Nano Letters

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.

A comprehensive understanding of the mechanisms underlying biological systems requires correlative analysis of multiple complementary molecular properties through multidimensional measurements. High-speed atomic force microscopy (HS-AFM) is a powerful tool for elucidating biomolecular structural dynamics at the single-molecule level with high spatiotemporal resolution. However, structural information alone is often insufficient for fully understanding the biological function mechanisms. Here, we report high-speed atomic force-Raman microscopy (HS-AFRM), which enables multimodal measurements combining video-rate structural imaging with chemical-bond analysis. Raman spectroscopy is a powerful, non-invasive technique that probes molecular vibrations to provide chemical information. We achieved several key technical developments that facilitated the seamless integration of HS-AFM and micro-Raman spectroscopy, allowing reliable correlative measurements of structural and chemical information. We validated the versatility of the developed system using representative samples, including two-dimensional materials and a protein. Furthermore, we demonstrate probing of changes in the surrounding environment, which are inaccessible by HS-AFM alone. Multimodal measurements incorporating fluorescence spectroscopy were also demonstrated as an additional practical extension. This multimodal approach substantially enhances the analytical capability of HS-AFM, providing a powerful platform for revealing correlated structural and chemical properties across diverse research fields.

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.

The molecular programmability of nucleic acids has facilitated the development of architected DNA/RNA nanostructures and their applications in novel materials and technologies. We report how different types of DNA and RNA nanoaggregates, bundling digital information encoded into oligo libraries, can be formed by manipulating the ionic strength of the solution. As DNA or RNA suspensions are immersed in solutions of increasing salt concentrations, we observe the onset of aggregation. Further increase in ionic strength leads to the formation of stable, reproducible, and well-defined aggregates. We show that these nanoaggregates are kinetically trapped at room temperature, stably partition DNA libraries that encode image files, and support file-specific random access by bundling DNA libraries with unique address oligos. The nanoaggregate files can be disrupted and reformed into scrambled bundles using simple external fluid shear or temperature annealing, rapidly obfuscating the data. We term these nanoaggregates nucleic acid PACKeTs: Partitioned Aggregates of Colloidal DNA/RNA through Kinetic Trapping. Overall, the results demonstrate how gaining fundamental insights into ionic colloidal aggregation enables new forms of manipulation of DNA and RNA libraries. This understanding could lead to novel functionalities including kinetically trapped data partitioning, random access, and data encryption or obfuscation.

Super-resolution fluorescence microscopy overcomes the optical diffraction limit and has significantly advanced our understanding of biological complexity within the framework of fluorescence labelling (1). Fluorescence labelling underpins this capability, enabling high photon budgets, superior signal contrast, and tuneable photophysical properties essential for diverse super-resolution modalities (2,3). In contrast, label-free Raman imaging offers intrinsic chemical specificity (4), supporting applications ranging from biomolecular fingerprinting to cell metabolic mapping (5-8) and histopathological tissue characterization (9,10). Despite its label-free advantage and chemical specificity, Raman imaging remains fundamentally limited in both spatial resolution and imaging contrast due to inherently low signal throughput and weak intrinsic Raman contrast (11-13). Here, we introduce spatial-filtering nanoscopy (SFN), a physics-driven super-resolution strategy that achieves resolution enhancement through targeted signal purification instead of signal amplification, offering a conceptually distinct pathway beyond the diffraction limit. SFN synergistically integrates a sub-millimetre microsphere lens (SMML) with a standard confocal Raman microscope, harnessing two complementary physical effects: (i) the photonic redistribution effect (PRE), which narrows the lateral excitation profile, and (ii) the three-dimensional spatial filtering (3D-SFE), which effectively suppresses both lateral and axial background. We demonstrate SFN-enabled super-resolution Raman imaging of silicon nanostructures, intact cells, and tissue sections, achieving an effective lateral resolution of approximately 40 nm, and chemically resolving subcellular features including organelles and pseudopodia without exogenous labels. These findings establish SFN as a broadly generalizable hardware-based super-resolution strategy, readily deployable on standard confocal platforms and extensible across diverse optical imaging modalities.

