Nanoscale

We studied the friction dynamics of icosahedral viruses adsorbed to solid surfaces to probe their adhesion. Using the lateral torsion of cantilevers in atomic force microscopy to move individual capsids in a liquid environment, we found that the virions tend to roll rather than slide on the surface. In contrast, rigid, ligand-stabilized gold nanoparticles are more likely to combine rolling with sliding under the same conditions. The experiments indicate that the force required to drag the viruses on the surface is four times less than that of AuNPs, while the lateral force work needed to induce virus movement was [~] 104 kT, ten times less than that of the rigid gold nanoparticles. These results go beyond the paradigm that adhesion of nanoparticles is mainly governed by geometrical factors, such as size and area of contact, highlighting the need to amend modeling approaches to account for mechanically-compliant tribological response of biologically derived nanoparticles.

We designed a strategy, based on a careful examination of the activation capabilities of proteins and antibodies used as substrates for adhering T cells, coupled to protein microstamping. This allowed us to control at the same time the position, shape, mechanics and activation state of T cells. Once adhered on shaped patterns we examined the capacities of T cells to be activated with soluble aCD3, in comparison to T cells adhered to a continuously decorated substrate with the same density of ligands. We show that, in our hand, adhering onto an anti CD45 (aCD45) antibody decorated surface is not affecting T cell calcium fluxes, even adhered on variable size micro-patterns. We further demonstrate this by expressing MEGF10 as a non immune adhesion receptor in T cells to obtain the very same spreading area on PLL substrates and Young modulus than immobilized cells on aCD45, while retaining similar activation capabilities using soluble aCD3 or through model APC contacts. We propose that our system is a way to test activation or anergy of T cells with defined adhesion and mechanical characteristics, and may allow to dissect fine details of these mechanisms since it allows to observe homogenised populations in standardized T cell activation assays.

The catch bond complex between serine-aspartate repeat protein G (SdrG) from Staphylococcus epidermidis and the beta chain of fibrinogen (Fg{beta}) exhibits two distinct rupture populations when dissociated under tensile force. Such complexes present exciting possibilities for developing dynamic biomaterials due to their unique response to shear force. However, the environmental responsiveness of this complex and its influence on adhesion behaviour in multi-valent systems remain underexplored. Using AFM-single molecule force spectroscopy (AFM-SMFS) and spinning disk adhesion (SDA) assays, we examined how protein orientation, mutations, and environment influence the stability and scaling behaviour of this catch bond system. Our findings confirmed that anchor point location (i.e., the direction from which the protein is pulled) strongly influences catch bond behaviour, while an S338H mutation in the binding domain destabilised the interaction in both single-molecule and multi-valent adhesion assays. This research examines how catch bond behaviour translates from the nanoscale to microscale using single molecule and multi-valent cell adhesion measurements and provides a toolkit for exploiting catch bonds towards macroscale material applications.

The human cathelicidin host defense peptide LL-37 forms complexes with nucleic acids that can have either beneficial or detrimental health effects. We suggest that these differential impacts are directly connected to dsDNA binding by LL-37 and to complex formation between protomers. Here, we show using phage {lambda} DNA that LL-37 binds non-specifically to dsDNA, condensing it, followed by complex formation between LL-37 peptides. We find that complex formation is concentration-dependent, with low LL-37 amounts yielding loosely aggregated DNA structures, while higher LL-37 concentrations lead to well-defined, disc-like structures of about 150 nm in diameter. The condensation of the nucleic acids, which causes a loss of the characteristic B-DNA features, results from interactions of the phosphodiester backbone with protonated amino acid side chains of the peptide at physiological pH, predominantly in A-T rich sequences of the nucleic acid. However, in our studies, electrostatic interactions did not appear to be the driving force for complexation, but rather we found the -helical structure of the peptide with its amphipathic and hydrophobic surfaces to be essential. Further, we show that LL-37 also interacts with nucleic acids from neutrophil extracellular traps (NETs) in a concentration-dependent way, causing a reduction in NET aggregate area, which may offer new biophysical insights into diseases such as systemic lupus erythematosus (SLE), which involve slower-than-normal NET clearance. Our results indicate the key importance of LL-37 expression levels for regulation of the innate immune system for optimal human health, since the relative amounts of expressed LL-37 present to interact with extracellular DNA will determine the extent to which the DNA can be condensed, which in turn will affect the ability of the body to clear the NETs before they can cause inflammatory conditions.

