Nature Nanotechnology

Selectively perturbing pathogenic membranes while preserving host-cell integrity is a fundamental challenge in the design of membrane-active biomaterials. This challenge is particularly acute for enveloped viruses because their lipid membranes are derived from host cells and therefore share many of the same molecular constituents. Here, we demonstrate that engineered nanochirality provides a structural parameter for distinguishing virus-organized membrane nanodomains from host-cell membranes. Histidine-functionalized graphene quantum dots (His-GQDs) form enantiomeric nanoscale structures with distinct interfacial topologies despite closely matched size, composition, and surface charge. D-His-GQDs preferentially interact with cholesterol-rich ordered membrane nanodomains associated with S-acylated coronavirus Spike, penetrate more deeply into raft-like lipid bilayers, and induce lipid disordering, membrane leakage, and viral-envelope disruption. Reducing Spike S-acylation or depleting membrane cholesterol attenuated this stereoselective interaction, while molecular simulations revealed asymmetric lipid organization surrounding S-acylated Spike and preferential insertion of D-His-GQDs into the ordered membrane environment. Functionally, D-His-GQDs directly inactivated human coronavirus OC43 (HCoV-OC43), inhibited infection with an EC50 of 0.92 g/mL and exhibited a selectivity index of 361. Stereoselective antiviral activity extended to human cytomegalovirus, demonstrating activity across distinct enveloped-virus families, and intranasal D-His-GQDs protected HCoV-OC43-challenged mice under both prophylactic and early post-exposure regimens. Overall, these findings establish structural nanochirality as a biomaterial design parameter for recognizing higher-order membrane organization and converting virus-associated membrane states into selectively addressable therapeutic vulnerabilities.

Circulating microRNAs (miRNAs) have emerged as promising liquid biopsy markers; however, their clinical translation is restricted by low abundance, biological heterogeneity, and the complexity of multiplex profiling. Herein, we develop a targeted cycling-mediated duplex-specific nuclease (DSN)-nanozyme signal amplification platform (TC-DSN-Nano) that establishes a compartmentalized catalytic transduction strategy for multiplexed miRNA analysis. By encapsulating gold nanozymes within liposomal nanoreactors, this platform spatially separates molecular recognition from catalytic signal generation, preserving nanozyme activity during sensor construction. Upon target miRNA recognition, DSN-mediated recycling induces regenerative release of nanozyme-loaded liposomes from magnetic substrates, converting individual miRNA molecules into amplified catalytic outputs without conventional nucleic acid amplification. The platform enables programmable profiling of six prostate cancer (PCa) associated miRNAs in serum samples. Furthermore, machine learning-assisted integration of multiplexed miRNA signatures enables accurate discrimination among healthy donors, nonmetastatic PCa patients, and metastatic PCa patients, achieving an overall classification accuracy of 90.5%. This work establishes a general compartmentalized nanozyme transduction framework that integrates molecular recycling, catalytic amplification, and computational analysis for liquid biopsy-based cancer stratification.

Cell membrane-coated nanoparticles (CNPs) are a potentially transformative biomimetic targeted-delivery platform, which can engage therapeutic targets through source-cell membrane protein functions or homotypic interactions. However, their therapeutic development is limited by the current inability to accurately resolve membrane coating completeness, and CNP particle-to-particle variation. Here, we introduce a STochastic Optical Reconstruction Microscopy (STORM)-based approach that resolves population composition and quantifies membrane coating coverage at the single-particle level. Applying it to dendritic cell membrane- and HeLa cell membrane-coated nanoparticles (DCmPs and HeLamPs), we revealed substantial heterogeneity within both particle types and a cell-type dependence of coating outcomes, corroborated by confocal imaging and flow cytometry. We also quantified membrane protein composition, including peptide major histocompatibility complex class I (pMHC-I) among DCmPs, and confirmed that DCmPs preferentially engaged antigen-specific T cells in coculture. This single-particle framework establishes a quantitative standard for CNP characterization, providing a foundation for quality control and rational design.

