The CD8+ T cell tolerance checkpoint triggers a distinct differentiation state defined by protein translation defects.

Peripheral CD8+ T cell tolerance is a checkpoint in both autoimmune disease and anti-cancer immunity. Despite its importance, the relationship between tolerance-induced states and other CD8+ T cell differentiation states remains unclear. Using flow cytometric phenotyping, single-cell RNA sequencing (scRNA-seq), and chromatin accessibility profiling, we demonstrated that in vivo peripheral tolerance to a self-antigen triggered a fundamentally distinct differentiation state separate from exhaustion, memory, and functional effector cells but analogous to cells defectively primed against tumors. Tolerant cells diverged early and progressively from effector cells, adopting a transcriptionally and epigenetically distinct state within 60 h of antigen encounter. Breaching tolerance required the synergistic actions of strong T cell receptor (TCR) signaling and inflammation, which cooperatively induced gene modules that enhanced protein translation. Weak TCR signaling during bystander infection failed to breach tolerance due to the uncoupling of effector gene expression from protein translation. Thus, tolerance engages a distinct differentiation trajectory enforced by protein translation defects.

High-Speed Atomic Force Microscopy Imaging of DNA Three-Point-Star Motif Self Assembly Using Photothermal Off-Resonance Tapping.

High-speed atomic force microscopy (HS-AFM) is a popular molecular imaging technique for visualizing single-molecule biological processes in real-time due to its ability to image under physiological conditions in liquid environments. The photothermal off-resonance tapping (PORT) mode uses a drive laser to oscillate the cantilever in a controlled manner. This direct cantilever actuation is effective in the MHz range. Combined with operating the feedback loop on the time domain force curve rather than the resonant amplitude, PORT enables high-speed imaging at up to ten frames per second with direct control over tip-sample forces. PORT has been shown to enable imaging of delicate assembly dynamics and precise monitoring of patterns formed by biomolecules. Thus far, the technique has been used for a variety of dynamic in vitro studies, including the DNA 3-point-star motif assembly patterns shown in this work. Through a series of experiments, this protocol systematically identifies the optimal imaging parameter settings and ultimate limits of the HS-PORT AFM imaging system and how they affect biomolecular assembly processes. Additionally, it investigates potential undesired thermal effects induced by the drive laser on the sample and surrounding liquid, particularly when the scanning is limited to small areas. These findings provide valuable insights that will drive the advancement of PORT mode's application in studying complex biological systems.

Balancing the Nanoscale Organization in Multivalent Materials for Functional Inhibition of the Programmed Death-1 Immune Checkpoint.

Dendritic cells (DCs) regulate immune priming by expressing programmed death ligand 1 (PD-L1) and PD-L2, which interact with the inhibitory receptor PD-1 on activated T cells. PD-1 signaling regulates T cell effector functions and limits autoimmunity. Tumor cells can hijack this pathway by overexpressing PD-L1 to suppress antitumor T cell responses. Blocking this inhibitory pathway has been beneficial for the treatment of various cancer types, although only a subset of patients responds. A deepened understanding of the spatial organization and molecular interplay between PD-1 and its ligands may inform the design of more efficacious nanotherapeutics. We visualized the natural molecular PD-L1 organization on DCs by DNA-PAINT microscopy and created a template to engineer DNA-based nanoclusters presenting PD-1 at defined valencies, distances, and patterns. These multivalent nanomaterials were examined for their cellular binding and blocking ability. Our data show that PD-1 nano-organization has profound effects on ligand interaction and that the valency of PD-1 molecules modulates the effectiveness in restoring T cell function. This work highlights the power of spatially controlled functional materials to unravel the importance of multivalent patterns in the PD-1 pathway and presents alternative design strategies for immune-engineering.

Stabilizing Polymer Coatings Alter the Protein Corona of DNA Origami and Can Be Engineered to Bias the Cellular Uptake.

