Nano Letters

Spatial organization and temporal regulation of membrane components are essential for achieving complex functions in artificial cells, such as cell division and signalling. DNA-based molecular tools provide a powerful means to control biomolecular interactions with high precision. Here, we investigate the phase behavior of cholesterol-modified, star-shaped DNA nanomotifs anchored to the lipid bilayers of giant unilamellar vesicles (GUVs), by using fluorescence confocal microscopy and cryo-electron microscopy. These motifs spontaneously anchor to the lipid bilayers via hydrophobic interactions and exhibit distinct spatial organization depending on their sticky end sequences. Motifs with complementary sticky end sequences interact and distribute uniformly, while orthogonal motifs with different sticky end sequences segregate into isolated gel-like domains with limited lateral mobility. Notably, the phase separation of motifs does not require lipid phase separation, indicating that DNA-driven organization can take place independently of lipid phase separation. The behavior of this system is governed by the interplay of three key parameters: (i) hydrophobic anchoring via cholesterol, (ii) electrostatic repulsion between negatively charged DNA nanomotifs, and (iii) sticky end interactions. The observed two-dimensional phase separation of orthogonal DNA nanomotifs at the GUV interface presents a novel strategy for controlling lateral membrane organization in GUV systems. This approach would offer flexibility in membrane composition and enables molecular positioning, thereby achieving a high degree of organization on the surface in artificial cell models.

Quantitative measurements performed directly in vivo are necessary to understand how forces shape living tissues, yet this remains challenging due to optical scattering and mechanical complexity. Here, we present a method for making absolute force measurements using nanoscopic optical tweezers with a sensitivity of 300 fN in optically turbid biological media. Our approach combines back focal plane interferometry operating within the optical memory effect regime with a global fluctuation-dissipation fitting framework that simultaneously calibrates position detection, trap stiffness, and viscoelastic response. This method overcomes aberration-induced biases by jointly fitting passive fluctuations and driven harmonic responses, enabling robust force reconstruction in thick, scattering tissues within the mechanically relevant frequency range below 300 Hz. We validate our approach using highly scattering Drosophila pupae and embryos, demonstrating reliable in vivo measurements of forces and mechanical properties. Operating at a 1 kHz acquisition bandwidth, the system captures relevant mechanical dynamics without requiring extended high-frequency detection. Using this framework, we quantify the increase in cortical tension during pupal morphogenesis, characterize tissue viscoelasticity, and reveal stage-dependent variations in nuclear membrane tension during embryogenesis, even in the presence of strong ATP-driven fluctuations. Beyond bulk measurements, our method enables the quantitative mechanical characterization of single cells within mechanically coupled tissues.

Biohybrid robots combining compliant synthetic support structures with biological actuators could enable future applications ranging from precision microsurgery to unmanned exploration. Machines actuated by living skeletal muscles are capable of adaptive behaviors, such as sensing and responding to environmental stimuli in real-time, offering functional advantages over non-biological actuators. However, typical skeletal muscle-powered biohybrid robots depend on 3D tissues which require large cell volumes and offer limited control of muscle fiber alignment, thus reducing efficiency of force generation and transduction. Here, we present a locomotive biohybrid robot powered by 2D monolayers, or thin films, of precisely aligned skeletal muscle fibers on a micropatterned hydrogel skeleton. We demonstrate how varying skeleton design parameters, ranging from material stiffness to microscale topology, impacts muscle fiber alignment and resultant actuation strains, generating forces 10X higher than previous 2D skeletal muscle actuators, improving untethered actuation longevity by [~]4500X from < 10 minutes to > 30 days, and increasing efficiency of muscle force output (force per unit volume of muscle) by 20X as compared to 3D muscles. Utilizing our optimized design for skeletal muscle thin films, we create a multi-limbed robot composed of independent muscle-powered fins capable of on/off control and frequency-dependent speed control. With these control inputs, we achieve steered multi-directional locomotion at speeds up to 4 body lengths per minute in straight movement and 1200 degrees per minute in rotational movement, highlighting potential for such actuators to be transformed into long-lasting functional soft robots.

