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DNA origami nanostructures are promising carries for drug delivery applications. However, their limited stability under relevant conditions often presents a challenge. Herein, the structural stability of DNA origami nanostructures is investigated in a setting compatible with their application in photodynamic therapy (PDT). To this end, DNA origami triangles and six‐helix bundles (6HBs) are loaded with the clinically tested photosensitizer methylene blue, which upon irradiation with red light generates reactive oxygen species (ROS) that attack the DNA origami nanostructures. ROS‐induced structural damage is observed to depend on the ionic composition of the surrounding medium and becomes more severe at low ionic strength. Mg2+ ions can efficiently protect the DNA origami nanostructures from ROS‐induced damage and may even heal some of the damage obtained under Mg2+‐free conditions when added after irradiation. Finally, the employed DNA origami 6HBs are more resistant toward ROS‐induced structural damage than the triangles, which is attributed to their markedly different mechanical properties. These results thus provide some fundamental insights into the stabilizing role of DNA origami superstructure that may guide the selection or design of DNA origami nanocarriers with optimized stability for their application in PDT.

Plasmonic nanoparticles play a pivotal role in various research areas due to their exceptional optical and thermo‐optical properties, like high spectral tunability and efficient light‐to‐heat conversion. Gold, with its biocompatibility, low cytotoxicity, and tunable resonances , makes gold nanoparticles ideal for photothermal therapies. Geometries, including spheres, core–shells, rods, disks, stars, nanocages, and nanotoroids, are extensively studied, with the gold nanodoughnut emerging as one of the most promising ones due to its ability to produce high temperatures and rotational stability. Nevertheless, the fabrication of metallic toroidal shapes remains a challenge. Recent advances in DNA‐based nanotechnology, especially DNA‐origami techniques, provide feasible route for the fabrication of this geometry through metallization reactions or attachment of metal nanoparticles. However, particles manufactured using this method possess a DNA core that influences their thermoplasmonic performance. In this work, a theoretical investigation is conducted on the thermoplasmonic response of DNA‐origami‐based core/shell toroids (CSTs) for photothermal applications. Key parameters that optimize the CST thermoplasmonic response are identified, and compared with their solid counterparts and discrete metallic coatings. Additionally, the CSTs tolerance to random rotations is assessed, providing insights into their behavior in fluidic environments and implications for its practical consideration.

Deoxyribonucleic acid (DNA) origami enables the synthesis of bespoke nanoscale structures suitable for diverse applications. Effective design requires preventing uncontrolled aggregation, while still facilitating directed multi‐subunit assembly. Uncontrolled aggregation is often caused by base‐stacking interactions between arrays of blunt‐ended helices, a problem which is typically mitigated by incorporating disordered single‐stranded DNA (ssDNA) (like scaffold loops or poly‐thymine brushes) at the end of double‐stranded DNA (dsDNA) helices. Such disordered regions are ubiquitous in DNA origami structures yet their optimal design requirements in various chemical environments remains unclear. This study systematically investigates scaffold loops and poly‐nucleotide brushes for aggregation prevention and control of multi‐subunit assembly, examining length, sequence, and MgCl2 concentration dependencies. Additionally, a novel strategy is introduced using orthogonal double‐stranded DNA helices to create a steric shield against base stacking. The results reveal key considerations. Poly‐thymine brushes excel in achieving monodispersity across diverse conditions whereas scaffold loops aid directed multi‐subunit assembly. Orthogonal DNA helices remove the need for disordered regions altogether, preventing aggregation over a broad range of MgCl2 concentrations and facilitating controlled multi‐subunit assembly. This study expands the toolbox for DNA origami design and enables a more informed approach for achieving control of monodispersity and multi‐subunit assembly in DNA origami structures.

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.

Abstract Self‐assembly of biological molecules and structures is a fundamental property of life. Whereas most biological functions are based on self‐assembled proteins and protein complexes, the self‐assembly of lipids is important for the spatial organization of heterogeneous cellular reaction environments and to catalyze cooperative interactions on/with membranes. Lipid domains or “rafts”, which are known to selectively recruit proteins, play an important functional role in sorting and trafficking of membrane components between subcellular organelles. However, how the recruitment and interactions of proteins in turn contributes to the formation and turnover of these structures has not been systematically addressed, due to the large variety in membrane–protein features and their spatiotemporal dynamics. The small size and transient nature of lipid domains adds to the complexity in visualizing them in living cells. Here, DNA origami is presented as a programmable tool to mimic protein clustering and assembly on membranes and illustrate how nanometer sized lipid domains coalesce into visible domains upon origami self‐assembly in defined patterns. Hence, the local membrane composition can be efficiently regulated by the self‐assembly of peripheral membrane binders. This reinforces the hypothesis that lipid rafts in cells occur as a result of membrane–protein interactions and, in particular, protein self‐assembly.

Mosaic nanoparticle vaccines with heterotypic antigens exhibit broad-spectrum antiviral capabilities, but the impact of antigen proportions and distribution patterns on vaccine-induced immunity remains largely unexplored. Here, we present a DNA nanotechnology-based strategy for spatially assembling heterotypic antigens to guide the rational design of mosaic nanoparticle vaccines. By utilizing two aptamers with orthogonal selectivity for the original SARS-CoV-2 spike trimer and Omicron receptor-binding domain (RBD), along with a DNA soccer-ball framework, we precisely manipulate the spacing, stoichiometry, and overall distribution of heterotypic antigens to create mosaic nanoparticles with average, bipolar, and unipolar antigen distributions. Systematic in vitro and in vivo immunological investigations demonstrate that 30 heterotypic antigens in equivalent proportions, with an average distribution, lead to higher production of broad-spectrum neutralizing antibodies compared to the bipolar and unipolar distributions. Furthermore, the precise assembly utilizing our developed methodology reveals that a mere increment of five Omicron RBD antigens on a nanoparticle (from 15 to 20) not only diminishes neutralization against the Omicron variant but also triggers excessive inflammation. This work provides a unique perspective on the rational design of mosaic vaccines by highlighting the significance of the spatial placement and proportion of heterotypic antigens in their structure-activity mechanisms., (© 2024 Wiley-VCH GmbH.)