With DNA-based nanomaterials being designed for applications in cellular environments, the need arises to accurately understand their surface interactions toward biological targets. As for any material exposed to protein-rich cell culture conditions, a protein corona will establish around DNA nanoparticles, potentially altering the a-priori designed particle function. Here, we first set out to identify the protein corona around DNA origami nanomaterials, taking into account the application of stabilizing block co-polymer coatings (oligolysine-1kPEG or oligolysine-5kPEG) widely used to ensure particle integrity. By implementing a label-free methodology, the distinct polymer coating conditions show unique protein profiles, predominantly defined by differences in the molecular weight and isoelectric point of the adsorbed proteins. Interestingly, none of the applied coatings reduced the diversity of the proteins detected within the specific coronae. We then biased the protein corona through pre-incubation with selected proteins and show significant changes in the cell uptake. Our study contributes to a deeper understanding of the complex interplay between DNA nanomaterials, proteins, and cells at the bio-interface.

Cellular Uptake of DNA Origami.

This chapter discusses the methods involved in achieving and analyzing cellular uptake of DNA origami. While cells naturally internalize substances from their surroundings, more than a simple addition of DNA origami in the surrounding cell medium is necessary to ensure DNA origami particles successfully enter the intracellular environment. Starting with the folding of the DNA, careful handling of sterile buffers and tools is essential, as well as the use of an endotoxin free scaffold. We explain how DNA origami needs a certain form of stabilization or protection to survive the degrading low-salt and high-nuclease environment of common cell culture media. Depending on the preferred method of post-uptake analysis (confocal), microscopy, or flow cytometry, we elaborate on the full protocols and crucial steps to prepare cell uptake experiments. Finally, notes are added on the intracellular fate (see Notes 14 and 15), and cellular retention of DNA origami (see Note 16) is discussed.

Biotechnological Frontiers of DNA Nanomaterials Continue to Expand: Bacterial Infection using Virus-Inspired Capsids.

The elegant geometry of viruses has inspired bio-engineers to synthetically explore the self-assembly of polyhedral capsids employed to protect new cargo or change an enzymatic microenvironment. Recently, Yang and co-workers used DNA nanotechnology to revisit the icosahedral capsid structure of the phiX174 bacteriophage and reloaded the original viral genome as cargo into their fully synthetic architecture. Surprisingly, when using a favorable combination of structural rigidity and dynamic multivalent cargo entrapment, the synthetic particles were able to infect non-competent bacterial cells and produce the original phiX174 bacteriophage. This work presents an exciting new direction of DNA nanotech for bio-engineering applications which involve bacterial interactions.

Interplay of the mechanical and structural properties of DNA nanostructures determines their electrostatic interactions with lipid membranes.

Nucleic acids and lipids function in close proximity in biological processes, as well as in nanoengineered constructs for therapeutic applications. As both molecules carry a rich charge profile, and frequently coexist in complex ionic solutions, the electrostatics surely play a pivotal role in interactions between them. Here we discuss how each component of a DNA/ion/lipid system determines its electrostatic attachment. We examine membrane binding of a library of DNA molecules varying from nanoengineered DNA origami through plasmids to short DNA domains, demonstrating the interplay between the molecular structure of the nucleic acid and the phase of lipid bilayers. Furthermore, the magnitude of DNA/lipid interactions is tuned by varying the concentration of magnesium ions in the physiologically relevant range. Notably, we observe that the structural and mechanical properties of DNA are critical in determining its attachment to lipid bilayers and demonstrate that binding is correlated positively with the size, and negatively with the flexibility of the nucleic acid. The findings are utilized in a proof-of-concept comparison of membrane interactions of two DNA origami designs - potential nanotherapeutic platforms - showing how the results can have a direct impact on the choice of DNA geometry for biotechnological applications.

Multivalent Pattern Recognition through Control of Nano-Spacing in Low-Valency Super-Selective Materials.

