Nano Letters

Developing insect cyborgs by integrating external components (optical, electrical or mechanical) with biological counterparts has a potential to offer elegant solutions for complex engineering problems.1 A key limiting step in the development of such biorobots arises at the nano-bio interface, i.e. between the organism and the nano implant that offers remote controllability.1,2 Often, invasive procedures are necessary that tend to severely compromise the navigation capabilities as well as the longevity of such biorobots. Therefore, we sought to develop a non-invasive solution using plasmonic nanostructures that can be photoexcited to generate heat with spatial and temporal control. We designed a nanotattoo using silk that can interface the plasmonic nanostructures with a biological tissue. Our results reveal that both structural and functional integrity of the biological tissues such as insect antenna, compound eyes and wings were preserved after the attachment of the nanotattoo. Finally, we demonstrate that insects with the plasmonic nanotattoos can be remote controlled using light and integrated with functional recognition elements to detect the chemical environment in the region of interest. In sum, we believe that the proposed technology will play a crucial role in the emerging fields of biorobotics and other nano-bio applications.

Atomic force microscopy (AFM) has demonstrated the ability to resolve single DNA molecules in liquid at a spatial resolution that is sufficient to visualize the double helix structure and variations therein. Such variations can be due to inherent configurational flexibility and may be related to, e.g., DNA sequence, ionic screening, supercoiling, or protein binding. These AFM experiments require DNA to be adhered to a solid and preferably flat support. For high-resolution, in-liquid AFM studies so far, such adhesion has commonly been achieved using Ni2+ ions to electrostatically bridge between the negatively charged DNA and a negatively charged, atomically flat mica surface, yet Ni2+ ions tend to cause precipitation of salts on the surface, increasing the risk of AFM tip contamination and increasing the corrugation of the support surface, making it harder to distinguish secondary DNA structure. Here, we report on a sample preparation protocol that, instead, relies on Co2+ ions to adhere DNA to mica. While the Co2+ is similarly effective as Ni2+ for facilitating DNA adsorption onto mica, it leads to significantly reduced salt precipitation with the potential to provide enhanced reproducibility in high-resolution DNA imaging by AFM. We expect this to substantially facilitate high-resolution AFM studies of DNA in aqueous solutions.

Atomic force microscopy (AFM) is a powerful technique for imaging molecules, macromolecular complexes, and nanoparticles with nanometer-resolution. However, AFM images are distorted by the shape of the tip used. These distortions can be corrected if the tip shape can be determined by scanning a sample with features sharper than the tip and higher than the object of interest. Here we present a 3D DNA origami structure as fiducial for tip reconstruction and image correction. Our fiducial is stable under a broad range of conditions and has sharp steps at different heights that enable reliable tip reconstruction from as few as ten fiducials. The DNA origami is readily co-deposited with biological and non-biological samples, achieves higher precision for the tip apex than polycrystalline samples, and dramatically improves the accuracy of the lateral dimensions determined from the images. Our fiducial thus enables accurate and precise AFM imaging for a broad range of applications.

Microtubules, a major component of the cytoskeleton consisting of tubulin dimers, are involved in various cellular functions, including forming axons and dendrites of neurons and retaining cell shapes by forming various accumulated superstructures such as bundles and doublets. Moreover, microtubule-accumulated structures like swarming microtubule assemblies are attractive components for dynamic materials, such as active matter and molecular robots. Thus, dynamic control of microtubule superstructures is an important topic. However, implementing stimulus-dependent control of superstructures remains challenging. This challenge can be resolved by developing designer protein approaches. We have previously developed a Tau-derived peptide (TP), which binds to the inner or outer surface of microtubules depending on the timing of the incubation. In this report, we designed the TP-fused photo-switchable protein Dronpa (TP-Dronpa) that reversibly photoconverts between monomeric and tetrameric states to photocontrol microtubule assemblies. The formation of microtubule superstructures, including bundles and doublets, was induced by tetrameric TP-Dronpa, whereas monomeric TP-Dronpa ensured that microtubules remained dispersed. Tetrameric TP-Dronpa also induced motile aster-like structures and swarming movement of microtubules on a kinesin-coated substrate. The formation/dissociation of these microtubule superstructures can be controlled by light irradiation. This system can generate and photocontrol various microtubule superstructures and provides an approach to facilitate the assembly of dynamic materials for various applications. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=131 SRC="FIGDIR/small/614838v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@fdcc92org.highwire.dtl.DTLVardef@81bf32org.highwire.dtl.DTLVardef@2f7f5eorg.highwire.dtl.DTLVardef@52fc9f_HPS_FORMAT_FIGEXP M_FIG C_FIG