Targeting the cell nucleus remains a challenge for drug delivery. Here we present a universal platform for smart design of nano-particles (NPs) decoration that allows recruitment of multiple dynein motors to drive their active motion towards the nucleus. The uniqueness of our approach is based on using: (i) a spacer polymer, commonly Biotin-Polyethylene-glycol-thiol (B-PEG-SH), whose grafting density and molecular weight can be tuned thereby allowing NP transport optimization, and (ii) protein binding peptides, like cell penetrating, NLS, or cancer targeting, peptides. Universal chemistry is employed to link peptides to the PEG free-end. To manifest our platform, we use a SV40T large antigen-originating NLS peptide. Our modular design allows tuning the number of recruited motors, and to replace the NLS by a variety of other localization signal molecules. Our control of the NP decoration scheme, and the modularity of our platform, carries great advantage for nano-carrier design for drug delivery applications.

Solid-state nanopores represent an emerging technology for the highly sensitive detection of biomolecular markers, but the detection of DNA point mutations is challenged by the high noise levels associated with solid-state nanopore reading. In contrast, barcoded DNA origami nanostructures can provide unique single-molecule nanopore fingerprints. In this work, we have integrated nanopore-barcoded DNA nanostructures with enzymatic DNA ligation, the latter of which is routinely involved in clinical protocols for DNA mutation detection. We designed two triangular DNA origami variants containing three elongated staples that provide strands extensions on one side that are complementary to a target sequence. Addition of the latter in solution promotes the formation of a DNA triangle dimer. Since T4 DNA ligase repairs a nick in a dsDNA segment only if there is Watson-Crick base-pairing at the nick, the two DNA triangles can be covalently linked only if the DNA sequence bridging the two triangles carries the targeted mutation. We have found striking differences between ligation detection by gel electrophoresis, AFM, and quartz capillary-based nanopores. The stacking interaction between DNA triangles is enhanced by the formation of dimers, and promote the formation of higher order nanostructure, which serve as molecular weight amplification for DNA ligation in gels. The triangle-triangle stacking dynamics presumably involves a clam-like folding mechanism, which is detectable by quartz nanopore analysis, and which hinders ligation by T4 DNA ligase. The results provide the basis for development of rapid, highly sensitive, and affordable high-throughput approaches for profiling genetic variations in point-of-care settings.

Radiation enhancement is a promising anti-cancer approach based on a local radiation dose increase due to the presence of metallic nanoparticles (NPs) within cancer cells. Depending on their composition, size and cellular properties, NPs can follow multiple cellular pathways and entry routes. We observed that gold, platinum and TiO2 NPs are internalized at higher levels in mesenchymal cells compared to epithelial cells in breast cancer models. A global survey of gene expression between epithelial and mesenchymal cells exposed to 4 different NP types revealed an involvement of membrane structure, and further experiments confirmed that the hyaluronic acid (HA) and its receptor CD44 are mediators of metallic NP uptake into cells. We extended our results to a larger panel of breast cancer cell lines and again showed a preferential uptake of all NPs tested in mesenchymal cells and relying on the HA/CD44 axis. These data provide considerations for the design of NP-based therapies targeting mesenchymal cancer cells, which are often resistant to treatment and correlate with poor prognosis and tumor recurrence.

Recent advances in lab automation have dramatically increased the throughput of material synthesis, enabling rapid screening of nanomaterials for specific applications in nanotechnology and nanomedicine. However, this progress highlights a key bottleneck: most high-resolution characterization techniques, such as electron microscopy, atomic force microscopy (AFM), and super-resolution microscopy, remain low-throughput and labor-intensive. To keep pace, characterization must evolve toward greater automation and scalability. Here, we present an integrated and automatable workflow for multicolor DNA point accumulation for imaging in nanoscale topography (DNA-PAINT) super-resolution microscopy tailored for nanoparticles. Our approach combines kinetic simulations, automated multiplexed imaging, and streamlined image analysis to enable end-to-end automation, from experimental design to quantitative data output. Simulations predict optimal experimental conditions, thus reducing the need for manual optimization. A fluidics system paired with a TIRF microscope is used to automate multiplexed imaging by rounds of imaging and probe exchange (exchange-PAINT) on multiple memorized positions without human oversight during the acquisition process. Finally, an image analysis pipeline tailored for NPs allows for the quantification of nanoparticle size and multiplexed ligand functionalization. This methodology improves the throughput and reproducibility of single-molecule localization microscopy (SMLM) using DNA-PAINT and lowers the entry barrier for non-expert users, thus paving the way for broader adoption in nanomedicine and materials discovery workflows.

