Spatial engineering for biocatalytic cascade control through biomolecular compartmentalization.

The rapid growth of data volume and computing technologies has exposed the limitations of traditional encryption in terms of physical protection, system flexibility, and key management. Confronted with increasingly sophisticated attack threats, the static defense paradigm based on a single-mechanism encryption has become inadequate for maintaining information security. Therefore, integrating multimechanism strategies for collaborative defense within a dynamic architecture is essential to enhance information security. Here, we develop an algorithm-empowered encryption strategy using photocontrolled DNA origami nanostructures, which deeply integrates algorithms with molecular-scale programmable structural reconfiguration to establish a bioalgorithmic collaborative security framework. Rectangular DNA origami nanostructures serve as encoding physical substrates, storing digits and alphabets through binary mapping for information steganography. Additionally, dynamic connectors constructed from azobenzene-modified DNA strands establish linkages between DNA origami nanostructures, with the interstructural states precisely regulated via ultraviolet-visible (UV-vis) irradiation. Meanwhile, embedding the classical substitution-permutation ciphers into dynamically controllable nanostructures, combined with computationally driven key management, enables molecular-level information encryption and programmatic control. The cooperative encryption mechanism of substitution and permutation ciphers enhances information confusion and diffusion, while computational algorithms assist in optimizing the key layout and performing heuristic searches, further ensuring the security of molecular-level information processing. This work offers a promising perspective for research in information security and biointegrated intelligent systems.

Detecting biomolecules at ultralow concentrations remains a fundamental challenge in optical biosensing, primarily due to weak light–matter interactions governing signal generation. To address this limitation, we introduce a nonlinear optical sensing platform that leverages second-harmonic generation (SHG) within two-dimensional materials, amplified through quantum-engineered energy transfer. Our approach integrates DNA-programmable nanostructures with monolayer MoS 2 to form disordered metasurfaces that significantly enhance nonlinear optical responses. By precisely positioning individual CdTe/ZnS quantum dots (QDs) at defined distances from the MoS 2 surface using DNA origami scaffolds, we establish highly efficient Förster resonance energy transfer (FRET) pathways, boosting SHG signals by 124.70 fold. Furthermore, the platform incorporates a clustered regularly interspaced short palindromic repeats (CRISPRs) system as a switch: target recognition induces conformational changes that modulate SHG intensity with 93.60% specificity. This integrated material-biology design achieves unprecedented detection limits of 168 zM for microRNAs (miRNAs), representing an improvement of over six orders of magnitude compared to conventional optical biosensors, while retaining single-base discrimination capability. Validation with clinical lung cancer patient samples demonstrated superior diagnostic performance relative to reverse transcription quantitative polymerase chain reaction (RT-qPCR), exhibiting significantly enhanced signal-to-noise ratios in complex biological matrices. Our work establishes a new paradigm in nonlinear optical sensing by demonstrating how engineered quantum interactions can overcome intrinsic limitations of optical detection. This co-design framework, synergizing materials, nanophotonics, and biology, paves the way for next-generation optical diagnostic platforms.

mRNA-DNA hybrid origami enables integration of the RNA functionality into programmable DNA nanostructures, yet robust design and assembly rules remain lacking. Here, we systematically define parameters governing the high-yield formation of compact mRNA-DNA hybrid origami. Using mature mRNAs encoding firefly luciferase, enhanced green fluorescent protein (EGFP), and mCherry as scaffolds, we designed five architectures spanning varied sizes, shapes, crossover geometries, and packing densities. We identify asymmetric A-form crossovers, monovalent-cation-rich buffers, and moderate-temperature annealing as critical for suppressing RNA degradation and kinetic trapping while accommodating RNA-DNA helical geometry. Atomic force microscopy confirms monodisperse, well-folded nanostructures with nanoscale precision comparable to that of DNA origami. These results establish generalizable design rules and a standardized synthesis protocol for mRNA-DNA hybrid origami.