3D single-particle tracking has the potential to resolve the molecular level forces which dictate particle motion in biological systems. However, the information gleaned from 3D single-particle tracking often cannot resolve underlying nanoscale potentials due to limited spatiotemporal resolution. To this end, we introduce an active-feedback 3D tracking microscope that utilizes silver nanoparticles (AgNPs) as probes to study intricate biophysical events in live cells at the nanometer and microsecond scales. Due to this extremely high and durable scattering photon flux of the plasmonic particles, 1 MHz sampling frequency at nanometer precision in all three dimensions can be achieved over an unlimited observation times. In this work, we applied microsecond-sampling, active-feedback 3D single-particle tracking to investigate the interaction between AgNPs and nanoscale filopodium on the live-cell surface. The nanometer precision and microsecond sampling revealed that TAT peptide modified particles visit and dwell at local "hot spots" on the filopodium surface. The high sampling rate further enabled the calculation of the local forces and potentials within these nanoscale hotspots on the cylindrical surface of live cell filopodia. This study presents a promising tool to investigate intracellular biophysical events with unprecedented spatiotemporal resolution and a pipeline to study nanoscale potentials on three-dimensional cellular structures.

DNA nanotechnology relies on programmable anchoring of regions of single-stranded DNA through base pair hybridization to create nanoscale objects such as polyhedra, tubes, sheets, and other desired shapes. Recent work from our lab measured the energetics of base-stacking interactions and suggested that terminal stacking interactions between two adjacent strands could be an additional design parameter for DNA nanotechnology. Here, we explore that idea by creating DNA tetrahedra held together with sticky ends that contain identical base pairing interactions but different terminal stacking interactions. Testing all 16 possible combinations, we found that the melting temperature of DNA tetrahedra varied by up to 10 {degrees}C from altering a single base stack in the design. These results can inform stacking design to control DNA tetrahedra stability in a substantial and predictable way. To that end, we show that a 4 bp sticky end with weak terminal stacking does not form stable tetrahedra, while strengthening the stacks confers high stability with a 46.8 {+/-} 1.2 {degrees}C melting temperature, comparable to a 6 bp sticky end with weak stacking (49.7 {+/-} 2.9 {degrees}C). The results likely apply to other types of DNA nanostructures and suggest that terminal stacking interactions play an integral role in formation and stability of DNA nanostructures.

Protein-protein complexes can vary in mechanical stability depending on the direction from which force is applied. Here we investigated the anisotropic mechanical stability of a molecular complex between a therapeutic non-immunoglobulin scaffold called Affibody and the extracellular domain of the immune checkpoint protein PD-L1. We used a combination of single-molecule AFM force spectroscopy (AFM-SMFS) with bioorthogonal clickable peptide handles, shear stress bead adhesion assays, molecular modeling, and steered molecular dynamics (SMD) simulations to understand the pulling point dependency of mechanostability of the Affibody:(PD-L1) complex. We observed diverse mechanical responses depending on the anchor point. For example, pulling from residue #22 on Affibody generated an intermediate unfolding event attributed to partial unfolding of PD-L1, while pulling from Affibodys N-terminus generated force-activated catch bond behavior. We found that pulling from residue #22 or #47 on Affibody generated the highest rupture forces, with the complex breaking at up to [~] 190 pN under loading rates of [~]104-105 pN/sec, representing a [~]4-fold increase in mechanostability as compared with low force N-terminal pulling. SMD simulations provided consistent tendencies in rupture forces, and through visualization of force propagation networks provided mechanistic insights. These results demonstrate how mechanostability of therapeutic protein-protein interfaces can be controlled by informed selection of anchor points within molecules, with implications for optimal bioconjugation strategies in drug delivery vehicles.