Precise organization of bioactive ligands at material-cell interfaces is critical for directing integrin-mediated cell behavior, yet tuning supramolecular organization commonly necessitates orchestrated redesign of molecular composition or ligand sequence. Here we establish terminal modification as a minimal, sequence-preserving strategy to reprogram the organization and function of self-assembling integrin ligands. Using FFFIKVAV as a model system, N- and C-terminal modifications generated distinct supramolecular states with markedly different integrin activation and cell migration responses. Notably, stronger intermolecular association did not produce stronger biological activity, as N-terminal acetylation promoted scaffold consolidation while reducing the configurational freedom of the exposed IKVAV ligand and diminishing integrin activation and cell migration. Alongside modulating migration magnitude, terminal chemistry also diversified the cellular programs supporting migration, with the parent peptide and its C-terminally amidated analogue favoring protrusion-dominant and stress-fiber-dominant adhesion-cytoskeletal programs, respectively. These functional states were reproduced when the assemblies were introduced directly to pre-adherent cells, extending their activity beyond preformed material coatings. Our findings identify terminal chemistry as a compact molecular design variable that links supramolecular organization to ligand function while diversifying the biological output of a shared bioactive sequence.

Membrane tension, defined as the energy cost to expand the membrane surface area, is a fundamental mechanical property of the lipid bilayer and an important modulator of various cellular processes. Liposomes provide model systems to study protein-mediated membrane dynamics, where membrane tension can be controlled by osmotic pressure and micropipette aspiration. However, it is challenging to individually control membrane tension of nanometer-sized liposomes, such as small unilamellar vesicles (SUVs), that are widely used for modeling subcellular membrane structures and drug delivery. To bridge this technological gap, we present reconfigurable DNA origami rings for dynamically controlling membrane tension of sub-100 nm liposomes. The DNA nanorings template the formation of uniformly sized SUVs and undergo trigger-responsive dilation and contraction, thus applying controlled and reversible mechanical stress to the SUV membranes. Electron microscopy analyses show deformed liposomes with ~2.5% expanded membrane area following ring dilation. The corresponding increase in membrane tension opens a mechanosensitive ion channel MscL with gating tension of ~10 mN/m. Selectively manipulating a mixture of SUV populations enables timed release of their distinct molecular cargos, either concurrently or one at a time. Furthermore, vesicles with expanded membranes are more conducive to SNARE-mediated fusion. The nanomechanical device thus provides a programmable platform for engineering the mechanics of synthetic nano-vesicles and studying membrane tension modulated processes at the molecular level.

Electrochemical aptamer-based (EAB) sensors have emerged as a promising framework for reagentless, real-time molecular monitoring in complex biological matrices. Signal transduction in a prominent benchmark system--targeting aminoglycoside antibiotics--has been nearly universally attributed to target-specific, binding-induced conformational changes that modulate the distance between a redox reporter and the electrode surface. Here, using a suite of interfacial electrochemical and optical characterization methods, we systematically show that charge-screening, not structure switching, dominates the signaling mechanism in the case of the aminoglycoside EAB sensor. First, we demonstrate that the legacy aminoglycoside sensor sequence exhibits apparent affinities and relative electron kinetic modulations similar to unrelated control sequences, including generic stem-loop hairpins, unstructured poly-T sequences, and short duplexes. Multi-modal validation via bio-layer interferometry (BLI), circular dichroism (CD), and fluorescence spectroscopy confirms that this promiscuous target association occurs across disparate DNA sequences and without detectable conformational reconfiguration. Further investigation via charge-scaling polyamine assays and on-electrode fluorescence imaging demonstrate that the dominant signal transduction mechanism in these sensors is instead driven by macro-scale electrostatic backbone charge-screening.