Multiplexed profiling of microRNAs (miRNAs) is central to refining diagnosis and prognosis, yet conventional fluorescence-based strategies remain vulnerable to environmental interference and signal crosstalk and rarely provide ground-truth validation of nanoprobe conformation. Here, we report pattern-verifiable heterowalkers (HW-TDONs) that integrate three DNAzyme walking modules confined on a triangular DNA origami nanosheet, each tethered to a size-distinct gold nanoparticle (10/15/20 nm) for the concurrent sensing of miRNA-10b, miRNA-21, and miRNA-155. Target recognition triggers autonomous DNAzyme walking that cleaves substrate strands, activating orthogonal fluorophores, and reconfiguring the origami pattern via AuNP detachment from predesignated sites. This dual-mode output quantitatively couples tricolored fluorescence with atomic force microscopy (AFM)─resolvable nanopattern statistics, thereby self-validating signal generation and mitigating false interpretations. This reliable and versatile platform enables combinatorial miRNA quantification in cell lysates and permits simultaneous imaging of multiple miRNAs in living cells, thereby supporting precise cell subtype discrimination. By coupling nanoscale conformational inspection to orthogonal optical readouts, HW-TDONs provide a programmable and scalable toolkit for facilitating multiplexed nucleic acid analysis and marker-associated pathological exploration with expansible potential for integrating patient-specific biomarkers in biomedical applications.

Dynamic DNA nanodevices, inspired by macroscopic machines, provide a versatile platform for constructing nanoscale mechanisms with specialized functionalities. In this study, we developed a DNA origami-based rotating nanodevice for continuous nucleic acid sensing, achieving a detection limit in the low nanomolar range. The nanodevice exhibits reversibility, enabling multiple rounds of target detection through toehold-mediated strand displacement. This regeneration capability was demonstrated at both the ensemble and single-molecule levels, with the latter offering high-resolution insights into dynamic conformational changes. By exploiting the spatial precision and programmability of DNA origami, we designed two distinct systems: a single-mode device that generates a Förster Resonance Energy Transfer (FRET) signal, and a dual-mode device capable of producing either a FRET or quenched signal, depending on the target detected, and enabling multiplexed measurements. Using these setups both at the ensemble and single-molecule level, we systematically explored various parameters to enhance binding efficiency, providing insights for optimizing and fine-tuning future designs.

DNA origami has become a ubiquitous platform because it enables straightforward design of nanostructures that self-assemble with high yield. The interactions between the cooperative effects involved in its assembly are currently not well understood. Fortunately, the nearly infinite number of choices available to the origami designer provides a rich environment in which to explore cooperativity. The DNA domains comprising origami have predictable energetics, and the sources of cooperativity are conceptually straightforward, and the difficulty in predicting assembly comes from their large number of cooperative interactions. We are able to probe cooperativity by using design variations and measuring their effect on assembly yield. We employ an accelerated assembly protocol that increases the sensitivity of structural perfection, or lack thereof, to design variation, and apply this approach to survey a broad set of design features. Using the resulting dataset, we develop metrics to correlate thermal stability, beneficial cooperativity from short folds, and detrimental cooperativity from long folds, with defectivity. Surprisingly, these metrics can be combined to create a single parameter with a clear correlation to yield, which serves as a useful starting place for a predictive understanding of the interplay between cooperativity and design. In doing so, we also identify qualitative trends that provide useful insight into design best practice.

Priming rare subdominant precursor B cells in germinal centers (GCs) is a central goal of vaccination to generate broadly neutralizing antibodies (bnAbs) against HIV. Multivalent immunogen display on protein nanoparticle scaffolds can promote such responses, but it also generates scaffold-specific B cells that could theoretically limit bnAb precursor expansion in GCs. We rationally designed DNA origami-based virus-like particles (DNA-VLPs) displaying a germline-targeting HIV envelope protein immunogen, which elicited no scaffold-specific antibody responses. Compared with a state-of-the-art clinical protein nanoparticle, these DNA-VLPs increased the expansion of epitope-specific GC B cells relative to off-target B cells and enhanced expansion of bnAb-lineage B cells in a humanized mouse model of CD4 binding site priming. Thus, minimizing off-target responses enhances bnAb priming and indicates that DNA-VLPs are a promising vaccine platform.

DNA origami scaffolds displaying HIV antigens stimulate focused antibody responses in mice.

Stimulated emission depletion (STED) microscopy enables super-resolution imaging of complex biological samples in 3D, in large volumes, and live. However, molecular quantification with STED has remained underexplored. Here, we present a straightforward approach for quantitative STED that enables molecule counting. For this purpose, we designed DNA-fluorophore labels that enable signal amplification and allow for reliable intensity-based quantitative imaging. We demonstrate accurate molecule counting on DNA origami. Furthermore, we visualized and quantified EGF receptor monomers and dimers in cells. In summary, we introduce a robust, fast, and easy-to-implement tool for quantitative STED microscopy with single-protein resolution.