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.

Mucus is a viscoelastic gel secreted by the pulmonary epithelium in the tracheobronchial region of the lungs. The coordinated beating of cilia in contact with the gel layer moves mucus upwards towards pharynx, removing inhaled pathogens and particles from the airways. The efficacy of this clearance mechanism depends primarily on the rheological properties of mucus. Here we use a magnetic wire based microrheology technique to study the viscoelastic properties of human mucus collected from human bronchus tubes. The response of wires between 5 and 80 {micro}m in length to a magnetic rotating field is monitored by optical time-lapse microscopy and analyzed using constitutive equation models of rheology, including Maxwell and Kelvin-Voigt. The static shear viscosity and elastic modulus can be inferred from low frequency (10-3 - 10 rad s-1) measurements, leading to the evaluation of the mucin network relaxation time. This relaxation time is found to be widely distributed, from one to several hundred seconds. Mucus is identified as a viscoelastic liquid with an elastic modulus of 2.5 {+/-} 0.5 Pa and a static viscosity of 100 {+/-} 40 Pa s. Our work shows that beyond the established spatial variations in rheological properties due to microcavities, mucus exhibits secondary inhomogeneities associated with the relaxation time of the mucin network that may be important for its flow properties.

Single-molecule force spectroscopy (SMFS) is a powerful method for studying folding states and mechanical properties of proteins, however, it requires surface immobilization of proteins onto force-transducing probes such as cantilevers or microscale beads. A common immobilization method relies on coupling surface-exposed lysine residues to carboxylated surfaces using 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide and N-hydroxysuccinimide (EDC/NHS). Because proteins typically contain many lysine groups, this strategy results in a heterogeneous distribution of tether positions in the molecule. Genetically encoded peptide tags (e.g., ybbR) provide alternative chemistries for achieving site-specific immobilization, but thus far a direct comparison of site-specific vs. lysine-based immobilization strategies to assess effects on the observed mechanical properties was lacking. Here, we directly compared lysine- vs. ybbR-based protein immobilization in SMFS assays using several model polyprotein systems. Our results show that lysine-based immobilization results in significant signal deterioration for monomeric streptavidin-biotin interactions, and loss of the ability to correctly classify unfolding pathways in a multipathway Cohesin-Dockerin system. We developed a mixed immobilization approach where a site-specifically tethered ligand was used to probe surface-bound proteins immobilized through lysine groups, and found partial recovery of specific signals. The mixed immobilization approach represents a viable alternative for mechanical assays on in vivo-derived samples or other proteins of interest where genetically encoded tags are not feasible.

T cells must reliably discriminate between foreign-derived antigens that require an adaptive immune response from non-specific self-antigens that do not. This discrimination is highly specific to the affinity of the bond between ligand and T cell receptors (TCRs), as well as highly sensitive to the concentration of ligand. In this study, we examined these features of T cell mediated immunity in the context of multivalent ligand-receptor interactions between clusters of TCRs with pMHC-coated nanoparticles (NPs). Using Monte Carlo simulations of NP-T cell surface interactions, we compared the effect of TCR clustering on the dose-response profiles of various NP designs. These simulations revealed a trade-off between sensitivity and specificity, mediated by the spatial clustering of TCRs and the geometry of the NP. In particular, large clusters of TCRs were more sensitive to both NP valence and ligand concentration at the expense of antigen specificity. Conversely, uniformly distributed TCR landscapes were better suited to affinity-based ligand discrimination, while sacrificing sensitivity to ligand concentration. These features of NP-mediated T cell activation depended significantly on NP size and valence rather than on the average ligand concentration. Furthermore, we demonstrated how kinetic proofreading mechanisms may help compensate for the limitations associated with TCR clustering. These findings thus highlight the importance of interacting geometries of NP design and TCR landscape in modulating the specificity and sensitivity of the T cell response. SignificanceT cells rely on surface T cell receptors (TCRs) to recognize foreign antigens presented as peptide-major histocompatibility complex (pMHC) molecules. TCR clustering is crucial for T cell activation, though its full role remains not entirely clear. Using Monte Carlo simulations, we demonstrate that TCR clustering profoundly influences both surface binding dynamics of multivalent pMHC-coated nanoparticles, used in autoimmune disease therapies, as well as downstream intracellular signals leading to T cell activation. Our findings thus provide important insights into the role of interaction geometries in shaping T cell response, with implications for optimizing nanoparticle design to enhance their therapeutic efficacy.