Phe-Gly-rich nucleoporins (FG-Nups) within the nuclear pore complex (NPC) form a diffusion barrier that controls the exchange of macromolecules between the nucleus and cytoplasm. The behaviors of these intrinsically disordered FG-Nups are fundamental to the NPC function but remain difficult to probe within the dynamic, native nuclear pore. To bridge this knowledge gap, here we build expandable DNA-origami rings with tunable elasticity and study FG-Nups site-specifically tethered in such mechanically responsive NPC mimics. The elastic deformation of DNA rings, measured by single-particle TEM, reveals the type (attraction or repulsion) and strength of FG-Nup interactions that shape the collective morphology of these unstructured proteins. Analyzing homotypic interactions of five FG-Nup species finds diverse FG-Nup behaviors that can be classified as repulsive/extended (Nsp1), moderately cohesive/extended (Nup62, Nup153, and Nup214), and strongly cohesive/compact (Nup98). Furthermore, we examine how FG-Nups' primary sequence, O-glycosylation, grafting density, and binding to nuclear transport receptors (NTRs) impact their biophysical state. For example, O-GlcNAcylation reduced FG-Nup cohesiveness, switching Nup98 from a collapsed state to more extended conformations. Our work thus elucidates spatially confined Nup-Nup and Nup-NTR interactions within an NPC-like nanopore, suggests a previously underappreciated role of FG-Nups in modulating NPC dilation, and establishes a generalizable method for studying multivalent interactions among disordered proteins.

Focusing antibody responses towards known neutralizing epitopes is a major strategy for protective immunization. Targeting flexible antigenic regions represents a unique challenge because functional antibodies must be able to bind the epitope of interest in multiple conformations. The fusion peptide (FP) of HIV-1 is a flexible peptide critical to viral entry of host cells for which broadly neutralizing antibodies have been identified. Because elicitation of potent, functional antibodies toward this epitope remains an unmet challenge in HIV-1 vaccination, we introduce a designed immunogen platform capable of raising anti-FP-antibodies with increased conformational tolerance relative to a traditional carrier protein. Antibodies induced by these immunogens bind heterologous FP sequences when elicited in mice and infant non-human primates. This platform shows promise for preclinical development to prime antibody responses toward FP of the HIV-1 envelope and can likely be translated to peptide immunofocusing for other viral pathogens.

A broad-spectrum antiviral is required to enable rapid intervention against current, newly emerging and mutating viruses. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus (IAV), Sindbis virus (SINV) and chikungunya virus (CHIKV) all engage negatively charged moieties on host cells, such as heparan sulfate (HS) and sialic acid, to facilitate attachment for entry and infection. We designed HS-mimetic nanoparticles in the form of polymer vesicles (polymersomes) by blending membrane-forming and sulfonated copolymers. These biocompatible polymersomes successfully inhibited host cell infection of HS-dependent SARS-CoV-2 and HS-independent IAV in vitro with sub-nanomolar potency. Multivalent interaction of several virus ligands with a single polymersome as quantified by fluorescence correlation spectroscopy likely contributes to their potency. The sulfonated polymersomes also functioned in vivo as demonstrated by a reduction in the spread of SINV, an alphavirus model for CHIKV, in zebrafish embryos, which led to a significantly increased survival. Hence, our polymersomes could form the basis for an urgently required multifunctional broad-spectrum antiviral nanomedicine.

Glial cells play essential roles in maintaining neural tissue homeostasis and limiting the spread of injury-induced neuroinflammation, yet their dysfunction can also exacerbate neurodegenerative, neuroimmune, and neurodevelopmental disorders. Therapeutic targeting of specific glial cell populations would open up new treatment possibilities, but currently available viral and non-viral delivery vehicles are limited by carrier toxicity as well as poor delivery efficiency and specificity. To work towards new glia-targeted, non-viral delivery vehicles, we performed an in silico analysis on neural cell specific RNA-Seq datasets in mice to identify surface molecules differentially enriched on neurons, astrocytes, and microglia. We identified Slc2a5 (Glut5) as a microglia-enriched transporter and validated its microglia specific expression in healthy murine neural tissue using RNA in situ hybridization. By employing a sequential thiol-ene and enzymatic catalyzed reaction scheme, we synthesized polyelectrolytic dendrimers with multivalent presentation of fructose, the natural ligand for Glut5, and formulated nano-sized polyionic complexes (PICs) loaded with protein or nucleic acid cargo by mixing oppositely charged dendrimers. Fructose functionalized PICs lacking cationic surface charge enabled fructose-dependent intracellular protein delivery to Glut5-expressing human breast cancer cells (MCF-7) and mouse fibroblasts, while restricting uptake by Glut5-negative mouse astrocytes, confirming transporter-dependent selectivity in vitro. Fructose functionalized PICs resulted in minimal neuroinflammation when injected into mouse striatum at 1 mg/ml but elicited concentration-dependent neural tissue toxicity at higher doses. Fructose-PICs failed to achieve microglia specific intracellular delivery of functional proteins when injected into healthy neural tissue but aided in directing protein delivery to microglia and astrocytes at ischemic stroke lesions. Together, our findings demonstrate proof-of-principle for using in silico RNA-seq screening workflows to identify cell specific surfaceome targets and incorporating that insight into a modular biomaterial platform for targeted drug delivery.