Single-walled carbon nanotubes (SWCNTs) show promise for optical biosensing, imaging, and drug delivery, but turning them into safe, precision nanomedicine tools requires understanding how nanotube surface chemistry dictates recognition and processing by cells. Like other nanomaterials, carbon nanotubes acquire a biomolecular corona on contact with biological fluids, and corona identity is increasingly recognized as central to sensor performance and drug delivery efficacy. However, whether corona identity also governs the intracellular fate of carbon nanotubes remains largely unknown. Here, we show that the single-stranded DNA wrapping of (6,5)-enriched single-walled carbon nanotubes reprograms their protein corona, intracellular trafficking, and macrophage response. By profiling (AT)15, (GT)15, and (CT)15 wrapped SWCNTs, we show that the wrapping sequence programs both the protein corona and the resulting proteostatic stress on macrophages. Photoluminescence imaging and confocal Raman measurements reported that (AT)15 is internalized the most yet leaves the proteome and nanotube structure largely undisturbed, whereas (CT)15, taken up the least, undergoes the most aggressive intracellular degradation and drives the highest oxidative and proteostatic stress. Corona proteomics indicated that all three tested nanotubes form coronas with distinct functional identities that are responsible for divergent intracellular routes. Time-resolved intracellular proteomics combined with functional assays resolved how the host cell reorganizes its biomolecular complexity over time, including oxidative outputs, aside from a sequence-independent core response involving particle engagement, phagosomal sorting, and lysosomal processing. These findings provide mechanistic insight into nanomaterial-cell interactions and the wrapping sequence as a tunable, nucleotide-level design handle for controlling the intracellular fate of carbon nanomaterials, with potential implications for safe and effective nanomedicine platforms.

Precise drug delivery within anatomically complex tissues demands systems capable of both active navigation and deep tissue access, properties that have remained difficult to reconcile in existing nanocarriers and microrobots. Here we introduce Dynabots, a dynamic microrobotic assembly constructed from multifunctional nanoparticles covalently linked by thermally cleavable molecular connectors. This nanoparticle-rich architecture enables the integration of magnetic, imaging, and therapeutic components while preserving a high content of functional material. Collective assembly imparts enhanced magnetic responsiveness and maneuverability, enabling controlled navigation through tortuous biological environments. Upon exposure to mild thermal stimuli, the assemblies undergo programmed disassembly, releasing individual nanoparticles that can diffuse through tissue for localized therapeutic action. We establish the programmable transitions, biocompatibility, and therapeutic efficacy of this process across in vitro and in vivo models, including real-time fluoroscopic guidance within anatomically realistic phantoms and live rodent and porcine systems. By integrating magnetic control, reconfigurable architecture, and stimulus-triggered disassembly, Dynabots unite navigational precision with tissue permeability, providing a versatile platform for adaptive and deep-tissue drug delivery.

Subcellular thermal engineering provides a powerful approach for investigating and manipulating biological processes. However, existing subcellular heating platforms capable of combining spatially confined heating, quantitative thermometry and simultaneous imaging of cellular responses remain limited. We developed a quantitative nanoheater-thermometer (qNanoHT), a polymeric nanoparticle integrating a temperature-sensitive fluorescent, dye and a photothermal dye. qNanoHT determines local temperature from fluorescence lifetime using fluorescence lifetime imaging microscopy (FLIM), thereby reducing susceptibility to photobleaching, focal drift and variations in probe concentration compared with intensity-based methods. The platform enabled real-time measurement at a subcellular heat spot while the dynamics of heat shock factor 1 (HSF1) were monitored in living cells. Heating at a single intracellular site was sufficient to induce HSF1 foci. Foci induced by mild heating at approximately 38 {degrees}C dissolved after heating ceased, whereas those induced by stronger heating at approximately 41 {degrees}C persisted and were associated with caspase-3/7 activation and apoptosis. Notably, qNanoHT-mediated subcellular heating induced HSF1 foci at a lower measured temperature than uniform whole-cell heating approximately 38 {degrees}C versus 39 {degrees}C indicating that the spatial extent of heating influences the HSF1 activation threshold. qNanoHT therefore provides a quantitative platform for relating local intracellular temperature to cellular stress responses and subsequent cell fate.