Multivalency as an interaction principle is widely utilized in nature. It enables specific and strong binding by multiple weak interactions through enhanced avidity and is a core process in immune recognition and cellular signaling and a current concept in drug design. Rapid binding and unbinding of monovalent constituent interactions during multivalent binding creates dynamics that require a single-molecule approach to be studied. Here, we use the high signals from plasmon enhanced fluorescence of nanoparticles to extract binding kinetics and dynamics of multivalent interactions on the single-molecule level and in real-time. We study mono-, bi-and trivalent binding interactions using a DNA Holliday Junction as a model construct with programmable valency. Furthermore, we introduce a model framework for binding kinetics that involves the binding restriction during multivalent interactions to take into account the structural conformation of multivalent molecules allowing quantitative comparison. We used this approach to explore how length and flexibility of the DNA ligands affect binding restriction and binding strength, where overall binding strength decreased with spacer length. For trivalent systems increasing spacer length was found to activate binding in the trivalent state giving insight into the design of multivalent drug or targeting moieties. Interestingly we could exploit the rapidly decaying near fields of the plasmon that induce a strong dependence of the signal to position of the fluorophore to observe binding dynamics during single multivalent binding events.

The structural integrity of the chromosomes is essential to every functional process within the eukaryotic nuclei. Chromosomes are DNA-histone complexes essential for the inheritance of genetic information to the offspring and any defect in it is linked to mitotic errors, cancer growth, and cellular aging. Changes in the mechanical properties of a chromosome could lead to its compromised function and stability, leading to chromosome breaks. Here, we studied the changes in chromosome physical properties using metaphase chromosomes isolated from human breast cancer cells (MCF7) exposed to Valproic Acid (VPA), a known epigenetic modifier drug involved in histone hyperacetylation and DNA demethylation. Due to chromosomal structural intricacy, preparative and technical limitations of analytical tools, we employed a label-free atomic force microscopy approach for simultaneously visualizing and mapping single chromosome elasticity. Additionally, we performed electron transport characteristics of metaphase chromosomes to elucidate the effect of VPA. Our multi-parametric strategy of probing physical properties of chromosomes offers a new scope in terms of analytical tools for studying chromosomal structural changes/aberrations and associated structure-function relationships pertinent to cancer.

Molecular mechanotyping, the quantification of changes in the stability of supramolecular interactions and chemical bonds under the action of mechanical forces, is an essential tool in the field of mechanochemistry. This is conventionally done in single-molecule force-spectroscopy (smFS) assays, for example with optical tweezers or Atomic Force Microscopy. While these techniques provide detailed mechanochemical insights, they are time-consuming, technically demanding and expensive; as a result, high-throughput screening of the mechanochemical properties of molecules of interest is challenging. To resolve this, we present a rapid, simple and low-cost mechanotyping assay: microfluidic force spectroscopy ({micro}FFS), which probes force-dependent bond stability by measuring the detachment of microparticles, bound to microfluidic channels by the interaction of interest, under hydrodynamic forcing. As this allows the simultaneous observation of hundreds of microparticles, we obtain a quantitative mechanotype in a single measurement, using readily available equipment. We validate our method by studying the stability of DNA duplexes, previously characterized through smFS. We further show that we can quantitatively describe the experimental data with simulations, which allows us to link the {micro}FFS data to single-bond mechanochemical properties. This opens the way to use ({micro}FFS) as a rapid molecular mechanotyping tool.