Directional long-distance signal transmission on two-dimensional DNA origami assemblies

Inspired by the natural ability of bacteriophages to deliver genetic material directly into host cells, we employed a bottom-up approach to construct a multifunctional synthetic DNA origami needle-like structure. This origami is functionalized with trastuzumab antibodies, cholesterol, protective polymers, and two dyes, which together enable selective targeting and insertion into SKBR3 cancer cells. A disulfide-linked dye payload was attached to the apex of the needle, allowing controlled release in the cytoplasm triggered by the high intracellular glutathione concentration. Real-time tracking of the payload confirmed both successful targeting of the origami structure and subsequent direct cytosolic delivery. By mimicking fundamental mechanisms of bacteriophages, we propose that this artificial needle structure can serve as a prototypical device for the targeted delivery of small-molecule drugs directly into the cytosol.

Reconfigurable DNA nanostructures have emerged as a promising research area with applications in drug delivery, molecular computing, biosensing, and stimuli-responsive soft nanomaterials. While significant progress has been made in creating novel DNA nanostructures and exploring their applications, comparatively little effort has focused on developing new methodologies to confirm their folding. Conventional imaging approaches typically rely on sophisticated microscopy techniques including atomic force and transmission electron microscopy. Alternative low-cost methods for verifying DNA nanostructure assembly and shape sorting are thus highly valuable. Here, we present a fingerprinting nanosensor array integrated with machine learning (ML) to distinguish between two DNA origami shapes (triangle and nanotube) and to differentiate them from an unfolded scaffold strand. The nanosensor array, consisting of 11 nanoassemblies, termed nanosensors, is prepared by complexing graphene oxide nanosheets (nGO) with 11 fluorophore-labeled single-stranded DNA probes. Upon complexation, the fluorescence of the DNA probes is quenched through graphene oxide-mediated fluorescence quenching. Adding the DNA nanostructures to each nanosensor displaced a fraction of the surface-adsorbed fluorescent DNA probes, producing unique fluorescence recovery signatures that were subsequently processed through feature engineering for accurate ML-assisted classification. Using this approach, we achieved 94% prediction accuracy in discriminating DNA origami triangle, DNA origami nanotube, and unassembled M13 scaffold. Our strategy provides a new and generalizable platform for shape sorting in DNA origami field, offering new avenues for high-throughput, label-free classification.

ABSTRACT Single‐photon emitters radiate as electric dipoles, which limits light collection efficiency and complicates integration into flat photonic devices. Developing nanophotonic structures capable of directing photon emission with tunable angular distributions in the visible spectrum has been pursued for applications ranging from integrated optical systems to discrimination of molecular species. To date, such directional control has been achieved using components whose overall footprint is larger than the emission wavelength and often rely on lossy plasmonic components. Here, we employ the DNA origami technique for deterministic nanoscale assembly, positioning single fluorophores in nanometric proximity to a single silicon spherical nanoparticle (SiNP) and demonstrate unidirectional emission with forward‐to‐backward intensity ratios up to ∼7 dB. Furthermore, we show that a single silicon nanosphere antenna can function as a color router or a beam steerer depending on its size, emitter spectral range and emitter‐nanoparticle distance, enabling the use of these structures as versatile functional components in photonic devices.

ABSTRACT Lipid rafts cluster at the immunological synapse during activation and serve as signaling hubs in T cells. While cholesterol plays a crucial role in lipid raft formation, the impact of their spatial configuration remains less understood. Here, we introduce programmable DNA origami nanostructures, cholesterol nano‐patch (CNP), for nanoscale spatial control of cholesterols on live T cell membranes, enabling us to elucidate their roles in lipid raft formation, receptor activation, and intracellular signaling. We demonstrate that CNPs with high‐density cholesterol arrangements efficiently bind to the T cell plasma membrane and form a large and polarized coalescence, leading to the stabilization of ordered lipid membrane domains by increasing the local concentration of cholesterols. These synthetic lipid rafts colocalize with flotillin‐1, a raft‐associated protein, and promote the membrane reorganization and physical segregation of key signaling molecules, such as T cell receptors, Lck, LAT, and CD45. Consequently, this leads to early T cell activation in the absence of antigenic stimulation. Our DNA origami approach demonstrates the nanoscale arrangement of cholesterol plays significant roles in lipid raft formation, membrane phase separation, and protein reorganization, which are sufficient to induce early T cell activation.