Despite their growing popularity, many fundamental properties and applications of MXene materials remain underexplored. Here, the nonradiative energy transfer properties of 2D titanium carbide MXene are investigated and their application in single-molecule biosensing is explored for the first time. DNA origami positioners are used for single dye placement immobilized by a specific chemistry based on glycine-MXene interactions, allowing precise control of their orientation on the surface. Each DNA origami structure carries a single dye molecule at predetermined heights. Single-molecule fluorescence confocal microscopy reveals that energy transfer of an organic emitter (ATTO 542) on transparent thin films made of spincast Ti 3 C 2 T x flakes follows a cubic distance dependence, where 50% of energy transfer efficiency is reached at 2.7 nm (d 0 ). MXenes are applied as short-distance spectroscopic nanorulers, determining z distances of dye-labeled supported lipid bilayers fused on MXene's hydrophilic surface. Hydration layer (2.1 nm) and lipid bilayer thickness (4.5 nm) values that agree with the literature are obtained. These results highlight titanium carbide MXenes as promising substrates for single-molecule biosensing of ultrathin assemblies, owing to their sensitivity near the interface, a distance regime that is typically inaccessible to other energy transfer tools., (© 2024 The Author(s). Advanced Materials published by Wiley‐VCH GmbH.)

Conventional cryptographic methods rely on increased computational complexity to counteract the threat posed by growing computing power for sustainable protection. DNA cryptography circumvents this threat by leveraging complex DNA recognition to maintain information security. Specifically, DNA origami has been repurposed for cryptography, using programmable folding of the long scaffold strand carrying additional tagged strands for information encryption. Herein, a subtraction-based cryptographic strategy is presented that uses structural defects on DNA origami to contain encrypted information. Designated staple strands are removed from the staple pool with "hook" strands to create active defect sites on DNA origami for information encryption. These defects can be filled by incubating the structures with the intact pool of biotinylated staple strands, resulting in biotin patterns that can be used for protein-binding steganography. The yields of individual protein pixels reached over 91%, and self-correction codes are implemented to aid the information recovery. Furthermore, the encrypted organization of defective DNA origami structures is investigated to explore the potential of this method for scalable information storage. This method uses DNA origami to encrypt information in hidden structural features, utilizing subtraction for robust cryptography while ensuring the safety and recovery of data., (© 2024 Wiley‐VCH GmbH.)

DNA nanostructures have been utilized to study biological mechanical processes and construct artificial nanosystems. Many application scenarios necessitate nanodevices able to robustly generate large single molecular forces. However, most existing dynamic DNA nanostructures are triggered by probabilistic hybridization reactions between spatially separated DNA strands, which only non-deterministically generate relatively small compression forces (≈0.4 piconewtons (pN)). Here, an intercalator-triggered dynamic DNA origami nanostructure is developed, where large amounts of local binding reactions between intercalators and the nanostructure collectively lead to the robust generation of relatively large compression forces (≈11.2 pN). Biomolecular loads with different stiffnesses, 3, 4, and 6-helix DNA bundles are efficiently bent by the compression forces. This work provides a robust and powerful force-generation tool for building highly chemo-mechanically coupled molecular machines in synthetic nanosystems., (© 2024 Wiley‐VCH GmbH.)

Interacting with living systems typically involves the ability to address lipid membranes of cellular systems. The first step of interaction of a nanorobot with a cell will thus be the detection of binding to a lipid membrane. Utilizing DNA origami, we engineered a biosensor with single-molecule Fluorescence Resonance Energy Transfer (smFRET) as transduction mechanism for precise lipid vesicle detection and cargo delivery. The system hinges on a hydrophobic ATTO647N modified single-stranded DNA (ssDNA) leash, protruding from a DNA origami nanostructure. In a vesicle-free environment, the ssDNA coils, ensuring high FRET efficiency. Upon vesicle binding to cholesterol anchors on the DNA origami, hydrophobic ATTO647N induces the ssDNA to stretch towards the lipid bilayer, reducing FRET efficiency. As the next step, the sensing strand serves as molecular cargo that can be transferred to the vesicle through a triggered strand displacement reaction. Depending on the number of cholesterols on the displacer strands, we either induce a diffusive release of the fluorescent load towards neighboring vesicles or a stoichiometric release of a single cargo-unit to the vesicle on the nanosensor. Ultimately, our multi-functional liposome interaction and detection platform opens up pathways for innovative biosensing applications and stoichiometric loading of vesicles with single-molecule control., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Biological nanopores crucially control the import and export of biomolecules across lipid membranes in cells. They have found widespread use in biophysics and biotechnology, where their typically narrow, fixed diameters enable selective transport of ions and small molecules, as well as DNA and peptides for sequencing applications. Yet, due to their small channel sizes, they preclude the passage of large macromolecules, e.g., therapeutics. Here, the unique combined properties of DNA origami nanotechnology, machine-inspired design, and synthetic biology are harnessed, to present a structurally reconfigurable DNA origami MechanoPore (MP) that features a lumen that is tuneable in size through molecular triggers. Controllable switching of MPs between 3 stable states is confirmed by 3D-DNA-PAINT super-resolution imaging and through dye-influx assays, after reconstitution of the large MPs in the membrane of liposomes via an inverted-emulsion cDICE technique. Confocal imaging of transmembrane transport shows size-selective behavior with adjustable thresholds. Importantly, the conformational changes are fully reversible, attesting to the robust mechanical switching that overcomes pressure from the surrounding lipid molecules. These MPs advance nanopore technology, offering functional nanostructures that can be tuned on-demand - thereby impacting fields as diverse as drug delivery, biomolecule sorting, and sensing, as well as bottom-up synthetic biology., (© 2024 The Author(s). Advanced Materials published by Wiley‐VCH GmbH.)