Cathodoluminescence (CL) microscopy has the potential to achieve a key goal in biological imaging: the simultaneous visualization of proteins and cellular ultrastructure. This goal can be attained by tagging proteins of interest with spectrally distinct cathodoluminescent probes for detection in electron microscopy. To this end, lanthanide nanoparticles (LNPs) are promising probe candidates due to their stability under the electron beam and their distinct ion-dependent emission spectra suitable for multiplexed detection. However, the hydrophobic surface chemistry of LNPs limits their use in biological samples and requires surface functionalization compatible with aqueous environments and EM sample preparation protocols. Here, we use a DNA-based ligand exchange strategy that renders cathodoluminescent LNPs hydrophilic and compatible with further functionalization for specific protein labeling. We characterize the CL emission of DNA-functionalized LNPs following aqueous transfer and common EM preparation steps, including osmium tetroxide staining and drying protocols based on hexamethyldisilazane and critical point drying, and show that LNPs retain their CL emission under all tested conditions. Finally, we demonstrate multicolor CL imaging of spectrally distinct, DNA-functionalized LNPs on the surface of mammalian cells, enabling simultaneous visualization of cellular ultrastructure via secondary electrons and LNPs via multiple CL color channels.

Targeted lipid nanoparticles (tLNPs) represent the next frontier in nucleic acid therapeutics, enabling cell-specific delivery through covalent attachment of targeting ligands that drive receptor-mediated uptake. tLNPs are particularly promising for pregnancy-associated applications where precise on-target delivery is required to minimize maternal toxicity and protect fetal health. Yet, their rational design is limited by an incomplete understanding of how tLNP physicochemical properties influence biological performance. Conventional LNPs already exhibit pronounced heterogeneity in size, composition, and RNA loading, which is further amplified in tLNPs by variability in ligand attachment and surface density. Because traditional analytical methods report only ensemble-averaged properties, the nanoscale diversity of tLNPs remains unresolved. Here, we find that tLNP functional behavior is governed by previously inaccessible, structurally distinct tLNP subpopulations that are not captured by bulk measurements. We utilize asymmetric flow field-flow fractionation integrated with in-line UV spectral analysis, light scattering, and synchrotron small-angle X-ray scattering (AF4-UV-DLS-MALS-SAXS) to resolve ligand-dependent tLNP subpopulations that differ in size, shape, composition, and relative abundance. We find that protein conjugation preserves the internal lipid-RNA nanostructure of base LNPs but substantially increases particle heterogeneity, particularly for larger and multivalent targeting ligands. Despite increased heterogeneity, tLNPs functionalized with higher-avidity ligands achieve more effective targeted placental RNA delivery in mice, suggesting that binding avidity can offset the functional consequences of polydispersity. Chemometric SAXS analyses reveal that only SAXS-resolved tLNP subpopulations, not ensemble-averaged parameters, correlate with targeted placental transfection in vivo, whereas bulk-derived physicochemical metrics more strongly associate with nonspecific hepatic delivery. Together, this work harnesses a separation-coupled biophysical platform to resolve previously inaccessible tLNP subpopulations and demonstrates that subpopulation nanoscale structure, rather than bulk-averaged properties, dictates targeted RNA delivery. These insights provide a mechanistic foundation for rational engineering of next-generation precision targeted RNA LNP therapeutics.

Immune cell receptor - ligand interactions are key to cancer immunotherapy. However, receptor-ligand affinities often fail to predict T-cell mediated cancer killing, while immune-target cell binding strength measurements are limited by low precision and high non-specific binding. Here we present bilayer acoustic force spectroscopy (BAFS), a method to quantify the binding strength of receptors in immune synapses that virtually eliminates non-specific binding and increases the resolving power by up to 50-fold. By replacing target cells with a supported lipid bilayer functionalized with antigens, BAFS avoids antigen-independent interactions and target cell heterogeneity, while maintaining the spatial self-organization of receptors that typifies active immune synapses. We demonstrate the high sensitivity and control by showing how CAR T-cell synapse strength depends on CD19 antigen density, and by revealing that CD8 synergistically strengthens {beta}TCR-pMHC synapses independently of Lck recruitment to CD8. BAFS is a general method that can be used broadly in immunotherapy screening and to dissect the complex molecular interactions that underpin immune synapse activation.