Protein interactions with DNA often result in rotational movements. These movements underlie many genetic processes such as DNA transcription by RNA polymerase (RNAP). To illuminate these movements, we previously developed Origami Rotor-Based Imaging and Tracking (ORBIT), a fluorescence-based method that enables high-speed tracking of DNA rotation. When used to track DNA rotation during transcription by E. coli RNAP, ORBIT enabled detection of single base-pair steps. However, the duration of ORBIT experiments was limited due to fluorescence photobleaching. To overcome this limitation and extend the total possible observation time, we here introduce dye-cycling ORBIT (dcORBIT), a method that enables observation of protein-DNA interactions for over 10 minutes at 20 Hz while maintaining single base-pair precision. dcORBIT thereby opens new possibilities to study the fundamental rotational movements underlying gene expression.

Optically trapped microscopic probes with precisely defined size, shape, and composition can be used for quantitative environmental sensing and for parametric investigation of fundamental physical phenomena at the classical-quantum boundary. The preparation of uniform ensembles of such probes is challenging, particularly considering probes with controlled shape or material asymmetries. We report a bottom-up strategy for fabricating the optomechanical probes using DNA nanotechnology. Specifically, we synthesize Janus-type colloidal heterodimers comprising two microspheres of different materials and sizes interconnected by 24HB DNA origami nanostructures. The interconnecting DNA origami scaffolds both facilitate the heterodimer assembly and enable their functionalization with other optical components. The utility of the fully assembled probes is then demonstrated by their 2D and 3D manipulation with optical tweezers. The versatility of the presented approach opens up the way toward fabricating novel custom-tailored probes for optomechanical experiments.

T cell-engaging therapy represents a cutting-edge approach in immuno-oncology that harnesses the power of the immune system to combat cancer. Despite its promise, challenges related to efficacy and safety limit its broader clinical application. Here we introduce SpTCE, a spatially controllable T-cell cluster engager based on DNA origami technology. SpTCE features an elegant two-layer architecture built on a DNA origami chassis. The inner functional layer is engineered to organize multiple, multivalent engaging antibodies and complementary hand-in-hand strands, which work together to enhance T-cell clustering and activation. The outer shielding layer integrates albumin binding domains with i-motif switches, which form a pH-responsive albumin coat and serve two functions: isolating the inner layer from healthy tissues and improving the structural stability of the DNA origami through an albumin coat. We validate acidic pH-triggered, specific T-cell cytotoxicity of SpTCE against tumor cells in vitro and observe the substantial intratumoral accumulation and improved physiological stability in vivo. As a proof of concept, we show that SpTCE effectively redirects T cells to eliminate tumors with high specificity and a more favorable off-tumor toxicity profile than a clinical-stage bispecific T cell engager. Collectively, these findings highlight the potential of SpTCE as a promising T-cell engager, offering precise control over T-cell activation while balancing efficacy and safety, and expanding the possibilities for advanced immuno-oncology treatments.

DNA nanotechnology has rapidly evolved, leading to the development of dynamic nanoscale and microscale devices that mimic natural molecular machinery. This Review explores the latest advancements in DNA-based machines, motors and switches, emphasizing the need for clear definitions to distinguish between these often-interchanged terms. By analysing key performance metrics such as speed, force generation, efficiency and autonomy, we provide a framework for evaluating these devices against their biological counterparts, including motor proteins such as myosin and kinesin. We highlight innovative design strategies such as strand displacement, DNA origami and hybrid systems, which enhance the functionality of DNA-based constructs and bridge the gap between synthetic and natural systems. These advancements have promising applications in areas such as targeted drug delivery, biosensing and nanofabrication, although challenges in achieving the high performance and efficiency seen in biological systems remain. Through a synthesis of current research, this Review outlines the opportunities and challenges in the development of DNA-based nanoscale and microscale devices.

DNA origami holds great potential for advancing therapeutics, but the lack of methods for the precise assessment of structural integrity in vivo prevents its translation. Here we introduce proximity ligation assay for structural tracking and integrity quantification (PLASTIQ) for resolving origami structural integrity with only 1 µl of blood sample and with a detection limit of 0.01 fM. Through PLASTIQ, we could observe and quantify the dynamics of DNA origami degradation during blood circulation and evaluate the effectiveness of PEGylation for slowing this process in a murine model. Additionally, by using a double-layered barrel-like origami structure, we found distinct degradation kinetics of DNA helices depending on their specific location, revealing the slower degradation of internal helices compared with the outer ones. Our results suggest that PLASTIQ offers a quantitative approach for assessing DNA origami integrity in vivo by longitudinal sampling, providing dynamic pharmaceutical-level insights for accelerating the development of DNA-nanostructure-based therapeutic molecules and drugs.