DNA origami is a flexible platform for the precise organization of nano-objects, enabling numerous applications from biomedicine to nano-photonics. Its huge potential stems from its high flexibility that allows customized structures to meet specific requirements. The ability to generate diverse final structures from a common base by folding significantly enhances design variety and is regularly occurring in liquid. This study describes a novel approach that combines top-down lithography with bottom-up DNA origami techniques to control folding of the DNA origami with the adsorption on pre-patterned surfaces. Using this approach, tunable plasmonic dimer nano-arrays are fabricated on a silicon surface. This involves employing electron beam lithography to create adsorption sites on the surface and utilizing self-organized adsorption of DNA origami functionalized with two gold nanoparticles (AuNPs). The desired folding of the DNA origami helices can be controlled by the size and shape of the adsorption sites. This approach can for example be used to tune the center-to-center distance of the AuNPs dimers on the origami template. To demonstrate this technique's efficiency, the Raman signal of dye molecules (carboxy tetramethylrhodamine, TAMRA) coated on the AuNPs surface are investigated. These findings highlight the potential of tunable DNA origami-based plasmonic nanostructures for many applications., (© 2024 The Authors. Small published by Wiley‐VCH GmbH.)

Abstract Organization of elongated particles into ordered phases on 2D surfaces and interfaces has been extensively studied during the last decades both theoretically and experimentally. For mutually repulsive particles on solid nondeformable substrates, the process is controlled only by the aspect ratio and the surface density of the adsorbed particles. The local elastic response of soft substrates to particle adhesion can drastically change the collective behavior of adsorbed rod‐like particles resulting in their self‐organization via substrate‐mediated interparticle attraction. Here, high‐speed atomic force microscopy is used to study the organization of DNA origami particles on locally responsive supported lipid bilayers (SLBs) in comparison with that on nondeformable solid mica surfaces. At high surface coverage, the aspect ratio‐dependent anisotropic phases expected for densely packed particles are observed. At intermediate and low surface densities, however, a drastically different phenomenology is observed: surprisingly strong surface‐mediated interparticle attraction of DNA origami particles is found on SLBs resulting in their self‐organization compared to their purely repulsive interaction on a mica surface. The formation of organized aggregates of elongated DNA origami particles on SLBs is explained by exceptionally strong nanoparticle adhesion to the membrane that responds with a local deformation in spite of the presence of the solid support.

Structural DNA nanotechnology enables custom fabrication of nanoscale devices and promises diverse biological applications. However, the effects of design on DNA nanostructure (DN)-cell interactions in vitro and in vivo are not yet well-characterized. origamiFISH is a recently developed technique for imaging DNs in cells and tissues. Compared to the use of fluorescent tags, origamiFISH offers label-free and structure-agnostic detection of DNs with significantly improved sensitivity. Here, the origamiFISH technique is extended to quantify DNs in single-cell suspensions, including in nonadherent cells such as subsets of immune cells, via readout by flow cytometry. This method, referred to as origamiFISH-Flow, is high-throughput (e.g., 10 000 cells per second) and compatible with immunostaining for concurrent cell-type and cell-state characterization. It is shown that origamiFISH-Flow provides 20-fold higher signal-to-noise ratio for DN detection compared to dye labeling approaches, leading to the capture of >25-fold more DN + cells under single-picomolar DN uptake concentrations. Additionally, the use of origamiFISH-Flow is validated to profile the uptake of various DN shapes across multiple cell lines and splenocytes, as well as to quantify in vivo DN accumulation in lymphoid organs. Together, origamiFISH-Flow offers a new tool to interrogate DN interactions with cells and tissues, while providing insights for tailoring their designs in bio-applications., (© 2024 The Authors. Small published by Wiley‐VCH GmbH.)

Chemistry has the power to endow supramolecular nanostructures with new biomedically relevant functions. Here it is reported that DNA nanostructures modified with cholesterol tags disrupt bacterial membranes to cause microbial cell death. The lipidated DNA nanostructures bind more readily to cholesterol-free bacterial membranes than to cholesterol-rich, eukaryotic membranes. These highly negatively charged, lipidated DNA nanostructures cause bacterial cell death by rupturing membranes. Strikingly, killing is mediated by clusters of barrel-shaped nanostructures that adhere to the membrane without the involvement of expected bilayer-puncturing barrels. These DNA nanomaterials may inspire the development of polymeric or small-molecule antibacterial agents that mimic the principles of selective binding and rupturing to help combat antimicrobial resistance., (© 2024 The Authors. Small published by Wiley‐VCH GmbH.)

Plasmonic nanomaterials bearing targeting ligands are of great interest for surface-enhanced Raman scattering (SERS)-based bioimaging applications. However, the practical utility of SERS-based imaging strategies has been hindered by the lack of a straightforward method to synthesize highly sensitive SERS-active nanostructures with high yield and efficiency. In this work, leveraging DNA origami principles, we report the first-in-class design of a SERS-based plasmonically coupled nanoprobe for targeted cancer imaging (SPECTRA). The nanoprobe harnesses a cancer cell targeting DNA aptamer sequence and vibrational tag with stretching frequency in the cell-silent Raman window. Through the integration of aptamer sequence specific for DU145 cells, we show the unique capabilities of SPECTRA for targeted imaging of DU145 cells. Our results demonstrate that the scalability, cost-effectiveness, and reproducibility of this method of fabrication of SERS nanoprobes can serve as a versatile platform for creating nanoprobes with broad applications in the fields of cancer biology and biomedical imaging., Competing Interests: Conflict of interest The authors declare no conflict of interest.

