Nature Nanotechnology

DNA nanotechnology offers precise, biocompatible structures with strong potential for targeted drug delivery, yet current discovery approaches rely on testing individual designs, limiting exploration of structural diversity. Here, we introduce an evolutionary selection strategy that screens large libraries of DNA nanostructures, each folded from a single-stranded structure genome compatible with amplification and sequencing. Cellular internalization is used as the selection pressure: libraries are incubated with mammalian cells, internalized structures are recovered from lysates, and the process is iterated across multiple rounds in HEK293T and RAW264.7 cells. High-throughput sequencing of recovered structure genomes reveals cell-type-specific enrichment patterns, enabling the identification of individual nanostructures with preferential uptake. Selected candidates were synthesized and evaluated as purified structures, confirming differential internalization by quantitative flow cytometry and microscopy. Turning DNA nanostructure discovery into a selection-based process, could enable high-throughput exploration of structural diversity and provide an alternative route to identify nanostructures with cell-specific uptake properties for biomedical applications.

Intricate self-organization is essential in many biological processes, underpinning vital functions and interactions. In an effort to mimic such processes, synthetic biology aims to engineer dynamic structures with controllable functions using nanotechnological tools. A key requirement of engineered building blocks is the ability to assemble and disassemble hierarchically with precision. Using the DNA origami technique, we here present the moDON, a modular DNA origami nanostructure, which is capable of assembling into almost 20 000 diverse monomers, forming complex and controlled superstructures in three dimensions. While shape and addressability of DNA origami are nearly arbitrary, its overall size is limited by the scaffold size. Previous methods of extending the size of DNA origami (e.g. hierarchical assembly, modified scaffolds, etc.), either led to loss over control of shape and addressability beyond monomers or to proportionally increased cost and design effort. With the moDON we were able to overcome both issues. The modular design combines xy- and z-plane assembly methods, enabling the construction of finite and periodic structures beyond 1 GDa. We demonstrate xy-z orthogonality, by enabling controlled selective or parallel assembly and disassembly via distinct orthogonal triggers. The kinetic profile of assembly and disassembly aligns with biological time scales, paving the way for applications in dynamic nanomachinery and advanced biomaterials. Finally, we showcase the conjugation of gold nanoparticles to specific positions within superstructures, underscoring the efficacy of this approach for creating intricate and orthogonal nanoscale architectures with preserved site-specific addressability. The moDON thus offers an efficient, cost-effective solution for constructing large, precisely organized, and fully addressable structures with vast potential in synthetic cellular systems design.

The persistent immunosuppression and spatial heterogeneity of advanced solid tumors necessitate next-generation neoadjuvant platforms capable of reprogramming tumor microenvironments while overcoming resistance to immune checkpoint blockade. Addressing this unmet clinical challenge, we report a {beta}-glucan-empowered nano-neoadjuvant agents (NNAs) engineered through topology-guided supramolecular assembly of pathogen-mimetic {beta}-glucan motifs onto photoresponsive single-wall carbon nanotubes. This biomimetic architecture capitalizes on three synergistic mechanisms: (1) Dectin-1-mediated pathogen-associated molecular pattern recognition to rewire myeloid cell functionality, (2) endogenous leukocyte-hitchhiking delivery paradigm, and (3) spatiotemporally controlled photothermal ablation via near-infrared transducing SWNT cores. NNAs also orchestrates a proinflammatory cascade involving IL-12-dependent Th1 polarization and activation, effectively converting "cold" tumors into immunogenic niches. This strategy redefines preoperative therapeutic paradigms, offering a multimodal platform to harness the proinflammatory adjuvant effect of SWNTs for preoperative immunomodulation in advanced-stage malignancies. By bridging innate immune recognition with nanotechnology-enabled precision, this paradigm establishes a new roadmap for preoperative conditioning of advanced malignancies, offering a theranostic framework to transform surgical outcomes through immunologically informed nanomedicine design.