Super-selective multivalent ligand-receptor interactions display a signature step-like onset in binding when meeting a characteristic density of target receptors. Materials engineered for super-selective binding generally display a high number of flexible ligands to enhance the systems' avidity. In many biological processes, however, ligands are present in moderate copy numbers and arranged in spatio-temporal patterns. In this low-valency regime, the rigidity of the ligand-presenting architecture plays a critical role in the selectivity of the multivalent complex through decrease of the entropic penalty of binding. Exploiting the precision in spatial design inherent to the DNA nanotechnology, we engineered a library of rigid architectures to explore how valency, affinity, and nano-spacing control the presence of super-selectivity in multivalent binding. A micromolar monovalent affinity was required for super-selective binding to be observed within low-valency systems, and the transition point for stable interactions was measured at hexavalent ligand presentation, setting the limits of the low-valency regime. Super-selective binding was observed for all hexavalent architectures, and, more strikingly, the ligand pattern determined the selectivity onset. Hereby, we demonstrate for the first time that nano-control of geometric patterns can be used to discriminate between receptor densities in a super-selective manner. Materials that were indistinguishable in their molecular composition and ligand valency bound with various efficacies on surfaces with constant receptor densities. We define this new phenomenon in super-selective binding as multivalent pattern recognition.

Selective Integrin α5ß1 Targeting through Spatially Constrained Multivalent DNA-Based Nanoparticles.

Targeting cells specifically based on receptor expression levels remains an area of active research to date. Selective binding of receptors cannot be achieved by increasing the individual binding strength, as this does not account for differing distributions of receptor density across healthy and diseased cells. Engaging receptors above a threshold concentration would be desirable in devising selective diagnostics. Integrins are prime target candidates as they are readily available on the cell surface and have been reported to be overexpressed in diseases. Insights into their spatial organization would therefore be advantageous to design selective targeting agents. Here, we investigated the effect of activation method on integrin α5ß1 clustering by immunofluorescence and modeled the global neighbor distances with input from an immuno-staining assay and image processing of microscopy images. This data was used to engineer spatially-controlled DNA-scaffolded bivalent ligands, which we used to compare trends in spatial-selective binding observed across HUVEC, CHO and HeLa in resting versus activated conditions in confocal microscopy images. For HUVEC and CHO, the data demonstrated an improved selectivity and localisation of binding for smaller spacings ~7 nm and ~24 nm, in good agreement with the model. A deviation from the mode predictions for HeLa was observed, indicative of a clustered, instead of homogeneous, integrin organization. Our findings demonstrate how low-technology imaging methods can guide the design of spatially controlled ligands to selectively differentiate between cell type and integrin activation state.

Strategic Insights into Engineering Parameters Affecting Cell Type-Specific Uptake of DNA-Based Nanomaterials.

DNA-based nanomaterials are gaining popularity as uniform and programmable bioengineering tools as a result of recent solutions to their weak stability under biological conditions. The DNA nanotechnology platform uniquely allows decoupling of engineering parameters to comprehensively study the effect of each upon cellular encounter. We here present a systematic analysis of the effect of surface parameters of DNA-based nanoparticles on uptake in three different cell models: tumor cells, macrophages, and dendritic cells. The influence of surface charge, stabilizing coating, fluorophore types, functionalization technique, and particle concentration employed is found to cause significant differences in material uptake among these cell types. We therefore provide new insights into the large variance in cell type-specific uptake, highlighting the necessity of proper engineering and careful assay development when DNA-based materials are used as tools in bioengineering and as future nanotherapeutic agents.

Spatially Controlled Activation of Toll-like Receptor 9 with DNA-Based Nanomaterials.

First evidence of geometrical patterns and defined distances of biomolecules as fundamental parameters to regulate receptor binding and cell signaling have emerged recently. Here, we demonstrate the importance of controlled nanospacing of immunostimulatory agents for the activation of immune cells by exploiting DNA-based nanomaterials and pre-existing crystallography data. We created DNA origami nanoparticles that present CpG-motifs in rationally designed spatial patterns to activate Toll-like Receptor 9 in RAW 264.7 macrophages. We demonstrated that stronger immune activation is achieved when active molecules are positioned at the distance of 7 nm, matching the active dimer structure of the receptor. Moreover, we show how the introduction of linkers between particle and ligand can influence the spatial tolerance of binding. These findings are fundamental for a fine-tuned manipulation of the immune system, considering the importance of spatially controlled presentation of therapeutics to increase efficacy and specificity of immune-modulating nanomaterials where multivalent binding is involved.

Significance of Receptor Mobility in Multivalent Binding on Lipid Membranes.