DNA nanostructures exhibit versatile geometries and possess sophisticated capabilities not found in other nanomaterials. They serve as customizable nanoplatforms for orchestrating the spatial arrangement of molecular components, such as biomolecules, antibodies, or synthetic nanomaterials. This is achieved by incorporating oligonucleotides into the design of the nanostructure. In the realm of drug delivery to cancer cells, there is a growing interest in active targeting assays to enhance efficacy and selectivity. The active targeting approach involves a "key-lock" mechanism where the carrier, through its ligand, recognizes specific receptors on tumor cells, facilitating the release of drugs. Various DNA nanostructures, including DNA origami, Tetrahedral, nanoflower, cruciform, nanostar, nanocentipede, and nanococklebur, can traverse the lipid layer of the cell membrane, allowing cargo delivery to the nucleus. Aptamers, easily formed in vitro, are recognized for their targeted delivery capabilities due to their high selectivity for specific targets and low immunogenicity. This review provides a comprehensive overview of recent advancements in the formation and modification of aptamer-modified DNA nanostructures within drug delivery systems., (© 2024 The Authors. Advanced Science published by Wiley‐VCH GmbH.)

The 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., (© 2024 Wiley-VCH GmbH.)

DNA origami synthesis is a well-established technique with wide-ranging applications. In most cases, the synthesized origami must be purified to remove excess materials such as DNA oligos and other functional molecules. While several purification techniques are routinely used, all have limitations, and cannot be integrated with robotic systems. Here the use of solid-phase reversible immobilization (SPRI) beads as a scalable, high-throughput, and automatable method to purify DNA origami is demonstrated. Not only can this method remove unreacted oligos and biomolecules with yields comparable to existing methods while maintaining the high structural integrity of the origami, but it can also be integrated into an automated workflow to purify simultaneously large numbers and quantities of samples. It is envisioned that the SPRI beads purification method will improve the scalability of DNA nanostructures synthesis both for research and commercial applications., (© 2023 The Authors. Small published by Wiley‐VCH GmbH.)

DNA molecules that store genetic information in living creatures can be repurposed as building blocks to construct artificial architectures, ranging from the nanoscale to the microscale. The precise fabrication of self-assembled DNA nanomaterials and their various applications have greatly impacted nanoscience and nanotechnology. More specifically, the DNA origami technique has realized the assembly of various nanostructures featuring rationally predesigned geometries, precise addressability, and versatile programmability, as well as remarkable biocompatibility. These features have elevated DNA origami from academic interest to an emerging class of drug delivery platform for a wide range of diseases. In this minireview, the latest advances in the burgeoning field of DNA-origami-based innovative platforms for regulating biological functions and delivering versatile drugs are presented. Challenges regarding the novel drug vehicle's safety, stability, targeting strategy, and future clinical translation are also discussed., (© 2023 Wiley‐VCH GmbH.)

DNA origami nanodevices achieve programmable structure and tunable mechanical and dynamic properties by leveraging the sequence-specific interactions of nucleic acids. Previous advances have also established DNA origami as a useful building block to make well-defined micron-scale structures through hierarchical self-assembly, but these efforts have largely leveraged the structural features of DNA origami. The tunable dynamic and mechanical properties also provide an opportunity to make assemblies with adaptive structures and properties. Here the integration of DNA origami hinge nanodevices and coiled-coil peptides are reported into hybrid reconfigurable assemblies. With the same dynamic device and peptide interaction, it is made multiple higher-order assemblies (i.e., polymorphic assembly) by organizing clusters of peptides into patches or arranging single peptides into patterns on the surfaces of DNA origami to control the relative orientation of devices. The coiled-coil interactions are used to construct circular and linear assemblies whose structure and mechanical properties can be modulated with DNA-based reconfiguration. Reconfiguration of linear assemblies leads to micron scale motions and ≈2.5-10-fold increase in bending stiffness. The results provide a foundation for stimulus-responsive hybrid assemblies that can adapt their structure and properties in response to nucleic acid, peptide, protein, or other triggers., (© 2024 The Authors. Advanced Science published by Wiley‐VCH GmbH.)

Single-stranded DNA oligonucleotides wrapping on the surface of single-walled carbon nanotubes (SWCNTs), described as DNA corona, are often used as a dispersing agent for SWCNTs. The uneven distribution of DNA corona along SWCNTs is related to the photoelectric properties and the surface activity of SWCNTs. An ionic strength-mediated "DNA corona defects" (DCDs) strategy is proposed to acquire an exposed surface of SWCNTs (accessible surface) as large as possible while maintaining good dispersibility via modulating the conformation of DNA corona. By adjusting the solution ionic strength, the DNA corona phase transitioned from an even-distributed and loose conformation to a locally compact conformation. The resulting enlarged exposed surface of SWCNTs is called DCDs, which provide active sites for molecular adsorption. This strategy is applied for the arrangement of SWCNTs on DNA origami. SWCNTs with ≈11 nm DCD, providing enough space for the adsorption of "capture ssDNA" (≈7 nm width required for 24-nt) extended from DNA origami structures are fabricated. The DCD strategy has potential applications in SWCNT-based optoelectronic devices., (© 2024 The Authors. Advanced Science published by Wiley‐VCH GmbH.)