The persistent immunosuppression and spatial heterogeneity of advanced solid tumors necessitate next-generation neoadjuvant platforms capable of reprogramming tumor microenvironments while overcoming resistance to immune checkpoint blockade. Addressing this unmet clinical challenge, we report a {beta}-glucan-empowered nano-neoadjuvant agents (NNAs) engineered through topology-guided supramolecular assembly of pathogen-mimetic {beta}-glucan motifs onto photoresponsive single-wall carbon nanotubes. This biomimetic architecture capitalizes on three synergistic mechanisms: (1) Dectin-1-mediated pathogen-associated molecular pattern recognition to rewire myeloid cell functionality, (2) endogenous leukocyte-hitchhiking delivery paradigm, and (3) spatiotemporally controlled photothermal ablation via near-infrared transducing SWNT cores. NNAs also orchestrates a proinflammatory cascade involving IL-12-dependent Th1 polarization and activation, effectively converting "cold" tumors into immunogenic niches. This strategy redefines preoperative therapeutic paradigms, offering a multimodal platform to harness the proinflammatory adjuvant effect of SWNTs for preoperative immunomodulation in advanced-stage malignancies. By bridging innate immune recognition with nanotechnology-enabled precision, this paradigm establishes a new roadmap for preoperative conditioning of advanced malignancies, offering a theranostic framework to transform surgical outcomes through immunologically informed nanomedicine design.

Causal manipulation of vagal gut-brain pathways empowers studies of metabolism and interoception. However, the anatomy and cytoarchitecture of vagal circuits pose challenges to deployment of optical or electrical stimulation probes. We present a wireless modulation of vagal circuits via magnetite nanodiscs (MNDs) targeted to specific nodose ganglia neurons via genetically delivered anchoring moieties. Under slow-varying magnetic fields, membrane-bound MNDs transduce mechanical torques that trigger depolarization mediated by endogenous mechanoreceptors in sensory neurons. When targeted to neurons expressing oxytocin or glucagon-like peptide 1 receptors in the left nodose ganglia, MND stimulation activates downstream hindbrain satiety circuits and reduces food intake. These findings establish MND-mediated stimulation as a targeted, implant-free platform for modulating gut-brain neural circuits and beyond.

Quantum biological tunnelling for electron transfer (QBET) is involved in controlling cellular behaviour. Control of electrical-molecular communication could revolutionise the development of disruptive technologies for understanding and modulating electrically induced molecular signalling. Current communication technology is not appropriate for interfacing with cells at a spatial/temporal level equivalent to the native biological signalling. This limits our ability to tune cell function by controlling single molecular events. Here, we merge wireless nano-electrochemical tools with cancer cells. Gold-bipolar nanoelectrodes functionalised with redox active species were developed as electric field stimulated bio-actuators, that we term bio-nanoantennae. We show that a remote electrical input regulates electron transport between the redox molecules on the bio-nanoantennae in a selective manner. The wireless modulation of electron transport results in QBET triggering apoptosis in patient-derived cancer cells, representing electrical-induced induced controlled molecular signalling. Transcriptomics data highlight the electric field-induced nanoantenna targets the cancer cells in a unique manner. The insight concerning action and functional nanomaterials opens a plethora of applications in healthcare. This approach may lead to new quantum-based medical diagnostics and treatments, as well as a fundamental understanding of biological physics.

Tissue barriers must be rapidly restored after injury to promote regeneration. However, the mechanism behind this process is unclear, particularly in cases where the underlying extracellular matrix is still compromised. Here, we report the discovery of matrimeres as constitutive nanoscale mediators of tissue integrity and function. We define matrimeres as non-vesicular nanoparticles secreted by cells, distinguished by a primary composition comprising at least one matrix protein and DNA molecules serving as scaffolds. Mesenchymal stromal cells assemble matrimeres from fibronectin and DNA within acidic intracellular compartments. Drawing inspiration from this biological process, we have achieved the successful reconstitution of matrimeres without cells. This was accomplished by using purified matrix proteins, including fibronectin and vitronectin, and DNA molecules under optimal acidic pH conditions, guided by the heparin-binding domain and phosphate backbone, respectively. Plasma fibronectin matrimeres circulate in the blood at homeostasis but exhibit a 10-fold decrease during systemic inflammatory injury in vivo. Exogenous matrimeres rapidly restore vascular integrity by actively reannealing endothelial cells post-injury and remain persistent in the host tissue matrix. The scalable production of matrimeres holds promise as a biologically inspired platform for regenerative nanomedicine.

Chirality in two-dimensional nanomaterials provides a powerful lever to control biological interfaces, yet the structural origins of nanoscale chirality remain poorly understood. Here, we systematically investigate chiral ligand modulated graphene quantum dots (GQDs) and reveal how ligand stereochemistry and edge chemistry modulate the formation of diverse chiral or achiral nanostructures. Spectroscopy, microscopy, and density functional theory with ring-puckering analysis identified six structural motifs, twisted-, twisted-boat, saddle-shaped, hybrid, unbuckled, and random. Among these, twisted-, twisted-boat, and saddle-shaped GQDs exhibited genuine nanoscale structural chirality, while unbuckled, hybrid, and random conformations lacked organized distortion. Importantly, structural chirality governed passive permeation into biological membrane (e.g. lipid membrane of extracellular vesicles), whereas achiral variants relied mainly on hydrophobic interactions. In contrast, active transport across biological membrane (e.g. endocytosis) is insensitive to nanoscale structural chirality but strongly influenced by chiral ligand identity and transporter recognition. Collectively, these results establish chiral ligand conjugation as a modular route to program both chiral and achiral motifs in graphene nanostructures and highlight nanoscale structural chirality as a design principle for engineering bio-nano interactions.