Microtubules are central components of cytoskeletal transport systems and have been widely repurposed as active elements in motor-driven nanodevices. However, site-specific functionalization of stabilized microtubules remains a fundamental challenge, as the tubulin lattice presents chemically indistinguishable binding sites along its length. Here we report a strategy for selective end-functionalization of stabilized microtubules using DNA origami nanostructures. By coupling DNA origami to Fab fragments targeting acetylated -tubulin Lys40 within the microtubule lumen, and exploiting steric exclusion of the origami from the lattice interior, binding is confined to accessible sites at microtubule ends and lattice defects. Using a six-helix bundle origami as a minimal construct, we demonstrate selective tip labelling of gliding microtubules without perturbing kinesin-driven motility. The same structures additionally mark lattice defects, enabling dynamic visualization of defect sites during transport. Furthermore, we show that tip-bound origami can hybridize with complementary DNA strands to capture cargo from surfaces in motion, establishing programmable, end-specific loading. This approach introduces a generalizable route to spatially controlled functionalization of cytoskeletal filaments, enabling new capabilities in molecular transport, nanoscale assembly, and the study of microtubule integrity and repair.

Proteins in solution adsorb to the corona of nanoparticles such as single-walled carbon nanotubes (SWCNTs), but these interactions are difficult to predict and analyze due to ambiguities in the structure of the latter. In this work, we employ ss(GT)15-DNA wrapped SWCNTs, a commonly used fluorescent sensor construct, to examine protein adsorption by quantifying binding dissociation constants and characterizing the corresponding photophysical effects. A library of 20 proteins are used to evaluate adsorption-induced changes in photoluminescence (PL) intensity ({Delta}I/I0) and emission wavelength upon solution phase binding. We find that 15 proteins produce monotonic dose-response behavior well described using a single-site Langmuir model. Alternatively, five proteins exhibited more complex, non-monotonic behavior consistent with a two-step binding model representing protein-protein interactions coupled to adsorption. The study reveals that metalloproteins, which comprised 12 of the 20 proteins in the library, induced greater PL quenching compared with metal-free proteins for this system, with maximum binding-associated quenching ({Delta}I/I0) of 94% for metalloproteins versus 20% for metal-free proteins. For metalloproteins, we introduce a proximity-based quenching framework in which protein size provides a coarse proxy for cofactor-SWCNT separation, offering a mechanistic interpretation of the observed quenching variation across proteins. Together, these results establish the use of metal coordination sites, such as those in metalloproteins, to assist the transduction of certain nanoparticle fluorescent sensors, helping with sensor probe design and interpretation in biological environments.

The bottom-up construction of synthetic cells based on giant unilamellar vesicles (GUVs) is a central goal in synthetic biology. Achieving targeted changes in membrane and cytoplasmic composition with temporal control remains challenging however. DNA-mediated fusion with small vesicles ([~]100 nm large unilamellar vesicles; LUVs) has been proposed as a strategy to deliver lipids and cytosolic contents in a programmable manner. However, in vitro, membrane fusion is generally found to be inefficient and poorly controllable for reasons that are poorly understood. Here, we present an approach based on lipid-conjugated DNA (LiNA) to mediate programmable fusion between LUVs and micron-sized GUVs, which we quantitatively monitor with confocal microscopy at the single-GUV level. We show that lipid and content mixing both occur with high efficiency over a wide range of LiNA concentrations, demonstrating that LiNAs indeed induce robust membrane fusion. Furthermore, we show that LiNA-mediated fusion provides a powerful tool to deliver cytosolic biomolecules, enabling control over internal activities. Our findings establish a quantitative framework for studying fusion-driven processes in synthetic cells and provide a versatile platform for the programmable delivery of lipids and cytosolic cargoes - thus advancing the development of synthetic cells that can grow and adapt through fusion-based uptake of molecular building blocks.

Intratumoral (i.t.) delivery of nanoparticles (NPs) is widely used to achieve high local NP concentrations. However, the temporal fate of i.t.-injected NPs remains poorly understood. We present a quantitative approach using whole-body magnetic particle imaging (MPI) to track magnetic NPs (MNPs) following i.t. injection. Using fiducial-calibrated imaging, we quantified MNP mass over time in subcutaneous 4T1 breast tumors. Longitudinal imaging revealed progressive loss of i.t. MNP content and heterogeneous systemic redistribution across animals despite standardized delivery conditions. Ex vivo MPI confirmed off-target accumulation primarily in the liver and spleen, consistent with reticuloendothelial clearance pathways. Histological analysis demonstrated spatially heterogeneous i.t. MNP deposition, potentially associated with local vascular features and tumor microenvironmental heterogeneity that may influence i.t. MNP retention or MNP clearance from the tumor. These findings highlight the importance of quantitative longitudinal whole-body MPI for understanding the fate of MNPs for informing localized nanotherapy.