With its unique spatiotemporal resolution at the single-molecule level, Forster resonance energy transfer (FRET) is a powerful tool for investigating biomolecular conformational dynamics and function. However, early photobleaching remains a key limitation restricting the achievable observation time and therefore the information gain and application range of single-molecule FRET. Here, we establish nanophotonic DyeCycling, which overcomes photobleaching by reversible fluorophore binding and efficient background suppression using zero-mode waveguides. Nanoscale conformational changes are sensitively resolved from milliseconds up to the hour range, enabling robust kinetic analyses at the single-molecule level. As a result, outlier molecules reflecting static heterogeneity and time-dependent kinetics within individual molecules reflecting dynamic heterogeneity are reliably resolved. The versatility of DyeCycling is demonstrated using DNA and protein systems. Altogether, nanophotonic DyeCycling extends smFRET observations beyond the conventional photobleaching limit, revealing previously inaccessible kinetic effects and providing deeper insight into biomolecular systems with multiple states and rates.

Adoptive cellular therapies can be effective against solid tumors but rely on the identification and expansion of rare tumor-reactive T cells (TRTs). Here, we utilize antigen-presenting cell-mimetic scaffolds (APC-ms), a modular biomaterial platform that presents defined peptide-HLA and co-stimulatory ligands on a fluid lipid bilayer to selectively recover rare antigen-specific T cells from peripheral blood. Independent variation of peptide-HLA, co-stimulatory ligands, and cytokine support defined a formulation that enriched MART-1-specific T cells to above 2% of CD8+ T cells in 35 of 50 HLA-A*02:01+ healthy donors from low or undetectable baseline frequencies. Compared with mo-DCs, APC-ms achieved greater fold expansion of MART-1-specific T cells with increased functional avidity and preservation of an early differentiation phenotype, and generated a higher number of MART-1-specific clonotypes. In five of eight patients with treatment-naive melanoma, APC-ms could enrich MART-1-specific T cells to a median of 13.7% of CD8+ cells (range 0.30 - 57.2%) Moreover, APC-ms could expand T cells specific for an HLA-A*24:01-restricted personal neoantigen that were previously undetectable in peripheral blood of a patient with melanoma. These findings establish APC-ms as a tunable platform for the recovery of rare TRTs from peripheral blood, supporting its further development for therapeutic TCR discovery and personalized adoptive cell therapy.

Engineered human neuromuscular models are limited by a trade-off between biological fidelity and architectural control: self-assembled organoids organize stochastically, while engineered 3D tissues impose anisotropy through passive tension at the edges without internal guidance. Here we encode architectural guidance into human neuromuscular tissues by coupling a three-dimensional, cell-laden biomatrix to a xolographically printed hydrogel substrate featuring sub-millimeter surface topography. We demonstrate that grooved substrates increase human primary myotube alignment and maturation in the overlying 3D tissue, while guiding directional axon extension from human iPSC-derived motor neuron spheroids. We show that combining both cell types along a shared alignment axis establishes functional neuromuscular connectivity, evidenced by acetylcholine receptor clustering, glutamate-evoked contraction and its blockade by d-tubocurarine (curare), and yields innervation coverage of centimeter scale tissues. Overall, this platform combines compliant substrates, mesoscale tissue architectural guidance, and human-relevant cell biology in a single untethered construct for studies of neuromuscular tissue development and function.