In the quest to create increasingly complex synthetic cell-mimicking systems, a wide range of DNA nanostructures have been developed to coat, permeabilize, sculpt, or otherwise functionalize lipid vesicles. In a complementary strategy, DNA architectures have been used as scaffolds to direct the growth of lipid membrane vesicles. Here we introduce a simple and broadly applicable method to realize freestanding, membrane-mimicking DNA shells: DNA shells are first assembled on the outer surface of giant unilamellar vesicles and then liberated by surfactant-mediated liposome removal. The resulting structures faithfully retain the geometry of their membrane template. We demonstrate the approach with two distinct classes of DNA tectons: a complex barrel-shaped DNA origami with programmable inter-subunit interactions, and a simple nanostar-inspired motif composed of only eleven oligonucleotides. The site-specific addressability of the former enable the rational design of binding interfaces, as demonstrated by controlled multilayer formation. The success of both strategies underscores the generality of our approach and the feasibility of creating shell-like compartments from different DNA architectures. This method enables the construction of tunable, DNA-only containers spanning the size range of eukaryotic cells, offering a fundamentally new type of compartmentalization for bottom-up synthetic biology.

Membrane fusion is a fundamental yet transient process that has long resisted direct structural and kinetic dissection. Here we introduce a DNA framework vesicle (DFV) nanoreactor that transforms this elusive biological phenomenon into a programmable, visualizable, and quantifiable process-a nanoreaction confined in space yet extended in time. By embedding lipid membranes and SNARE proteins within precisely defined DNA apertures, DFVs convert stochastic vesicle collisions into geometry-controlled fusion events with tunable kinetics. Cryo-electron microscopy resolves a complete sequence of six intermediates, revealing how nanoscale confinement reshapes the energetic landscape of bilayer merger. Quantitative fluorescence and nano-flow cytometry further establish a direct link between spatial design and fusion probability. Extending this concept to living cells, DFVs enable controllable membrane fusion and augmentation on VAMP2-expressing membranes, achieving direct cytosolic delivery of functional siRNA via fusion-driven transfer rather than endocytosis. This framework bridges structural precision and functional mimicry, offering a unified platform to reconstruct, quantify, and harness membrane fusion as a programmable process for synthetic biology and nanomedicine.

In cells, proteins rapidly self-assemble into sophisticated nanomachines. Bio-inspired self-assembly approaches, such as DNA origami, have achieved complex 3D nanostructures and devices. However, current synthetic systems are limited by lack of structural diversity, low yields in hierarchical assembly, and challenges in reconfiguration. Here, we develop a modular system of DNA origami voxels with programmable 3D connections. We demonstrate multifunctional pools of up to 12 unique voxels that can assemble into many shapes, prototyping 50 structures. Multi-step assembly pathways with sequential reduction in conformational freedom were then explored to increase yield. Voxels were first assembled into flexible chains and then folded into rigid structures, increasing yield 100-fold. Furthermore, programmable switching of local connections between flexible and rigid states achieved rapid and reversible reconfiguration of global structures. We envision that foldable chains of DNA origami voxels can be integrated with scalable assembly methods to achieve new levels of complexity in reconfigurable nanomaterials.

The delivery of nanotherapeutics to specific tissues relies on bespoke targeting strategies or invasive surgeries. Conversely, adeno-associated viruses (AAVs) can target specific tissues following intravenous injections. Here we show that cell-targeting properties of AAVs could be broadly conferred to nanomaterials. We develop a strategy to couple AAV capsids to nanoparticles that is invariant of viral serotype or nanomaterial chemistry and permits control over stoichiometry of the AAV-nanoparticle chimeras. The chimeras selectively escort nanoparticles into cell classes governed by AAV serotypes. When applied to magnetic nanoparticles, the AAV-nanoparticle chimeras enable magnetically localized gene delivery. In vivo, we show that leveraging the brain-targeting AAV serotype CAP-B10 achieves nanoparticle delivery to the parenchyma with [~]10% efficiency (% injected dose/g[brain]) while avoiding accumulation in the liver. The enhanced delivery efficiency and tissue specificity highlight the potential of AAV-chimeras as a versatile strategy to escort broad classes of nanotherapeutics to the brain and beyond.