DNA nanostructures have considerable biomedical potential as intracellular delivery vehicles as they are highly homogeneous and can be functionalized with high spatial resolution. However, challenges like instability under physiological conditions, limited cellular uptake, and lysosomal degradation limit their use. This paper presents a bio-reducible, cationic polymer poly(cystaminebisacrylamide-1,6-diaminohexane) (PCD) as a reversible DNA origami protector. PCD displays a stronger DNA affinity than other cationic polymers. DNA nanostructures with PCD protection are shielded from low salt conditions and DNase I degradation and show a 40-fold increase in cell-association when linked to targeting antibodies. Confocal microscopy reveals a potential secondary cell uptake mechanism, directly delivering the nanostructures to the cytoplasm. Additionally, PCD can be removed by cleaving its backbone disulfides using the intracellular reductant, glutathione. Finally, the application of these constructs is demonstrated for targeted delivery of a cytotoxic agent to cancer cells, which efficiently decreases their viability. The PCD protective agent that is reported here is a simple and efficient method for the stabilization of DNA origami structures. With the ability to deprotect the DNA nanostructures upon entry of the intracellular space, the possibility for the use of DNA origami in pharmaceutical applications is enhanced., (© 2023 The Authors. Small published by Wiley-VCH GmbH.)

Hierarchical DNA nanostructures offer programmable functions at scale, but making these structures dynamic, while keeping individual components intact, is challenging. Here we show that the DNA A-motif-protonated, self-complementary poly(adenine) sequences-can propagate DNA origami into one-dimensional, micron-length fibrils. When coupled to a small molecule pH regulator, visible light can activate the hierarchical assembly of our DNA origami into dissipative fibrils. This system is recyclable and does not require DNA modification. By employing a modular and waste-free strategy to assemble and disassemble hierarchical structures built from DNA origami, we offer a facile and accessible route to developing well-defined, dynamic, and large DNA assemblies with temporal control. As a general tool, we envision that coupling the A-motif to cycles of dissipative protonation will allow the transient construction of diverse DNA nanostructures, finding broad applications in dynamic and non-equilibrium nanotechnology., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

DNA nanostructures have played an important role in the development of novel drug delivery systems. Herein, we report a DNA origami-based CRISPR/Cas9 gene editing system for efficient gene therapy in vivo. In our design, a PAM-rich region precisely organized on the surface of DNA origami can easily recruit and load sgRNA/Cas9 complex by PAM-guided assembly and pre-designed DNA/RNA hybridization. After loading the sgRNA/Cas9 complex, the DNA origami can be further rolled up by the locking strands with a disulfide bond. With the incorporation of DNA aptamer and influenza hemagglutinin (HA) peptide, the cargo-loaded DNA origami can realize the targeted delivery and effective endosomal escape. After reduction by GSH, the opened DNA origami can release the sgRNA/Cas9 complex by RNase H cleavage to achieve a pronounced gene editing of a tumor-associated gene for gene therapy in vivo. This rationally developed DNA origami-based gene editing system presents a new avenue for the development of gene therapy., (© 2023 Wiley-VCH GmbH.)

The cellular compartment plays an essential role in organizing the complex and diverse biochemical reactions within the cell. By mimicking the function of such cellular compartments, the challenge of constructing artificial compartments has been taken up to develop new biochemical tools for efficient material production and diagnostics. The important features required for the artificial compartment are that it isolates the interior from the external environment and is further functionalized to control the transport of target chemicals to regulate the interior concentration of both substrate and reaction products. In this study, an artificial compartment with size-selective molecular transport function was constructed by using a DNA origami-guided liposome prepared by modifying the method reported by Perrault et al. This completely isolates the liposome interior, including the DNA origami skeleton, from the external environment and allows the assembly of a defined number of molecules of interest inside and/or outside the compartment. By incorporating a bacterial membrane protein, OmpF, into the liposome, the resulting artificial compartment was shown to transport only the molecule of interest with a molecular weight below 600 Da from the external environment into the interior of the compartment., (© 2023 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

Bacteria infection is a significant obstacle in the clinical treatment of exposed wounds facing widespread pathogens. Herein, we report a DNA origami-based bactericide for efficient anti-infection therapy of infected wounds in vivo. In our design, abundant DNAzymes (G4/hemin) can be precisely organized on the DNA origami for controllable generation of reactive oxygen species (ROS) to break bacterial membranes. After the destruction of the membrane, broad-spectrum antibiotic levofloxacin (LEV, loaded in the DNA origami through interaction with DNA duplex) can be easily delivered into the bacteria for successful sterilization. With the incorporation of DNA aptamer targeting bacterial peptidoglycan, the DNA origami-based bactericide can achieve targeted and combined antibacterial therapy for efficiently promoting the healing of infected wounds. This tailored DNA origami-based nanoplatform provides a new strategy for the treatment of infectious diseases in vivo., (© 2023 Wiley-VCH GmbH.)

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., (© 2023 Wiley-VCH GmbH.)

The world of 2D materials is steadily growing, with numerous researchers attempting to discover, elucidate, and exploit their properties. Approaches relying on the detection of single fluorescent molecules offer a set of advantages, for instance, high sensitivity and specificity, that allow the drawing of conclusions with unprecedented precision. Herein, it is argued how the study of 2D materials benefits from fluorescence-based single-molecule modalities, and vice versa. A special focus is placed on DNA, serving as a versatile adaptor when anchoring single dye molecules to 2D materials. The existing literature on the fruitful combination of the two fields is reviewed, and an outlook on the additional synergies that can be created between them provided., (© 2023 The Authors. Advanced Materials published by Wiley-VCH GmbH.)