Whole-cell biosensors can detect many types of chemicals in complex environments, but existing designs force a tradeoff in multiplexed sensing: separating sensor strains into individual reservoirs limits how many analytes can be monitored at once, while pooling strains in open co-culture lets faster-growing strains take over within days, corrupting the signal. The microfluidic "mother machine" has been used for over a decade, mostly to study individual bacterial strains with single-cell resolution, but never to house multiple engineered strains simultaneously for sensing applications. We repurpose this device to hold a library of genetically distinct biosensor strains, physically isolating each strain in its own growth channel while continuous flow keeps every strain fed. Each strain reports on a target chemical through a unique combination of three fluorescent proteins, so seven distinguishable signals emerge from only three optical channels. We characterized turn-on and turn-off kinetics of several biosensors in the mother machine. Over a multi-day experiment with repeated chemical exposures, biosensors housed this way matched the expected on/off state and held stable population ratios, while biosensors in an open batch co-culture drifted and lost reliable signal. This establishes mother-machine-based multiplexing as a scalable route to long-term chemical monitoring with living sensors.

Unlike nucleic acids, proteins cannot be directly amplified. Ultrasensitive detection must therefore couple efficient analyte-to-signal conversion with strong suppression of target-independent background. Consistently detecting ~100 molecules (below which Poisson sampling noise alone causes CV to exceed 10%) across protein targets in a format as scalable and accessible as PCR remains unsolved. Separation-based ultrasensitive immunoassays require target binding stable enough to survive stringent washing and are therefore constrained by wash-induced signal loss and, more fundamentally, the relative scarcity of exceptionally tight binders. Performance consequently varies across targets; detection limits below 1,000 molecules remain rare. Conventional separation-free proximity assays avoid washing but incur background from random probe collisions, with reported detection limits frequently exceeding 100,000 molecules. Here we introduce Successive Proximity Extension Amplification Reaction (SPEAR), a separation-free architecture in which autonomous engineered DNA nanodevices emulate biological kinetic proofreading. Two successive irreversible proximity-extension steps compound discrimination between analyte-dependent and background reactions, suppressing spurious signals while preserving efficient analyte-to-signal conversion. This design reduces reliance on high-affinity binders and enables sub-fM detection from 1 uL samples through simple 3-step mixing followed by standard qPCR readout. Across 11 biomarkers tested, SPEAR consistently achieved sub-100-molecule detection limits. Where compared, it outperformed ultrasensitive separation-based platforms, analytically and clinically, including for low-abundance Alzheimer's disease biomarkers. SPEAR also precisely quantified SARS-CoV-2 neutralizing antibody titers from finger-prick dried blood spots, concordant with the cell-based reference assay on venous blood. It detected a distinct mixed phosphorylation state of Tau, and targeting this state resolved previously observed capillary-venous discordance and improved amyloid-status discrimination. These results establish engineered autonomous successive molecular discrimination as a practical strategy for near-single-molecule protein detection, extending the sensitivity, scalability and accessibility of PCR-based testing to non-amplifiable analytes.

Gram-positive bacteria use secreted peptides and peptide-responsive transcription factors for quorum sensing. Here, we demonstrate that intracellular expression of these peptide effectors with their cognate transcription factors leads to regulation of gene expression in both in vitro and in vivo systems. Because both effector and regulatory parts can be readily expressed, complex circuits, including auto-activation schemes, can be developed, with switch-like behaviors. Moreover, the peptide effectors can be fused to proteins, enabling a direct intracellular readout of translation and potentially allowing any protein to encode regulatory interactions in cells. Our results support a foundation for regulatory circuits that extend beyond canonical components such as LacI and TetR.