Rigid DNA nanostructures that bind to floppy bilayer membranes are of fundamental interest as they replicate biological cytoskeletons for synthetic biology, biosensing, and biological research. Here, we establish principles underpinning the controlled interaction of DNA structures and lipid bilayers. As membrane anchors mediate interaction, more than 20 versions of a core DNA nanostructure are built each carrying up to five individual cholesterol anchors of different steric accessibility within the 3D geometry. The structures binding to membrane vesicles of tunable curvature is determined with ensemble methods and by single-molecule localization microscopy. This screen yields quantitative and unexpected insight on which steric anchor points cause efficient binding. Strikingly, defined nanostructures with a single molecular anchor discriminate effectively between vesicles of different nanoscale curvatures which may be exploited to discern diagnostically relevant membrane vesicles based on size. Furthermore, we reveal anchor-mediated bilayer interaction to be co-controlled by non-lipidated DNA regions and localized membrane curvatures stemming from heterogenous lipid composition, which modifies existing biophysical models. Our study extends DNA nanotechnology to control interactions with bilayer membranes and thereby facilitate the design of nanodevices for vesicle-based diagnostics, biosensing, and protocells.

Protein secretion underpins diverse physiological processes in cell-to-cell communication, tissue homeostasis, and the onset of diseases. Mapping the secretomes from paired cells provides avenues for understanding their interactions. However, prevailing approaches yield only semi-quantitative endpoint data, lacking real-time and quantitative information. Here, we present real time spatiotemporal imaging of extracellular secretions from individual cells via a high-throughput integrative biosensing nanoplasmonic array (iBNA) with a microfluidic chamber. The self-assembled iBNA, composed of precisely arranged gold nanostructures and functionalized with aptamer receptors, enhances plasmonic resonance and significantly improves the spatiotemporal resolution and specificity of interleukin-6 (IL-6) imaging, surpassing gold-standard techniques. The molecular recognition of iBNA, and sensing mechanism exploits biomolecular surface binding-induced localized plasmonic resonance shifts, correlating with cytokine concentration and enabling optoelectronic detection of the transmitted light. Using this approach, we achieve spatiotemporally resolved visualization of IL-6 secretion dynamics at the single-cell level and unveil the temporal and polarized variation of cell-cell communications. This transformative platform holds significant potential to advance immunological research, cellular biology, and diagnostic applications for infectious diseases by enabling unprecedented insights into the spatiotemporal patterns of protein secretion in individual cells.

T cell proliferative capacity and persistence critically determine the therapeutic success of chimeric antigen receptor (CAR) T cells. However, it remains unknown if and how human CAR-T cells can be externally programmed to reach maximal proliferative capacity. Here, we use programmable PLGA microparticles functionalized with CAR-antigens and CD28-costimulatory antibodies (CAREp) to repeatedly stimulate human CD8+ CAR-T cells in vitro. CAREp-stimulated CAR-T cells expanded continuously for over 100 days--versus [~]30 days with tumor cell stimulation--and achieved up to 1018-fold cumulative expansion, greatly surpassing CD3/28-Dynabeads. Early-phase transcriptomic responses-- upregulation of DNA repair, cell cycle, telomere maintenance, and mitochondrial pathways--aligned with long-term outcomes: massive proliferation, telomere stability, robust respiration, and preserved progenitor phenotype by single-cell sequencing. Differentiation and exhaustion signals were broadly suppressed. Transient telomerase activity further supported physiologic expansion. These findings demonstrate that nanoscale-controlled extracellular cues can rewire intracellular signaling to drive durable, super-physiological expansion of functional CAR-T cells.

Biological cells use cations as signalling messengers to regulate a variety of responses. Linking cations to the functionality of synthetic membranes is thus crucial to engineering advanced biomimetic agents, such as synthetic cells. Here, we introduce bio-inspired DNA-based receptors that exploit non-canonical G-quadruplexes for cation-actuated structural and functional responses in synthetic lipid membranes. Membrane confinement grants cationdependent control over receptor assembly and, when supplemented with hemin co-factors, their peroxidase DNAzyme activity. Cationmediated control extends to receptor lateral distribution to localise DNA-based catalysis within phase-separated membrane domains of model synthetic cells, imitating the localisation of multimeric membrane complexes to signalling hubs in living cells. Our modular strategy paves the way for engineering from the bottom-up cation-responsive pathways for sensing, signalling, and communication in synthetic cellular systems.