Scanning ion conductance microscopy (SICM) provides gentle, non-contact cell surface imaging, but it has not been used to investigate intracellular structures because the plasma membrane restricts nanopipette access. Here, we combined SICM with microsonication-based cell de-roofing to expose intracellular actin stress fibers (SFs) in U2OS cells for nanotopographical and -mechanical characterization. Importantly, the de-roofing conditions preserved actomyosin contractility, allowing analysis of SF structural and biomechanical changes during ATP-induced contraction. Resting SFs displayed an average height of 203{+/-}38 nm and width of 357{+/-}73 nm, and a complex surface architecture characterized by regularly spaced long-range height modulations (~500 nm periodicity; Wq ~25 nm) and smaller irregular corrugations (Ra ~19.2 nm). ATP stimulation reduced SF height and width by ~39% and ~15%, respectively, while largely preserving surface corrugation patterns. During contraction, some SFs separated into two longitudinal strands. High-resolution SICM imaging also revealed filamentous crosslinks mechanically coupling neighboring SFs, and nanomechanical measurements demonstrated local stiffening during contraction. These findings provide new insight into the structural and mechanical regulation of SF contraction and highlight the potential of SICM combined with cell de-roofing as a powerful platform for studying dynamic intracellular processes at nanometer resolution.

Nanoparticles are crucial in diverse fields such as healthcare, electronics, and energy. Their small size allows them to cross biological barriers, enhancing drug delivery but also posing health risks. Accurate characterization of nanoparticles is essential for assessing their safety and efficacy, particularly in medical applications. Traditional methods such as dynamic light scattering and mass spectrometry have limitations in sensitivity and range of application. To address these challenges, we introduce the interferometric Concentration Analyzer and Ultrasmall Nanoparticle Tracker (iCAUNT), a technique for detecting and quantifying nanoparticles smaller than one-tenth of the imaging light wavelength. As a non-invasive method, iCAUNT provides precise size and concentration measurements of biological and synthetic nanoparticles, offering significant potential for diagnostics, therapeutics, and broader nanoscience applications.

The protein corona influences the in vivo biodistribution of ionizable lipid nanoparticles (LNPs) in nucleic acid delivery, yet its structural architecture remains poorly defined. Using cryo-transmission electron microscopy, we visualized LNP-protein interactions in their native state. We show that, unlike the discrete "fuzzy" shells observed on hard nanoparticles, LNPs displayed no peripheral protein shell. Instead, controlled incubation and competitive "dual-particle" assays, supported by molecular dynamics simulations, indicate that LNP membranes undergo localized thickening and electron-dense remodeling consistent with lipoprotein integration rather than surface adsorption. Similar features were observed in extracellular vesicles, suggesting this behavior is shared among lipid-based carriers, and proteomic analysis identified apolipoproteins as the dominant associated proteins. Together, these findings support a model in which the biological identity of LNPs arises through membrane remodeling rather than shell-like adsorption, and provide a framework for the rational design of targeted nanomedicines. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=138 SRC="FIGDIR/small/695162v2_ufig1.gif" ALT="Figure 1"> View larger version (79K): org.highwire.dtl.DTLVardef@5275d3org.highwire.dtl.DTLVardef@1b59ae4org.highwire.dtl.DTLVardef@1cc290eorg.highwire.dtl.DTLVardef@9b7bfb_HPS_FORMAT_FIGEXP M_FIG C_FIG

Direct validation of the nanoscale structural organization of membrane proteins requires localization precision that matches their molecular dimensions. The sodium-potassium pump, or the Na,K-ATPase is an integral membrane protein responsible for maintaining electrochemical gradients and cellular energy homeostasis. Although its crystal structure is characterized, the organization of the Na,K-ATPase within native plasma membranes, particularly whether it forms functional oligomers, remains an open question. Here, we combined 3D MINFLUX nanoscopy with DNA-PAINT with sub-10 nm localization precision to map the clustering topology of the Na,K-ATPase in mammalian cells. By targeting EGFP-tagged Na,K-ATPase 1 and {beta}1 subunits using anti-GFP nanobodies, we obtained high-density 3D localization maps of the protein in the plasma membrane. To evaluate the point patterns, we developed a computational data-driven spatial point assignment approach that segments apical and basal localizations, mitigating clustering artifacts produced by imaging two membranes in close proximity. Furthermore, we used a spatial statistical approach analyzing sequential nearest-neighbour distances to elucidate supramolecular arrangement information. Our data reveal a preferential nearest-neighbour distance of approximately 7 nm, providing direct visual confirmation of Na,K-ATPase dimerization. Additionally, we identified higher-order nanoclusters composed of up to 21 proteins. These findings provide definitive structural evidence of the dimeric configuration of Na,K-ATPase, establishing a foundation for future research on the functional and regulatory implications of Na,K-ATPase clustering.