This study establishes and validates a series of three dimentional (3D) DNA origami frameworks (DOFs) carrying imaging probes to evaluate their pharmacokinetics and real-time bio-distribution in mice. Three typical DOFs with distinguished structural properties are subjected to mice intravenous injection to systematically investigate their in vivo behaviors. Tracing the radioisotope zirconium-89 ( 89 Zr) trapped at the inner space of the frameworks, positron emission tomography (PET) imaging is employed to record the real-time bio-distribution of the structures and acquire their pharmacokinetic parameters in the major metabolic organs. The 3D DOFs show different behavior compared to previous structures, with lower kidney accumulation and higher liver retention. Modifications to the structures, such as exposed ssDNA or polyethylene glycol (PEG) moieties, impact their behavior, but are structure-dependent. The 43 nm icosahedra framework among the DOFs perform the best in liver targeting, with the ssDNA extensions enhancing this tendency. The modification of triantennary N-acetylgalactosamine (GalNAc), further improves its uptake in liver cells, especially in hepatocytes over other cell types, discovered by flow cytometry analysis., (© 2023 Wiley-VCH GmbH.)

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., (© 2023 Wiley-VCH GmbH.)

While vaccines have been highly successful in protecting against various infections, there are still many high-priority pathogens for which there are no clinically approved formulations. To overcome this challenge, researchers have explored the use of nanoparticulate strategies for more effective antigen delivery to the immune system. Along these lines, nanotoxoids are a promising biomimetic platform that leverages cell membrane coating technology to safely deliver otherwise toxic bacterial antigens in their native form for antivirulence vaccination. Here, in order to further boost their immunogenicity, nanotoxoids formulated against staphylococcal α-hemolysin are embedded into a DNA-based hydrogel with immunostimulatory CpG motifs. The resulting nanoparticle-hydrogel composite is injectable and improves the in vivo delivery of vaccine antigens while simultaneously stimulating nearby immune cells. This leads to elevated antibody production and stronger antigen-specific cellular immune responses. In murine models of pneumonia and skin infection caused by methicillin-resistant Staphylococcus aureus, mice vaccinated with the hybrid vaccine formulation are well-protected. This work highlights the benefits of combining nanoparticulate antigen delivery systems with immunostimulatory hydrogels into a single platform, and the approach can be readily generalized to a wide range of infectious diseases., (© 2023 Wiley-VCH GmbH.)

Exploration of clinically acceptable blood glucose monitors has been engaging in the past decades, yet the ability to quantitatively detect blood glucose in a painless, accurate, and highly sensitive manner remains limited. Herein, a fluorescence-amplified origami microneedle (FAOM) device is described that integrates tubular DNA-origami nanostructures and glucose oxidase molecules into its inner network to quantitatively monitor blood glucose. The skin-attached FAOM device can collect glucose molecules in situ and transfer the input into a proton signal after the oxidase's catalysis. The proton-driven mechanical reconfiguration of DNA-origami tubes separates fluorescent molecules and their quenchers, eventually amplifying the glucose-correlated fluorescence signal. The function equation established on clinical examinees suggests that FAOM can report blood glucose in a highly sensitive and quantitative manner. In clinical blind tests, the FAOM achieves well-matched accuracy (98.70 ± 4.77%) compared with a commercial blood biochemical analyzer, fully meeting the requirements of accurate blood glucose monitoring. The FAOM device can be inserted into skin tissue in a trivially painful manner and with minimal leakage of DNA origami, substantially improving the tolerance and compliance of the blood glucose test., (© 2023 Wiley-VCH GmbH.)

DNA self-assembly provides a "bottom-up" route to fabricating complex shapes on the nanometer scale. However, each structure needs to be designed separately and carried out by professionally trained technicians, which seriously restricts its development and application. Herein, a point-and-shoot strategy based on enzyme-assisted DNA "paper-cutting" to construct planar DNA nanostructures using the same DNA origami as the template is reported. Precisely modeling the shapes with high precision in the strategy based on each staple strand of the desired shape structure hybridizes with its nearest neighbor fragments from the long scaffold strand. As a result, some planar DNA nanostructures by one-pot annealing the long scaffold strand and selected staple strands is constructed. The point-and-shoot strategy of avoiding DNA origami staple strands' re-designing based on different shapes breaks through the shape complexity limitation of the planar DNA nanostructures and enhances the simplicity of design and operation. Overall, the strategy's simple operability and great generality enable it to act as a candidate tool for manufacturing DNA nanostructures., (© 2023 Wiley-VCH GmbH.)

Through inducing death receptor (DR) clustering to activate downstream signaling, tumor necrosis factor related apoptosis inducing ligand (TRAIL) trimers trigger apoptosis of tumor cells. However, the poor agonistic activity of current TRAIL-based therapeutics limits their antitumor efficiency. The nanoscale spatial organization of TRAIL trimers at different interligand distances is still challenging, which is essential for the understanding of interaction pattern between TRAIL and DR. In this study, a flat rectangular DNA origami is employed as display scaffold, and an "engraving-printing" strategy is developed to rapidly decorate three TRAIL monomers onto its surface to form DNA-TRAIL3 trimer (DNA origami with surface decoration of three TRAIL monomers). With the spatial addressability of DNA origami, the interligand distances are precisely controlled from 15 to 60 nm. Through comparing the receptor affinity, agonistic activity and cytotoxicity of these DNA-TRAIL3 trimers, it is found that ≈40 nm is the critical interligand distance of DNA-TRAIL3 trimers to induce death receptor clustering and the resulting apoptosis.Finally, a hypothetical "active unit" model is proposed for the DR5 clustering induced by DNA-TRAIL3 trimers., (© 2023 Wiley-VCH GmbH.)