Rapid DNA transport limits the temporal resolution of solid-state nanopore sensing. Here we show that breaking pore symmetry through geometric inclination produces exceptionally prolonged DNA residence without surface functionalization or external trapping. For 500-base-pair double-stranded (ds) DNA ,13 nm diameter SiNx pores in tilted membranes exhibit a distinct long-lived population with a mean dwell time of 11.1 seconds at 600 mV bias, exceeding previously reported dsDNA dwell times by at-least 4 orders of magnitude. Controlled pore tilting reproduces the population of prolonged events, whose prevalence and duration depend on ionic strength. TEM-EELS based thickness measurements exclude substantial membrane-thickness differences, while continuum and atomistic simulations support a mechanism coupling electric-field redistribution to off-axis DNA motion, increased pore-wall contact and suppressed axial mobility. These findings establish geometric inclination as a passive design variable for engineering molecular residence in solid-state nanopores and provide a route towards longer observation windows for single-molecule sensing.

Efficient intracellular delivery of neoantigen-encoded mRNAs is central to the development of effective cancer vaccines and immunotherapies. However, how the nanoscale geometry of delivery carriers impacts mRNA delivery remains poorly understood, largely because conventional delivery platforms do not readily permit systematic variation of carrier architecture while maintaining comparable chemical composition. Here, we develop a modular RNA-DNA hybrid nanoplatform that enables controlled investigation of geometry-dependent mRNA delivery. Guided by a unified design principle, we construct a series of two- and three-dimensional nanostructures with programmable architectures and systematically compare their delivery performance using consistent functional readouts. We further investigate how mRNA embedding strategies and aptamer-mediated functionalization influence intracellular delivery efficiency. By providing precise control over carrier geometry, this platform establishes a framework for elucidating structure-function relationships in mRNA delivery and offers a versatile strategy for engineering targeted nucleic acid delivery systems.

Electroactive bacteria, like Shewanella oneidensis, can couple the oxidation of organic electron donors to the reduction of external conductive surfaces, such as minerals and electrodes, by utilizing multiheme cytochromes to carry charge from within the cell to external surfaces. Additionally, multiheme cytochromes facilitate gateable, long-distance (micrometer-scale) redox conduction along the outer membrane and across multiple cells bridging electrodes. While electroactive microbes are being used to develop bioelectrochemical devices, there have been limited efforts to use synthetic biology to exert additional control over microbes serving as device components. Thus, this work implements an optogenetic biofilm patterning gene circuit and a small molecule sensor in S. oneidensis to simultaneously control cell deposition and cytochrome expression. This allows for photolithographic patterning of biofilms possessing tunable electrical properties controlled with small molecules. This system demonstrates tunable electrochemical activity, redox conduction, intrinsic biofilm conductivity, and negative differential transconductance as a function of cytochrome expression. Additionally, temperature-dependent measurements of this tunable biofilm conduction reveal changes in activation energy as a function of cytochrome expression. Through this combination of synthetic biology and electrochemistry, simultaneous control over biofilm geometry and conductivity sheds light on fundamental microbial electron transport processes, and it enables the construction of living electronic devices.

Living cells are highly dynamic and densely crowded environments in which organelles such as vesicles undergo continuous motion that is essential for cellular processes. Therefore, accurate tracking of individual organelles is crucial for understanding intercellular dynamics and functions. However, precise tracking of individual organelles in living cells remains challenging due to high organelle densities, frequent particle overlap, and the coexistence of stationary and motile organelles. In particular, stationary organelles can obscure the trajectories of moving organelles, leading to tracking errors and fragmented tracks. To overcome these challenges, we developed Multiple Particle Tracking via Velocity Filtering (MPT-vVF), an unbiased, semi-automated tracking framework that incorporates a mathematically derived velocity-filtering algorithm to selectively identify and track moving organelles with high accuracy in crowded intracellular environments. MPT-vVF integrates denoising, background subtraction, and a velocity-matching detection step that discriminates true particle motion from noise based on spatiotemporal continuity, followed by robust trajectory linking. We demonstrate that MPT-vVF can accurately resolve nanometer-scale displacements of immobilized beads, highlighting its high tracking precision. We also validate the robustness of MPT-vVF by quantifying the transport of brain-derived neurotrophic factor (BDNF)-mRFP-containing vesicles in living hippocampal neurons. Furthermore, MPT-vVF reveals that exposure to 50-nm nanoplastics impairs vesicular transport, reducing both travel length and speed of BDNF-containing vesicles in living neurons. These findings establish MPT-vVF as a powerful method for quantitative analysis of intracellular organelles in crowded living cells and suggest its broad application to biophysics, cell biology, and soft matter research.