Noninvasive control of axonal cargos in live neurons is a challenging prospect that can enable novel research on the mechanisms of axonal cargo transport, cargo-mediated signaling and axonal traffic jams in neurons. However, conventional techniques for force manipulation such as optical traps are limited to a few micron-sized cargos and are not applicable to the small axonal cargos in live neurons. Here, we present a new methodology that permits the external control of axonal endosome transport via tailored magnetic forces. By culturing neurons in a microfluidic device made up of microfabricated magnetic arrays, we can exert 3 - 48 pN forces on retrograde axonal endosomes carrying fluorescent magnetic nanoparticles, 100 - 260 nm in size. The magnetic force counters the forces exerted by molecular motors driving the endosomes and results in a wide range of perturbations on endosome transport in axons. These perturbations, captured by oblique illumination fluorescence imaging, reveal new insights on the collective function of dyneins and the nature of paused and stationary states during retrograde endosome transport in axons. Most notably, we demonstrate controllable capture and release of retrograde endosomes in axons by toggling the external magnetic field. This technical advance has great potential to elucidate the spatiotemporal origins of long-distance endosome signaling pathways as well as the ramifications of axonal traffic jams in neurons.

The advancement of synthetic cells as drug delivery devices hinges on the development of targeting strategies, in particular the controlled synthesis of biomolecules in-situ using a deeply penetrative stimulus. To address this, we have designed spherical nucleic acids comprising DNA promoter sequences decorating magnetic nanoparticle cores. By harnessing the heat dissipated from magnetic hyperthermia (a clinically-approved anticancer therapy) we tightly controlled cell-free protein synthesis. We then deployed a tissue phantom that is impenetrable by current activation methods to demonstrate the potential of this technology for the remote control of synthetic cells using deeply tissue-penetrating magnetic fields. This paves the way for targeting and controlling the in-situ synthesis of biomolecules deep within the body. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=181 SRC="FIGDIR/small/608917v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@3cf352org.highwire.dtl.DTLVardef@18a8da3org.highwire.dtl.DTLVardef@159018aorg.highwire.dtl.DTLVardef@744529_HPS_FORMAT_FIGEXP M_FIG C_FIG

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.

DNA-Points Accumulation for Imaging in Nanoscale Topography (DNA-PAINT) enables multiplexed super-resolution imaging of biological samples. We expand the repertoire of speed-optimized DNA sequences for DNA-PAINT imaging to drive visualization of as many as twelve targets in a sequential manner with molecular resolution. By implementing Exchange-PAINT protocol, we demonstrate 12-plex super-resolved imaging of docking strand patterned DNA origami nanostructures within four hours with a localization precision of 3 to 5 nm. Using these sequences, we demonstrate 9-plex super-resolution imaging of diverse nuclear targets within four hours. Further, we present a comprehensive analysis pipeline to quantify nanoscale chromatin in single cells. The combination of multiplexed imaging and analysis pipeline enabled us to reveal the loss of chromatin contacts with nuclear speckles upon global transcription inhibition. This work highlights the versatility of our approach to simultaneously image multiple targets at accelerated speeds while maintaining precise spatial localization for each target, enabling in depth mapping of the nuclear landscape. These speed-optimized imager sequences for high multiplexed super-resolution imaging will drive its further adoption for diverse cellular imaging applications.

Rab-GTPases and their interacting partners are key regulators of secretory vesicle trafficking, docking, and fusion to the plasma membrane in neurons and neuroendocrine cells. Where and how these proteins are positioned and organized with respect to the vesicle and plasma membrane are unknown. Here, we use correlative super-resolution light and platinum replica electron microscopy to map Rab-GTPases (Rab27a and Rab3a) and their effectors (Granuphilin-a, Rabphilin3a, and Rim2) at the nanoscale in 2D. Next, we develop a targetable genetically-encoded electron microscopy labeling method that uses histidine based affinity-tags and metal-binding gold-nanoparticles to determine the axial location of exocytic proteins using electron tomography. Our data show that Rab-GTPases and their effectors are distributed across the entire surface of individual docked vesicles. This circumferential distribution likely aids in the efficient transport, capture, docking, and rapid fusion of vesicles in excitable cells. The nanoscale molecular model of dense core vesicles generated from our methods reveals how key proteins assemble at the plasma membrane to regulate membrane trafficking and exocytosis.