DNA nanotechnology allows for the fabrication of nanometer-sized objects with high precision and selective addressability as a result of the programmable hybridization of complementary DNA strands. Such structures can template the formation of other materials, including metals and complex silica nanostructures, where the silica shell simultaneously acts to protect the DNA from external detrimental factors. However, the formation of silica nanostructures with site-specific addressability has thus far not been explored. Here, it is shown that silica nanostructures templated by DNA origami remain addressable for post silicification modification with guest molecules even if the silica shell measures several nm in thickness. The conjugation of fluorescently labeled oligonucleotides is used to different silicified DNA origami structures carrying a complementary ssDNA handle as well as DNA-PAINT super-resolution imaging to show that ssDNA handles remain unsilicified and thus ensure retained addressability. It is also demonstrated that not only handles, but also ssDNA scaffold segments within a DNA origami nanostructure remain accessible, allowing for the formation of dynamic silica nanostructures. Finally, the power of this approach is demonstrated by forming 3D DNA origami crystals from silicified monomers. These results thus present a fully site-specifically addressable silica nanostructure with complete control over size and shape., (© 2023 The Authors. Advanced Materials published by Wiley-VCH GmbH.)

DNA origami molds allow a shape-controlled growth of metallic nanoparticles. So far, this approach is limited to gold and silver. Here, the fabrication of linear palladium nanostructures with controlled lengths and patterns is demonstrated. To obtain nucleation centers for a seeded growth, a synthesis procedure of palladium nanoparticles (PdNPs) using Bis(p-sulfonatophenyl)phenylphosphine (BSPP) both as reductant and stabilizer is developed to establish an efficient functionalization protocol of the particles with single-stranded DNA. Attaching the functionalized particles to complementary DNA strands inside DNA mold cavities supports subsequently a highly specific seeded palladium deposition. This provides rod-like PdNPs with diameters of 20-35 nm of grainy morphology. Using an annealing procedure and a post-reduction step with hydrogen, homogeneous palladium nanostructures can be obtained. With the adaptation of the procedure to palladium the capabilities of the mold-based tool-box are expanded. In the future, this may allow a facile adaptation of the mold approach to less noble metals including magnetic materials such as Ni and Co., (© 2023 The Authors. Small published by Wiley-VCH GmbH.)

DNA nanotechnology relies on the structural and functional information encoded in nucleic acids. Specifically, the sequence-guided reconfiguration of nucleic acids by auxiliary triggers provides a means to develop DNA switches, machines and stimuli-responsive materials. The present Review addresses recent advances in the construction and applications of dynamic reconfigurable DNA nanostructures, networks and materials. Dynamic transformations proceeding within engineered origami frames or between origami tiles, and the triggered dynamic reconfiguration of scaled supramolecular origami structures are addressed. The use of origami frameworks to assemble dynamic chiroplasmonic optical devices and to operate switchable chemical processes are discussed. Also, the dynamic operation of DNA networks is addressed, and the design of "smart" stimuli-responsive all-DNA materials and their applications are introduced. Future perspectives and applications of dynamic reconfigurable DNA nanostructures are presented., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Photodynamic therapy (PDT) used for treating cancer relies on the generation of highly reactive oxygen species, for example, singlet oxygen 1 O 2 , 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+ ., (© 2023 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

The elegant geometry of viruses has inspired bio-engineers to synthetically explore the self-assembly of polyhedral capsids employed to protect new cargo or change an enzymatic microenvironment. Recently, Yang and co-workers used DNA nanotechnology to revisit the icosahedral capsid structure of the phiX174 bacteriophage and reloaded the original viral genome as cargo into their fully synthetic architecture. Surprisingly, when using a favorable combination of structural rigidity and dynamic multivalent cargo entrapment, the synthetic particles were able to infect non-competent bacterial cells and produce the original phiX174 bacteriophage. This work presents an exciting new direction of DNA nanotech for bio-engineering applications which involve bacterial interactions., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Compartmentalization is the strategy evolved by nature to control reactions in space and time. The ability to emulate this strategy through synthetic compartmentalization systems has rapidly evolved in the past years, accompanied by an increasing understanding of the effects of spatial confinement on the thermodynamic and kinetic properties of the guest molecules. DNA nanotechnology has played a pivotal role in this scientific endeavor and is still one of the most promising approaches for the construction of nanocompartments with programmable structural features and nanometer-scaled addressability. In this review, the design approaches, bioapplications, and theoretical frameworks of self-assembled DNA nanocompartments are surveyed. From DNA polyhedral cages to virus-like capsules, the construction principles of such intriguing architectures are illustrated. Various applications of DNA nanocompartments, including their use for programmable enzyme scaffolding, single-molecule studies, biosensing, and as artificial nanofactories, ending with an ample description of DNA nanocages for biomedical purposes, are then reported. Finally, the theoretical hypotheses that make DNA nanocompartments, and nanosystems in general, a topic of great interest in modern science, are described and the progresses that have been done until now in the comprehension of the peculiar phenomena that occur within nanosized environments are summarized., (© 2022 The Authors. Small published by Wiley-VCH GmbH.)

Super-resolution microscopy has revolutionized how researchers characterize samples in the life sciences in the last decades. Amongst methods employing single-molecule localization microscopy, DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) is a relatively easy-to-implement method that uses the programmable and repetitive binding of dye-labeled DNA imager strands to their respective docking strands. Recently developed Peptide-PAINT replaces the interaction of oligonucleotides by short coiled-coil peptide sequences leading to an improved labeling scheme by reducing linkage errors to target proteins. However, only one coiled-coil pair is currently available for Peptide-PAINT, preventing multiplexed imaging. In this study, the initial Peptide-PAINT E/K coil is improved by modifying its length for optimized binding kinetics leading to improved localization precisions. Additionally, an orthogonal P3/P4 coil pair is introduced, enabling 2-plex Peptide-PAINT imaging and benchmarking its performance and orthogonality using single-molecule and DNA origami assays. Finally, the P3/P4 peptide pair is used to image the human epidermal growth factor receptors 2 (ErbB2/Her2) in 2D and 3D at the single receptor level using genetically encoded peptide tags., (© 2023 The Authors. Small published by Wiley-VCH GmbH.)