Molecular engineering seeks to create functional entities for the modular use in the bottom-up design of nanoassemblies that can perform complex tasks. Such systems require fuel-consuming nanomotors that can actively drive downstream passive followers. Most molecular motors are driven by Brownian motion, but the generated forces are scattered and insufficient for efficient transfer to passive second-tier components, which is why nanoscale driver-follower systems have not been realized. Here, we describe bottom-up construction of a DNA-nanomachine that engages in an active, autonomous and rhythmical pulsing motion of two rigid DNA-origami arms, driven by chemical energy. We show the straightforward coupling of the active nanomachine to a passive follower unit, to which it then transmits its own motion, thus constituting a genuine driver-follower pair. Our work introduces a versatile fuel-consuming nanomachine that can be coupled with passive modules in nanoassemblies, the function of which depends on downstream sequences of motion.

Techniques from structural DNA nanotechnology make it possible to assemble complex 3-dimensional nanostructures with virtually arbitrary control over their sizes, shapes and features at length scales of 3-100 nm, providing a flexible means for constructing nanoscale devices and machines. Here, we assemble micron-length DNA nanotubes and assess their performance as pipes for controlled ion transport. DNA nanotubes grow via assembly of DNA tiles from a seed pore, a 12-helix DNA origami cylinder functionalized with cholesterol, to form a DNA nanotube channel. The central channel of a nanotube can be obstructed via Watson-Crick hybridization of a channel cap, a second DNA origami structure, to the end of a nanotube channel or a nanotube seed pore. Single-channel electrophysiological characterization shows that both nanotube seed pores and nanotube channels display ohmic ion conductance consistent with their central channels diameters. Binding of the channel cap reduces the conductances of both DNA nanotube channels and seed pores, demonstrating control of ion-transport through these micron-length channels. Because these channels could be assembled into branched architectures or routed between specific molecular terminals, these results suggest a route to self-assembling nanofluidic devices and circuits in which transport can be controlled using dynamic biomolecular interactions.

Many cellular pathways rely on the active regulation of membrane composition to fine-tune key physico-chemical properties, including lipid packing, fluidity, and surface charge. While intrinsic membrane charge is often attributed to specific lipid headgroups and membrane-bound proteins, the contribution of acyl-chain chemistry to the electrostatic profile of membrane surfaces remains unexplored. Here, we systematically investigate how variations in acyl chain length and unsaturation modulate the biophysical and electrostatic properties of zwitterionic lipid membranes. Using amphiphilic DNA nanoprobes as model charged biomolecules, we reveal how lipid packing, fluidity, and membrane phase collectively govern surface charge and interactions with nanoprobes, delineating relationships that persist in the presence of anionic lipids. We further demonstrate that the identity and hydrophobicity of membrane anchors in nanoprobes significantly influence binding, providing a means to modulate membrane association through programmable strategies. By establishing design principles that link acyl chain chemistry to surface charge and biomolecular attachment, our findings provide a mechanistic framework to engineer selective membrane interactions. Beyond their direct applicability to biomimetic platforms and synthetic cell engineering, these insights hold broad relevance to lipid-based vaccine nanotechnologies and the fundamental understanding of membrane-biomolecule interactions in living cells.

Quantitative analysis of protein interactions and the formation of higher-order assemblies in living cells remains a major challenge. Here, we introduce a versatile nanopatterning toolbox that employs capillary nanostamping of functionalized polymers to generate high contrast bio-functionalized nanodot arrays (bNDAs) with diameters below 500 nm. By leveraging orthogonal adaptor designs, we achieve robust immobilization of diverse fluorescent protein fusions, enabling simultaneous and selective recruitment of cytosolic and membrane-associated proteins into discrete nanodomains. This approach of forming cytosolic nanodot arrays (cNDAs) provides striking capabilities for dissecting cytosolic multiprotein complexes with molecular precision. Focusing on the assembly of the multimeric myddosome complex, we demonstrate density-dependent recruitment and co-localization of the core components MyD88, IRAK4, IRAK1, and TRAF6 within cNDAs. Super-resolution microscopy reveals distinct nanoscale clustering of MyD88 and IRAK4 and uncovers the ultrastructural architecture of IRAK4 oligomers. These analyses highlight the spatial organization and hierarchical assembly of the myddosome at the nanoscale in the native cellular context. Collectively, our findings establish cNDAs as a powerful platform for reconstituting and analyzing intricate multiprotein assemblies in live cells, offering new opportunities for elucidating the principles of complex protein networks.