DNA Double Strand Breaks (DSBs) threaten genomic stability, leading to cell death, chromosomal rearrangements, and cancer-driving mutations. Therefore, the effective repair mechanisms are essential for maintaining genomic stability and ensuring cellular survival across diverse organisms. At the core of this process lies chromatin integrity, which facilitates the local DNA conformational changes, regulating accessibility to repair proteins and other biomolecules. To deepen our understanding of the DNA damage response pathway, the local molecular mechanisms regulating the interplay between DNA damage formation and alterations in chromatin conformation must be investigated at the nanoscale level. Here, we integrated atomic force microscope-infrared spectroscopy (AFM-IR) and confocal fluorescence microscopy to explore local chemical modifications in DNA structure and chromatin integrity in metaphase chromosomes and chromosomal aberrations isolated from cells treated with the chemotherapeutic agent, bleomycin. Our findings reveal changes in secondary protein structures, indicating the engagement of DNA repair proteins with high {beta}-sheet content. Nanospectroscopic mapping resolved the alterations in DNA condensation along the chromosomes. Moreover, we observed global DNA demethylation, particularly the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), correlating with increased DSBs. We conclude that these transitions in protein conformation and DNA methylation correlate with chromatin relaxation and enhanced accessibility for the repair protein.

DNA tetrahedra (DNA Td) are promising nanocarriers for drug delivery, but how hypoxia affects their cellular internalisation remains poorly understood. We synthesised and characterised Cy5-labelled DNA Td and established chemical hypoxia; in HeLa, MDA-MB-231, and MCF-7 cells. Hypoxia was confirmed by HIF-1; nuclear translocation. Confocal microscopy revealed significantly reduced DNA Td uptake under hypoxia, whereas transferrin and cholera toxin B uptake increased, indicating cargo-selective regulation. Temperature-arrest experiments confirmed reduced energy-dependent internalisation. Pharmacological profiling showed a shift from clathrin-mediated and galectin/glycan-dependent pathways toward lipid raft/cholesterol-dependent uptake. Hypoxia increased plasma membrane electronegativity, suggesting a biophysical barrier to DNA Td uptake. Importantly, DOTMA complexation restored uptake to normoxic levels, identifying electrostatic repulsion as a key determinant. In zebrafish larvae, hypoxia significantly enhanced whole-larva DNA Td accumulation. These findings highlight surface charge engineering as a strategy for improving DNA nanostructure delivery under hypoxic conditions.

DNA-based technologies rely on short, transient hybridization events, but selecting sequences with desired kinetic properties remains largely empirical because hybridization kinetics are difficult to predict from sequence and slow to measure one sequence at a time. Here, we introduce SPARXS-Hyb, an implementation of SPARXS (Single-molecule Parallel Analysis for Rapid eXploration of Sequence space) for multiplexed sequence-resolved screening of DNA hybridization. Using a surface-immobilized docking-strand library and a quencher-labelled imager-strand library, we screened 128 different DNA sequences in a single kinetic measurement, exposing all sequences to identical experimental conditions. This multiplexed approach removes a major confounding factor of serial measurements, allowing sequence-dependent differences to be compared directly. The resulting dataset reveals sequence-dependent transient binding behaviours and enabled us to identify a sequence with which an order-of-magnitude higher sampling rate can be achieved in DNA-PAINT (DNA points accumulation for imaging in nanoscale topography), a super-resolution microscopy technique based on DNA hybridization. By enabling multiplexed screening across a sequence library, SPARXS-Hyb provides a route to kinetics-guided sequence selection for programmable transient interactions in DNA nanotechnology.

The nuclear pore complex (NPC) is a massive protein system that controls all nucleocytoplasmic transport via a dynamic network of intrinsically disordered proteins called FG-Nups. We developed an NPC-mimicking DNA origami nanostructure that self-assembles into a tetrameric, octagonal nanopore with an inner diameter that, through addition of DNA oligonucleotides, can be tuned in a user-defined fashion from 57 to 66 nm - mimicking the contracted and dilated states of NPCs. We demonstrated that these origami nanopores can dilate within minutes and recontract at slower timescales. Both contracted and dilated pores can be inserted into lipid bilayers, where they maintain their conformation and allow transmembrane transport of fluorescent proteins. With outer diameters up to 87 nm, these are, to our knowledge, the largest DNA origami nanopores inserted into lipid membranes. Functionalization of the pores with the FG-Nup Nsp1 reduces non-specific cargo diffusion across the lipid membrane while facilitating translocation of the transport receptor Kap95, demonstrating transport selectivity much like biological NPCs. The work establishes a size-tunable platform to dissect nuclear transport mechanisms, with potential applications for engineering programmable artificial channels.