Optical heating coupled with near-infrared (NIR) light and photothermal materials enables thermal confinement within biospecimens, minimizing undesirable thermal damage. Here, we demonstrated that photothermally heating lipid bilayers embedded with a unique phthalocyanine dye (VPc) efficiently perturbs the bilayers, resulting in increased permeability. Notably, microscopic studies revealed that the mechanism causing changes in membrane permeability may not follow the conventional temperature-sensitive liposome model. Furthermore, the heat generated by NIR laser illumination rarely diffused into the surrounding environment, and the dye was located within the bilayers at the molecular level, where it effectively transferred heat to the lipid bilayer. We prepared VPc-embedded liposomes encapsulating acetylcholine (ACh) and demonstrated the NIR laser-triggered release of ACh, creating a concentration jump across a few cells or within a limited single cell region. This method induced Ca2+ flux through ACh receptor stimulation in thermally delicate biospecimens such as C2C12 myotubes and the Drosophila brain.

Nanopore electroporation (NanoEP) is an emerging transfection method that enables efficient and safe intracellular delivery and removal of biomolecular cargo for applications in disease modeling, tissue engineering, and therapeutic biologics manufacturing. Conventional device designs assume uniform vertical cargo flux across nanoporous membranes; however, we demonstrate that the lateral electric field distributions introduce a pronounced edge effect, with enhanced cargo delivery and depletion along the membrane perimeters. We identify and characterize the presence of this edge effect in NanoEP systems, and develop a modified Nernst-Planck model to guide the design of membrane geometries that either promote delivery uniformity or create prescribed spatial gradients within cell monolayers. By varying the internal angles formed by the membrane edges (60{degrees}C, 90{degrees}C, 120{degrees}C), we create predictable intracellular cargo gradients, while concave "serpentine" geometries with high perimeter-to-area ratios amplify delivery efficiency and minimize spatial heterogeneity compared to circular membranes. These findings establish membrane geometry as a tunable design parameter in NanoEP, enabling control over both uniform and patterned intracellular payload delivery or depletion. This geometric design principle offers a scalable strategy for next-generation transfection platforms and synthetic tissue constructs.

DNA has shown great biocompatibility, programmable mechanical properties, and structural addressability at the nanometer scale, making it a versatile material for building high precision nanorobotics for biomedical applications. Herein, we present design principle, synthesis, and characterization of a DNA nanorobotic hand, called the "NanoGripper", that contains a palm and four bendable fingers as inspired by human hands, bird claws, and bacteriophages evolved in nature. Each NanoGripper finger has three phalanges connected by two flexible and rotatable joints that are bendable in response to binding to other entities. Functions of the NanoGripper have been enabled and driven by the interactions between moieties attached to the fingers and their binding partners. We showcase that the NanoGripper can be engineered to interact with and capture various objects with different dimensions, including gold nanoparticles, gold NanoUrchins, and SARS-CoV-2 virions. When carrying multiple DNA aptamer nanoswitches programmed to generate fluorescent signal enhanced on a photonic crystal platform, the NanoGripper functions as a sensitive viral biosensor that detects intact SARS-CoV-2 virions in human saliva with a limit of detection of [~] 100 copies/mL, providing RT-PCR equivalent sensitivity. Additionally, we use confocal microscopy to visualize how the NanoGripper-aptamer complex can effectively block viral entry into the host cells, indicating the viral inhibition. In summary, we report the design, synthesis, and characterization of a complex nanomachine that can be readily tailored for specific applications. The study highlights a path toward novel, feasible, and efficient solutions for the diagnosis and therapy of other diseases such as HIV and influenza. One-sentence summaryDesign, synthesis, characterization, and functional showcase of a human-hand like designer DNA nanobot

Homopolymerization and cluster formation of cellular membrane receptors (MR) is closely related to their signaling activity. However, underlying mechanisms and effects of clustering are often hardly understood. This lack of knowledge is due to the lack of suitable tools which enable to specifically target and activate distinct MRs, without causing side-effects. In this study, we designed a fluorescent semisynthetic nanoparticle (NP) based on the iron-storage protein ferritin and S. aureus Protein A, that is readily equipped with a variety of antibodies with KD values below 5 nM. Specificity of the NP antigen recognition was evaluated in cell experiments with cells expressing Transferrin Receptor 1 or the death receptor CD95, both of which displayed rapid cluster formation upon contact with the NP. Lastly, it was possible to induce apoptosis solely by induced clustering of CD95 via our engineered NP.