DNA double-crossover (DX) molecules, comprising two Holliday junctions connected by two duplex arms, are fundamental building blocks of DNA nanostructures, but their mechanical properties remain poorly understood. Here we investigate the elasticity of isolated antiparallel DX motifs with 18 to 22 base pairs between the crossovers. Using mechanical models parameterized by extensive all-atom molecular dynamics simulations, we demonstrate that the bending rigidity of the duplexes within a DX motif is highly anisotropic, and that this anisotropy results from long-range elastic couplings involving all the duplex base pairs between the crossovers. The duplex stretch modulus decreases due to localized defects, while the twist stiffness is close to that of an isolated duplex. The DX core as a whole follows an analytical beam theory in bending but not in torsion. Our results extend beyond local elastic models of DNA nanostructures and pave the way for probing peculiar mechanical properties of other key motifs for DNA and RNA nanotechnology.

Transport in complex biological tissues is governed by local rheological heterogeneity at the nanoscale, yet probing such environments deep inside living systems remains challenging. Here, we introduce an orientation-sensitive single-particle tracking (SPoT) approach that simultaneously resolves translational and rotational dynamics of individual carbon nanotubes deep within biological tissue. By exploiting the intrinsic dipole-like emission and shortwave infrared luminescence of carbon nanotubes enhanced through the incorporation of quantum color-centers our method enables long-duration tracking with high signal-to-noise ratio in optically dense environments. Crucially, the length of these nanotubes can be precisely shortened down to a few tens of nanometers to adapt to diffusion environmental dimensions, further optimizing the tracking applicability. SPoT of single carbon nanotubes provides access to relative changes in local viscosity, steric constraints, and environmental anisotropy. When applied to the brain extracellular space, SPoT demonstrates that local variations in the translational and rotational diffusion of tracers are heterogeneous and not systematically correlated. This allows to disentangle the local effects of viscosity and spatial tortuosity within the brain extracellular space, which are distinct features that would otherwise remain undetected through translational diffusion analysis alone. By enabling combined translational and rotational tracking of nano-emitters over unprecedented depths and timescales, this work establishes a new framework for probing nanoscale transport and rheological heterogeneity in intact biological tissues and more generally in complex diffusive environments.

Mechanical forces at the cell-substrate interface govern processes from migration to differentiation, yet mapping these forces at high spatial resolution remains challenging. Traction force microscopy (TFM) addresses this by quantifying substrate deformations using fiducial markers, which are conventionally fluorescent beads. Here, we introduce fluorescently labeled DNA nanostructures (FluoroCubes) as alternative fiducials grafted onto polydimethylsiloxane (PDMS) substrates. Co-anchored with RGD peptides, FluoroCubes remain stably tethered, resist internalization, and enable dense, minimally perturbing labeling. This surface-functionalized platform is compatible with TIRF microscopy and leverages tunable biotin-NeutrAvidin chemistry for precise control of fiducial density. Using a modified multi-channel optical flow algorithm, we achieve improved displacement sensitivity and force reconstruction resolution compared to conventional algorithms. FluoroCube-functionalized substrates provide a reproducible, high-resolution method for traction force mapping and offer a versatile foundation for future integration with DNA-based molecular sensors to probe interfacial forces at biointerfaces.