Cell fusion enables cell-based applications, yet systematic optimization remains constrained by low throughput (microfluidic devices) and manual operation (bulk protocols). Here, an automated microfluidic platform is presented for cell pairing and chemical fusion at a statistically relevant scale. High throughput is achieved by arranging ~350,000 deformability-based cell traps as concentric circular arrays in a radial chip. Using tumor and dendritic cells as a model for cancer vaccine, the device reproducibly co-occupies ~75% of traps (~250,000) of which ~140,000 hold exactly one cell of each type. This exceeds the yield of previously reported in hydrodynamic pairing arrays by more than an order of magnitude. A concentration step-up-and-down PEG profile yielded a fusion efficiency of ~20% (range 17.7-23.4%, n =3) at viability above 90% that results in producing at least ~50,000 hybrids per chip. This efficiency is comparable to that reported for PEG-based fusion in small-scale microfluidic devices but obtained at higher throughput and approximately four-fold higher than the conventional benchtop protocol. Fusion efficiencies were quantified by image analysis and validated by flow cytometry. By combining automation, large-area circular trap arrays and reagent control, the platform is useful for data-rich optimization of cell engineering processes previously constrained by limited microfluidic throughput.

Self-labeling protein (SLP) tags enable versatile labeling of proteins in live cells, yet only two SLPs are commonly used, limiting multiplexing. Here, we repurposed the oncoprotein K-Ras(G12C) and its covalent inhibitors to create a third, orthogonal SLP system. We used a deep-learning-based approach to radically redesign the sequence of K-Ras(G12C) while preserving its covalent-inhibitor binding pocket. Our top design, LUCI-tag, is a 19 kDa, monomeric, thermostable SLP that rapidly and covalently reacts with commercially available K-Ras(G12C) inhibitors bearing diverse payloads. Unlike existing SLPs, LUCI-tag exhibited payload-agnostic labeling kinetics, outperforming HaloTag7 and SNAP-tag for a negatively charged payload. X-ray crystal structures of drug-bound and drug-free LUCI-tag showed the design was structurally accurate and contained a preorganized inhibitor-binding pocket that could explain its rapid labeling kinetics. Proteomics and cell-signaling experiments confirmed LUCI-tag is biologically inert. LUCI-tag enabled rapid, wash-free, live-cell imaging and simultaneous three-color multiplexed experiments with HaloTag7 and SNAP-tag. This work establishes LUCI-tag as an immediately useful orthogonal SLP and demonstrates that covalent drug-target pairs can be repurposed into a broadly applicable platform for protein labeling.

Macromolecular release from synthetic cells often relies on membrane pores or channels. We explored whether internally generated osmotic stress could increase membrane permeability and promote release. Using double-emulsion microfluidics, we encapsulated an Escherichia coli cell-free expression system in Pluronic L121 polymersomes to co-express the fluorescent protein mScarlet3 and the dextranase PsDex1711 in the presence of dextran. PsDex1711-mediated dextran hydrolysis produced a greater increase in bulk osmolality than reactions without PsDex1711 DNA. Tracking hundreds of individual polymersomes showed that PsDex1711-containing populations expanded, whereas control populations contracted. The distribution of normalized fluorescence revealed a subset with lower residual fluorescence than the main population. In selected expanded polymersomes, integrated internal mScarlet3 fluorescence declined to 18-22% of peak levels, without visible membrane disruption, consistent with protein release. These findings support a proof of concept for using internal gene expression to promote macromolecular release through osmotic actuation without introducing dedicated membrane pores.

Controlling protein localization through chemical biology tools is essential for mechanistic interrogations, functional perturbations, and therapeutic development. While existing tools cover a wide range of applications, no single approach simultaneously enables inducible trapping, controlled release, and visualization of a protein of interest (POI). To address this gap, we developed Biotin-Operated Ligand Trapping (BOLT), which combines a HaloTag-fused POI with a streptavidin trap. Desthiobiotin-conjugated Janelia Fluor HaloTag ligands enable POI visualization, trapping, and biotin-operated release. We demonstrate BOLT's capacity in synchronized secretory cargo release, microtubule-directed organelle movement and hitchhiking, and ectopically localizing nuclear transcription factor. Importantly, pulse-chase BOLT further reveals bidirectional endoplasmic reticulum-Golgi trafficking of an endogenous Golgi enzyme and distinguishes recycled from newly synthesized enzyme populations using distinct HTLs. Altogether, BOLT provides an all-in-one platform to interrogate how subcellular localization regulates protein function across HaloTag-compatible systems.