Adhesion of cells is the key factor determining functioning of multicellular organisms. Viscoelastic properties of cells can be studied by multiple methods. However, attractiveness of cells or extracellular matrix without the elastic component (dispersive adhesion) is not accessible. We present an extension of force spectrometry technology: the Multivalent Adhesive Probe Atomic Force Microscopy (MAPA) that delivers dispersive adhesion maps of live cells and biosurfaces, and identifies differences unresolved by viscoelastic probing.

Endosomal escape remains the central bottleneck limiting lipid nanoparticle (LNP)-mediated mRNA delivery. In this study, we use giant unilamellar vesicles (GUVs) mimicking the endosomal membrane to directly visualize the interaction and membrane disruptions caused by two model LNPs constituted by two different ionizable lipids: LP01 and BiP-20 (a Branched ionizable Phospholipid) that are both benchmark ionizable lipids for in vivo gene editing in liver. Despite identical TNS-measured apparent pKa (6.6) for both lipids in our conditions, LP01 LNP showed maximal membrane engagement at pH 5.8, while BiP20 LNP required pH 5.1, demonstrating that bulk pKa alone cannot predict the pH at which functional membrane interaction occurs. Using confocal microscopy, we show that the LNP engagement with the endosomal membrane proceeds through first electrostatic attraction, clustering, membrane disruption by forming highly curved morphologies, and finally leading to the rupture of the whole endosomal mimicking membrane. Notably, the mRNA cargo is released only upon complete lysis of the membrane. This indicates that the membrane destabilization and cargo release are mechanistically distinct steps. These findings establish a cell-free platform for dissecting individual sub-steps of endosomal escape.

Molecular transport through a nanopore determines the information that can be recovered from a translocation signal, yet it remains difficult to control in conventional solid-state nanopores. Rapid translocation reduces the information content and fixed nanopore geometries limit the dimensionality of the signal. Here, we control molecular transport through the development of the pipette-elastomer interfacial nanopore (PEIN), a dynamically reconfigurable solid-state nanopore which addresses these limitations. A PEIN is formed by depressing a glass nanopipette into a soft elastomer, progressively constricting its aperture and enabling continuous control over aperture size, while retaining the simplicity and favourable noise characteristics of glass nanopipette sensing. Using the dynamic aperture size control, DNA velocities could be controlled over more than a twofold range, with dwell times two orders of magnitude greater than observed in glass nanopipettes. DNA-origami rulers further revealed a progressive reduction in polymer velocity during translocation, indicating that hydrodynamic drag alone is insufficient to model forces on the DNA polymer. Finally, by using single- and double-stranded DNA and gold nanoparticles as molecular standards, we demonstrate reversible, size-selective molecular gating with sub-nanometre control. These results establish the PEIN as an accessible platform for controlling molecular transport and probing the relationships among biopolymer conformation, nanoscale confinement and translocation dynamics.

Single-particle tracking is widely used to probe nanoscale dynamics in biological systems, yet most approaches rely exclusively on translational motion, overlooking rotational dynamics that offer complementary information about the local physical environment. Here, we present a simultaneous rotational and translational single-particle tracking approach using a vortex-engineered point spread function in a single detection channel. We validate this approach with static and freely diffusing nanorods, demonstrating accurate orientation recovery and quantitative agreement with theoretical predictions of rotational diffusion. Using a biomimetic lipid bilayer system, we show that translational and rotational diffusion exhibit distinct sensitivities to environmental perturbations, confirming that these two modalities capture complementary local environment information. Applying this framework to living HeLa cells, we show that combined translational and rotational diffusion signatures define distinct biophysical fingerprints of cytoplasmic and endocytic compartments and reveal compartment-specific responses to metabolic perturbation. Finally, time-resolved analysis of individual endocytic compartments uncovers dynamic changes in the local physical environment that are inaccessible to conventional translational tracking. By coupling translational and rotational readouts, this framework opens a new dimension for probing the physical organization and dynamics of living systems at the nanoscale.