Epithelial ovarian cancer remains one of the most lethal malignancies among women, with late-stage diagnoses yielding 5-year survival rates below 30%. The metabolic heterogeneity of the tumor microenvironment (TME) highlights the need for methods capable of rapid, chemically specific phenotyping. Stimulated Raman scattering (SRS) microscopy when combined with deuterium labeled metabolites enables the non-invasive high contrast interrogation of cellular metabolic pathways. In this study, we used SRS microscopy to profile fatty acid and glycogen metabolism in epithelial ovarian cancer (SKOV-3) and cervical cancer (HeLa) cell models. Deuterium labeled glucose revealed striking differences in glycogen synthesis and intracellular distribution, with SKOV-3 cells exhibiting markedly greater single-cell heterogeneity than HeLa. Complementary measurements of lipid droplet (LD) synthesis and turnover under nutrient starvation further revealed cell-line-specific metabolic strategies, identifying LD and glycogen dynamics as a potential diagnostic marker of cancer metabolic phenotypes. These results demonstrate that SRS microscopy in the Raman silent region, paired with metabolic labeling, can sensitively resolve metabolic diversity across cancer cell subpopulations. Such metabolic phenotyping may inform both early diagnostic strategies and therapeutic approaches that combine cytotoxic treatment with targeted metabolic disruption.

A major limitation across nanoparticle delivery platforms is sequestration within endosomal compartments, which restricts access to intracellular targets despite efficient cellular uptake. Here, we show that peptide architecture can be used to control intracellular trafficking and reduce endosomal accumulation in lipid-protein nanocarriers. Specifically, we fuse R6W3 (RRWWRRWRR), an amphipathic cell penetrating peptide, to the N- or C- terminus of the nanodisc scaffold proteins and systematically evaluate its impact on membrane interactions and cellular behavior. Structural and biophysical characterization confirms that R6W3 incorporation preserves nanodisc assembly and protein-lipid interactions, enabling direct attribution of functional differences to peptide-driven interfacial effects. R6W3-functionalized nanodiscs exhibit enhanced binding and cellular uptake, with N-terminal fusion producing the strongest interfacial interactions. In live cells, R6W3-functionalization increases endocytic activity, evidenced by increased formation of clathrin-coated pits and intracellular colocalization with clathrin-coated vesicles. Notably, R6W3-funtionalized nanodiscs display reduced accumulation in early endosomes relative to unmodified nanodiscs, indicating decreased endosomal sequestration following endosomal uptake. These trafficking differences translate to functional outcomes, as doxorubicin-loaded, R6W3-functionalized nanodiscs achieve greater cytotoxicity than unmodified controls at equivalent concentrations. Together, these results establish peptide architecture as a design parameter for controlling intracellular trafficking and overcoming endosomal bottlenecks, providing a broadly applicable strategy for improving nanocarrier- based delivery systems.

Super-resolution microscopy with DNA-PAINT enables molecular-scale, multiplexed, and quantitative imaging, but its throughput is limited by slow binding kinetics and elevated background at high probe concentrations. Recent speed-optimized and fluorogenic probes improve performance but impose strong constraints on sequence design, revealing a fundamental tradeoff between fast binding and efficient quenching. Here, we introduce a modular probe architecture that spatially decouples binding kinetics from fluorophore-quencher interactions by integrating speed-optimized sequence motifs with PEG spacers. Using DNA origami nanostructures, we demonstrate enhanced localization rates, signal-to-background ratios, and imaging efficiency compared to state-of-the-art probes. We validate our approach in cells, demonstrating its capability to image nuclear targets and enabling three-dimensional imaging of the endoplasmic reticulum using standard widefield illumination. Our work establishes a general framework for fast, multiplexed, and low-background super-resolution imaging.

The organ-specific enrichment of drug delivery vehicles, such as lipid nanoparticles (LNPs), can be leveraged to concentrate drugs at disease sites to increase efficacy and limit toxicity. For immunostimulatory therapeutics, however, tissue accumulation beyond diseased sites may also shape drug activity by determining which organs and cell populations first sense the agonist and initiate downstream immune responses. Here, we show that the anticancer efficacy of an immunostimulatory duplex RNA (dsRNA) can be augmented using LNPs that are formulated to preferentially target the lung, which dictates the systemic pharmacodynamics of the cytokines it elicits. The immunostimulatory dsRNA was formulated into LNPs engineered for either enhanced liver-(LiverLNPs) or lung-(LungLNPs) based delivery, matched for size, encapsulation efficiency, and in vitro potency. In mice, delivery of dsRNA in LungLNPs enhanced uptake into endothelial, epithelial, and resident immune cells populations and induced substantially higher circulating levels of type I, type III interferons and proinflammatory cytokines than dsRNA formulated in LiverLNPs. This significant systemic response induced by lung-enhanced delivery required competent retinoic acid-inducible gene I and Toll-like receptor 7 signaling. Functionally, LNPs that preferentially targeted the lungs induced significantly greater suppression of tumor growth in both subcutaneous and metastatic models of melanoma. LungLNP/dsRNA also induced cytokine secretion and inhibited tumor cell proliferation in a human lung cancer-on-a-chip model. Together, these results establish that pulmonary exposure can alter systemic pharmacodynamics and therapeutic activity of immunostimulatory RNA.