Control over multiple optical elements that can be dynamically rearranged to yield substantial three-dimensional structural transformations is of great importance to realize reconfigurable plasmonic nanoarchitectures with sensitive and distinct optical feedback. In this work, we demonstrate a transformable plasmonic helix system, in which multiple gold nanoparticles (AuNPs) can be directly transported by DNA swingarms to target positions without undergoing consecutive stepwise movements. The swingarms allow for programmable AuNP translocations in large leaps within plasmonic nanoarchitectures, giving rise to tailored circular dichroism spectra. Our work provides an instructive bottom-up solution to building complex dynamic plasmonic systems, which can exhibit prominent optical responses through cooperative rearrangements of the constituent optical elements with high fidelity and programmability., (© 2022 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Viral genomes can be compressed into a near-spherical nanochamber to form infectious particles. In order to mimic the virus morphology and packaging behavior, we invented a programmable icosahedral DNA nanoframe with enhanced rigidity and encapsulated the phiX174 bacteriophage genome. The packaging efficiency could be modulated through specific anchoring strands adjustment, and the trapped phage genome remained accessible for enzymatic operations. Moreover, the packed complex could infect Escherichia coli (E. coli) cells through bacterial uptake to produce plaques. This rigid icosahedral DNA architecture demonstrated a versatile platform to develop virus mimetic particles for convenient functional nucleic acid entrapment, manipulation and delivery., (© 2022 Wiley-VCH GmbH.)

Introduction of the solid phase method to synthesize biopolymers has revolutionized the field of biological research by enabling efficient production of peptides and oligonucleotides. One of the advantages of this method is the ease of removal of excess production materials from the desired product, as it is immobilized on solid substrate. The DNA origami method utilizes the nature of nucleotide base-pairing to construct well-defined objects at the nanoscale, and has become a potent tool for manipulating matter in the fields of chemistry, physics, and biology. Here, the development of an approach to synthesize DNA nanostructures directly on magnetic beads, where the reaction is performed in heavy liquid to maintain the beads in suspension is reported. It is demonstrated that the method can achieve high folding yields of up to 90% for various DNA shapes, comparable to standard folding. At the same time, this establishes an easy, fast, and efficient way to further functionalize the DNA origami in one-pot, as well as providing a built-in purification method for easy removal of excess by-products such as non-integrated DNA strands and residual functionalization molecules., (© 2022 The Authors. Small published by Wiley-VCH GmbH.)

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., (© 2022 Wiley-VCH GmbH.)

DNA nanotechnology has provided credible approaches for assembly of three-dimensional (3D) lattices with complex patterns. However, the symmetries are strictly dependent on their initial configurations and difficult to alter via non-thermal treatments. While switchable nucleic acid structures have been employed to construct deformable DNA motifs, it remains challenging to arrange them anisotropically in 3D lattices to trigger directed collective shape transition and dynamic symmetry conversion. In this work, we used octahedral DNA origami frames to synthesize four DNA origami lattices by placing the pH-reactive i-motif sequences in the desired dimensions. Thereinto, lattices with an anisotropic design can switch between simple cubic (SC) and simple tetragonal (ST) upon pH change. Small angle X-ray scattering (SAXS) results reveal the feasibility of obtaining 3D lattices with sensitive responses to external stimuli, expanding the way to obtain low-symmetry lattices., (© 2022 Wiley-VCH GmbH.)

DNA origami has been widely used to construct nanophotonic structures due to its unparallel capability in assembling photonic nanomaterials with nanometer precision. Nevertheless, the majority of fabricated fluorescence-based nanophotonic systems so far operate in the ultraviolet-visible (UV-Vis) region, which are accompanied by limitations of shallow penetration and potential light damage. Herein, we constructed a reconfigurable nanophotonic heterostructure by integrating an upconversion nanoparticle (UCNP) and a gold nanorod (AuNR) on a DNA origami switch (DOS) template. With the unique anti-Stokes luminescence of UCNP whose excitation wavelength is mostly located in the near-infrared (NIR) region, the obtained reconfigurable nanophotonic system can be excited by 980 laser light and generates visible light emission. In our nanophotonic system, the DNA origami template serves as a switch to reversibly tune the optical properties of UCNP. The construction of this nanophotonic heterostructure expands the toolbox of DNA origami-based nanophotonic systems that may hold great potential in optical biosensing and imaging., (© 2022 Wiley-VCH GmbH.)

Repurposing the RNA-guided endonuclease Cas9 to develop artificial CRISPR molecular machines represents a new direction toward synthetic molecular information processing. The operation of CRISPR-Cas9-based machines, nevertheless, relies on the molecular recognition of freely diffused sgRNA/Cas9, making it practically challenging to perform spatially regulated localized searching or navigation. Here, we develop a DNA origami-based single-molecule CRISPR machine that can perform spatially resolved DNA cleavage via either free or localized searching modes. When triggered at a specific site on the DNA origami with nanoscale accuracy, the free searching mode leads to searching activity that gradually decays with the distance, whereas the localized mode generates spatially-confined searching activity. Our work expands the function of CRISPR molecular machines and lays foundations to develop integrated molecular circuits and high-throughput nucleic acid detection., (© 2022 Wiley-VCH GmbH.)