with high precision. Herein, the development of DNA origami technology is introduced, and the research progress of 2D and 3D DNA origami in the past two decades is systematically summarized. Then, the application of DNA origami template in nanomaterial preparation is emphasized. Finally, the main challenges and opportunities for nanomaterials fabrication based on DNA origami technology are illustrated.

A “turn on” fluorescent sensor for Hg2+ detection based on rolling circle amplification with DNA origami-assisted signal amplification strategy

As well as polluting the environment, mercury also causes diseases and poses a threat to human health. Thus, it is crucial to implement the rapid and sensitive determination of Hg2+. On the basis of assembling RCA (rolling circle amplification) scaffolds and complementary short chains to form ladder-nanoarrays based on the characteristics of RCA signal amplification, we have constructed a “turn on” fluorescence sensor with high selectivity and sensitivity for Hg2+ detection in environmental water samples. Nanoarray sensors assembled from DNA are biocompatible, programmable and addressable for target delivery and targeted detection, by labeling the thrombin-binding aptamer (TBA) exposed on the ladder with the FAM at the 5′ end and DABCYL labeling the quencher at the 3′ end, the fluorescence of FAM was weakened by fluorescence resonance energy transfer (FRET) when the two groups were close together. Upon addition of Hg2+, TBA combines with Hg2+ to form a hairpin structure to induce quencher release and fluorescence recovery. Consequently, the ladder-sensor performance is highly sensitive to Hg2+ in the concentration range of 1–1000 nM, with a limit detection (LOD) is 1.78 nM under the optimized conditions. In addition, fluorescence sensors built by combining DNA origami technology with aptamers has good optical performance, which opens up a new field for the identification of other biological samples.

Multiplexed Detection of Molecular Interactions with DNA Origami Engineered Cells in 3D Collagen Matrices.

The interactions of cells with signaling molecules present in their local microenvironment maintain cell proliferation, differentiation, and spatial organization and mediate progression of diseases such as metabolic disorders and cancer. Real-time monitoring of the interactions between cells and their extracellular ligands in a three-dimensional (3D) microenvironment can inform detection and understanding of cell processes and the development of effective therapeutic agents. DNA origami technology allows for the design and fabrication of biocompatible and 3D functional nanodevices via molecular self-assembly for various applications including molecular sensing. Here, we report a robust method to monitor live cell interactions with molecules in their surrounding environment in a 3D tissue model using a microfluidic device. We used a DNA origami cell sensing platform (CSP) to detect two specific nucleic acid sequences on the membrane of B cells and dendritic cells. We further demonstrated real-time detection of biomolecules with the DNA sensing platform on the surface of dendritic cells in a 3D microfluidic tissue model. Our results establish the integration of live cells with membranes engineered with DNA nanodevices into microfluidic chips as a highly capable biosensor approach to investigate subcellular interactions in physiologically relevant 3D environments under controlled biomolecular transport.

A universal way to enrich the nanoparticle lattices with polychrome DNA origami "homologs".

DNA origami technology has rapidly developed into an ideal means to programmably crystallize nanoparticles. However, most existing DNA origami three-dimensional platforms normally used a single type of DNA origami unit, which greatly limits the types of nanoparticle superlattices that can be synthesized. Here, we report a universal strategy to vastly enrich the library of nanoparticle superlattices, based on multiple-unit (≥4 units) DNA origami platforms, which were constructed by programmably cocrystallizing three different DNA origami octahedral "homologs." Through selectively inserting nanoparticles into DNA origami monomers, numerous nanoparticle superlattices can be synthesized on the basis of the same platform. In this work, we obtained 85 types of DOF/AuNP (DNA origami frame/gold nanoparticle) superlattices using three different DNA origami platforms as examples. We believe that our strategy can provide possible access to fabricate virtually endless types of nanoparticle superlattices and promote the construction of functional materials with special properties.

DNA Origami in the Quest for Membrane Piercing

The tool kit for label-free single-molecule sensing, nucleic acid sequencing (DNA, RNA and protein) and other biotechnological applications has been significantly broadened due to the wide range of available nanopore-based technologies. Currently, various sources of nanopores, including biological, fabricated solid-state, hybrid and recently de novo chemically synthesized ion-like channels have put in use for rapid single-molecule sensing of biomolecules and other diagnostic applications. At length scales of hundreds of nanometers, DNA nanotechnology, particularly DNA origami-based devices, enables the assembly of complex and dynamic 3-dimensional nanostructures, including nanopores with precise control over the size/shape. DNA origami technology has enabled to construct nanopores by DNA alone or hybrid architects with solid-state nanopore devices or nanocapillaries. In this review, we briefly discuss the nanopore technique that uses DNA nanotechnology to construct such individual pores in lipid-based systems or coupled with other solid-state devices, nanocapillaries for enhanced biosensing function. We summarize various DNA-based design nanopores and explore the sensing properties of such DNA channels. Apart from DNA origami channels we also pointed the design principles of RNA nanopores for peptide sensing applications.