Self-assembled DNA nanostructures show great promise as functional devices, highly configurable materials, and in nanorobotics. Magnetic control can provide a powerful actuation mechanism in a broad range of contexts, since it affords a high-level of external control, it is biocompatible, and orthogonal to chemical or electrical stimuli. Here we demonstrate magnetic molecular nanoactuators by leveraging the unique site-specificity of DNA origami to assemble highly anisotropic magnetic nanocubes on high-aspect ratio DNA origami bundles. We traced and controlled 100s of our DNA origami nanorotors at the single-rotor level and demonstrated their programmable magnetic clamping and controlled rotation under uniform and rotating magnetic fields. By varying the population and inter-particle spacing of the nanocubes, magnetic torque values in the order of 10-100 pN nm are achieved at field strengths < 10 mT. Monte Carlo simulations reveal that assembly of nanocubes on DNA origami rotors leads to collective magnetic properties, with numerically estimated torque values in good agreement with the experiments. Our magnetic nanorotors offer a foundation for biocompatible nanorobotics, as well as high-throughput magnetic force and torque tweezers.

Continuous biochemical sensing provides valuable insights into an individuals physiological state and the mechanisms underlying pathophysiological changes. However, most existing bioanalytical methods are not compatible with continuous biochemical sensing. A major technical challenge lies in achieving rapid measurement readouts while maintaining high specificity and sensitivity in complex biological fluids. Sensitive molecular detection typically requires slow analyte-binder dissociation and long incubation to reach equilibrium, whereas rapid and frequent measurements demand fast association-dissociation kinetics that are difficult to reconcile for low-abundance analytes. To address this challenge, we introduce a sensing mechanism termed photothermal recycling (PTR), which mimics the thermal cycling process in polymerase chain reaction. Using plasmonic photothermal effects, PTR rapidly recycles binders to enable frequent measurements. We demonstrate a digital PTR assay capable of multi-hour biochemical monitoring with subpicomolar(pM) sensitivity in buffer, diluted serum, and saliva. This approach leverages localized thermal energy to dynamically modulate biomolecular recognition, offering a new bioanalytical paradigm for continuous biochemical sensing across diverse application settings.

Tracking immune cells deep within living tissue remains a fundamental challenge due to the diffraction-limited resolution of ultrasound imaging and the inability to resolve dense cellular populations. Here, we introduce an intracellular super-resolution ultrasound imaging framework based on stochastic phase-changing nanodroplets (NDs) and ultrasound localization microscopy (ULM). We engineer [~]170 nm perfluorocarbon NDs that undergo reversible, stochastic liquid-gas transitions under acoustic excitation, generating temporally sparse "blinking" signals. Leveraging the intrinsic endocytic activity of macrophages, these NDs are internalized, enabling intracellular contrast generation independent of vascular flow. We validate this approach across imaging scales, from controlled phantoms and in vitro systems to in vivo tumor models, demonstrating robust intracellular blinking, high cell viability, and consistent super-resolution reconstruction in dense cellular environments. The stochastic blinking of internalized NDs provides the temporal separation required to localize individual sources, overcoming a central limitation of conventional ULM. Following systemic administration, ND-labeled macrophages are tracked in vivo after homing to the liver, where super-resolution ULM resolves cellular distributions with a spatial resolution of 26.3 {+/-} 3.2 {micro}m, corresponding to a 6.1-fold improvement over diffraction-limited imaging. This work establishes a previously unexplored paradigm for ultrasound-based intracellular super-resolution imaging, enabling non-invasive visualization of immune cell organization in deep tissue. By introducing spatiotemporally programmable intracellular contrast, this approach expands ultrasound beyond vascular imaging toward functional cellular imaging, with broad implications for immunology, diagnostics, and cell-based therapies.