Conjugation of proteins with gold nanoparticles (GNPs) is crucial in developing nanoparticle-based drugs, which are used in making targeted delivery systems for therapeutic and diagnostic use. This conjugation is often mediated by covalent gold-thiol (Au-S) bonds involving cysteine residues. However, the influence of other interactions, like electrostatics, on protein-GNP interactions remains poorly understood. While the independent roles of cysteine (thiol interactions) and lysine (electrostatics) are studied, how these two distinct mechanisms couple and mutually influence one another remains unknown. It is particularly unclear how the position and the electrostatics around cysteine residues within the protein structure modulate their interaction with GNPs. Hence, to isolate the electrostatic contribution from Au-S interaction, we systematically investigated the role of positively charged lysine in protein interaction with citrate-capped GNPs using protein L, a cysteine-free antibody-binding protein. Protein L has seven lysine residues, positively charged at pH 7.4. Each lysine was individually mutated to cysteine to create single-cysteine variants. The capping strength of these mutants on citrate-capped GNPs was assessed using various biophysical techniques. Surprisingly, not all cysteine mutants formed stable covalent conjugates. The K7C mutant, in particular, showed strong and irreversible GNP capping, not solely due to the introduced cysteine, but also because of nearby positively charged residues that promoted electrostatic attraction to the negatively charged citrate-capped GNP surface. This critical role of electrostatics was further confirmed using lysine acrylation, which effectively capped lysines and prevented their interaction. Our experimental plan was thus able to successfully decouple the contributions of Au-S bonding from lysine-driven electrostatics. These sequential and cooperative studies underscore that electrostatic and covalent factors are not independent but part of an interconnected process, thereby underscoring the need to consider local charge environments in protein engineering for nanoparticle conjugation.

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

Several biopersistent high aspect ratio nanomaterials show pronounced pathogenic effects, from chronic lung inflammation and fibrosis to cancer. For example, asbestos fibers are classified as carcinogens, whereas the carcinogenicity of highly inflammatory multi-wall carbon nanotubes (MWCNTs), and TiO2 nanomaterials is still being evaluated. The exact early mechanisms of their pathogenicity towards inflammation and cancer remains uncertain, but it is likely not due to genotoxic or mutagenic activity. A proposed early mode of action that might lead to the formation of cancerous cells for asbestos fibers is the formation of binucleated and multinucleated cells, resulting in genetic instability. Here, we show that two high aspect ratio nanomaterials, MWCNTs and TiO2 nanotubes, which both induce chronic lung inflammation, induce very different cancer-related changes in vitro. TiO2 nanotubes - but not low aspect-ratio nanocubes of the same crystalline structure - disrupt microtubule organization and prolong mitosis, as well as deform nuclear shape, induce the formation of binucleated cells, and downregulate the tumour suppressor protein p53, whereas only the MWCNTs activate the stimulator of the interferon genes (STING) pathway, a hallmark of lung cancer. The observed differences in cellular responses to different high aspect ratio materials imply the need for assessing each nanomaterial individually with a broad range of tests rather than relying solely on a single marker or pathway, or even morphological or bulk chemical properties to infer possible carcinogenic properties.

1Self-assembled monolayers (SAMs) have been widely utilized as a way of tailoring surface chemistry through the adsorption of organic molecules to different materials. SAMs are easy to prepare and offer a wide variety of organic molecules that afford additional or improved properties to the coated material. Spatial control of SAM placement has been achieved over many length-scales, even at the nanoscale. However, nanopatterned SAMs are usually prepared through serial processes utilizing atomic scanning probes or soft-lithography utilizing elastomeric masters. These techniques are expensive or not repeatable. Here we present the use of nanospheres for the creation of nanopatterned Au:Cu films which spatially control the grafting of a thermoresponsive SAM made from poly(N-isopropyl acrylamide) (PNIPAM). Chemical characterization validates the presence of PNIPAM and environmental atomic force microscopy showed its response to temperature which was evidenced by a change in stiffness. Our approach represents an affordable large area methodology for repeatable spatial control of SAMs at the nanoscale.

Insertion of hydrophobic nanoparticles into phospholipid bilayers is limited to small particles that can incorporate into the hydrophobic membrane core in between the two lipid leaflets. Incorporation of nanoparticles above this size limit requires development of challenging surface engineering methodologies. In principle, increasing membrane thickness should facilitate incorporation of larger nanoparticles. Here, we explore the effect of incorporating very long phospholipids (C24:1) into small unilamellar vesicles on the membrane insertion efficiency of hydrophobic nanoparticles that are 5-13 nm in diameter. To this end, we improved an existing vesicle preparation protocol and utilized cryogenic electron microscopy imaging to examine the mode of interaction and to evaluate the insertion efficiency of membrane-inserted nanoparticles. We also perform classical, coarse-grained molecular dynamics simulations to identify changes in lipid membrane structural properties that may increase insertion efficiency. Our results indicate that long-chain lipids increase the insertion efficiency by preferentially accumulating near membrane-inserted nanoparticles to reduce the thermodynamically unfavorable disruption of the membrane.