DNA-Assembled Chiral Satellite-Core Nanoparticle Superstructures: Two-State Chiral Interactions from Dynamic and Static Conformations.

A significant challenge exists in obtaining chiral nanostructures that are amenable to both solution-phase self-assembly and solid-phase preservation, which enable the observation of unveiled optical responses impacted by the dynamic or static conformation and the incident excitations. Here, to meet this demand, we employed DNA origami technology to create quasi-planar chiral satellite-core nanoparticle superstructures with an intermediate geometry between the monolayer and the double layer. We disentangled the complex chiral mechanisms, which include planar chirality, 3D chirality, and induced chirality transfer, through combined theoretical studies and thorough experimental measurements of both solution- and solid-phase samples. Two distinct states of optical responses were demonstrated by the dynamic and static conformations, involving a split or nonsplit circular dichroism (CD) line shape. More importantly, our study on chiral nanoparticle superstructures on a substrate featuring both a dominant 2D geometry and a defined 3D represents a great leap toward the realization of colloidal chiral metasurfaces.

DNA Kirigami Driven by Polymerase‐Triggered Strand Displacement

The precursors of functional biomolecules in living cells are synthesized in a bottom-up manner and subsequently activated by modification into a delicate structure with near-atomic precision. DNA origami technology provides a promising way to mimic the synthesis of precursors, although mimicking the modification process is a challenge. Herein, a DNA paper-cutting (DNA kirigami) method to trim origami into designer nanostructures is proposed, where the modification is implemented by a polymerase-triggered DNA strand displacement reaction. Six geometric shapes are created by cutting rectangular DNA origami. Gel electrophoresis and atomic force microscopy results demonstrate the feasibility and capability of the DNA paper-cutting method. The proposed DNA paper-cutting strategy can enrich the toolbox for dynamically transforming DNA origami and has potential applications in biomimetics. .

Self-assembled DNA origami-based duplexed aptasensors combined with centrifugal filters for efficient and rechargeable ATP detection

DNA origami technology has great potential for biosensor applications. Here, we described the construction of a self-assembled DNA origami biosensor for the precise localization of fluorescent aptamers. Due to the molecular weight difference between DNA origami and aptamer, centrifugal filters were used to quantitatively detect adenosine triphosphate (ATP). The ATP-specific aptamer labeled with fluorescence reporter 6-carboxyfluorescein FAM (FAM-aptamer) was selected as the recognition element and signal probe. ATP duplexed aptamers bound to triangular DNA origami by base-complementary pairing, resulting in high fluorescence signals on the origami arrays. The competitive binding of ATP toward the FAM-aptamer triggered the release of FAM-aptamer-ATP complexes from the surface of the origami array, resulting in weakened fluorescence signals. For ATP quantification, 100 kD centrifugal filters were employed, followed by measurement of the fluorescence signal trapped on the origami arrays of the filter device. The successful synthesis of origami-aptamer arrays was characterized by atomic force microscopy, laser confocal microscopy, and electrophoresis. Fluorescence measurements exhibited an excellent linear relationship with logarithms of ATP concentrations within 0.1–100 ng mL−1, with a detection limit of 0.29 ng mL−1. By replacing aptamers and complementary strands, we demonstrated the potential of this method for 17β-estradiol detection. Considering that the detection mechanism is based on the hybridization and displacement of DNA strands, the detection system had the potential for recharging. Our study provides new insights into applying DNA origami technology in small molecule detection.

Salting-Out of DNA Origami Nanostructures by Ammonium Sulfate.

DNA origami technology enables the folding of DNA strands into complex nanoscale shapes whose properties and interactions with molecular species often deviate significantly from that of genomic DNA. Here, we investigate the salting-out of different DNA origami shapes by the kosmotropic salt ammonium sulfate that is routinely employed in protein precipitation. We find that centrifugation in the presence of 3 M ammonium sulfate results in notable precipitation of DNA origami nanostructures but not of double-stranded genomic DNA. The precipitated DNA origami nanostructures can be resuspended in ammonium sulfate-free buffer without apparent formation of aggregates or loss of structural integrity. Even though quasi-1D six-helix bundle DNA origami are slightly less susceptible toward salting-out than more compact DNA origami triangles and 24-helix bundles, precipitation and recovery yields appear to be mostly independent of DNA origami shape and superstructure. Exploiting the specificity of ammonium sulfate salting-out for DNA origami nanostructures, we further apply this method to separate DNA origami triangles from genomic DNA fragments in a complex mixture. Our results thus demonstrate the possibility of concentrating and purifying DNA origami nanostructures by ammonium sulfate-induced salting-out.