Numerous key biological processes rely on the concept of multivalency, where ligands achieve stable binding only upon engaging multiple receptors. These processes, like viral entry or immune synapse formation, occur on the diffusive cellular membrane. One crucial, yet underexplored aspect of multivalent binding is the mobility of coupled receptors. Here, we discuss the consequences of mobility in multivalent processes from four perspectives: (I) The facilitation of receptor recruitment by the multivalent ligand due to their diffusivity prior to binding. (II) The effects of receptor preassembly, which allows their local accumulation. (III) The consequences of changes in mobility upon the formation of receptor/ligand complex. (IV) The changes in the diffusivity of lipid environment surrounding engaged receptors. We demonstrate how understanding mobility is essential for fully unravelling the principles of multivalent membrane processes, leading to further development in studies on receptor interactions, and guide the design of new generations of multivalent ligands.

Quantification of Strand Accessibility in Biostable DNA Origami with Single-Staple Resolution.

DNA-based nanostructures are actively gaining interest as tools for biomedical and therapeutic applications following the recent development of protective coating strategies prolonging structural integrity in physiological conditions. For tailored biological action, these nanostructures are often functionalized with targeting or imaging labels using DNA base pairing. Only if these labels are accessible on the structure's surface will they be able to interact with their intended biological target. However, the accessibility of functional sites for different geometries and environments, specifically after the application of a protective coating, is currently not known. Here, we assay this accessibility on the level of single handle strands with two- and three-dimensional resolution using DNA-PAINT and show that the hybridization kinetics of top and bottom sides on the same nanostructure linked to a surface remain unaltered. We furthermore demonstrate that the functionality of the structures remains available after an oligolysine-PEG coating is applied, enabling bioassays where functionality and stability are imperative.

Engineering the Dynamics of Cell Adhesion Cues in Supramolecular Hydrogels for Facile Control over Cell Encapsulation and Behavior.

The extracellular matrix (ECM) forms through hierarchical assembly of small and larger polymeric molecules into a transient, hydrogel-like fibrous network that provides mechanical support and biochemical cues to cells. Synthetic, fibrous supramolecular networks formed via non-covalent assembly of various molecules are therefore potential candidates as synthetic mimics of the natural ECM, provided that functionalization with biochemical cues is effective. Here, combinations of slow and fast exchanging molecules that self-assemble into supramolecular fibers are employed to form transient hydrogel networks with tunable dynamic behavior. Obtained results prove that modulating the ratio between these molecules dictates the extent of dynamic behavior of the hydrogels at both the molecular and the network level, which is proposed to enable effective incorporation of cell-adhesive functionalities in these materials. Excitingly, the dynamic nature of the supramolecular components in this system can be conveniently employed to formulate multicomponent supramolecular hydrogels for easy culturing and encapsulation of single cells, spheroids, and organoids. Importantly, these findings highlight the significance of molecular design and exchange dynamics for the application of supramolecular hydrogels as synthetic ECM mimics.

Complex multicomponent patterns rendered on a 3D DNA-barrel pegboard.

DNA origami, in which a long scaffold strand is assembled with a many short staple strands into parallel arrays of double helices, has proven a powerful method for custom nanofabrication. However, currently the design and optimization of custom 3D DNA-origami shapes is a barrier to rapid application to new areas. Here we introduce a modular barrel architecture, and demonstrate hierarchical assembly of a 100 megadalton DNA-origami barrel of ~90 nm diameter and ~250 nm height, that provides a rhombic-lattice canvas of a thousand pixels each, with pitch of ~8 nm, on its inner and outer surfaces. Complex patterns rendered on these surfaces were resolved using up to twelve rounds of Exchange-PAINT super-resolution microscopy. We envision these structures as versatile nanoscale pegboards for applications requiring complex 3D arrangements of matter, which will serve to promote rapid uptake of this technology in diverse fields beyond specialist groups working in DNA nanotechnology.

Endothelial extracellular vesicles contain protective proteins and rescue ischemia-reperfusion injury in a human heart-on-chip.

Extracellular vesicles (EVs) derived from various stem cell sources induce cardioprotective effects during ischemia-reperfusion injury (IRI). These have been attributed mainly to the antiapoptotic, proangiogenic, microRNA (miRNA) cargo within the stem cell-derived EVs. However, the mechanisms of EV-mediated endothelial signaling to cardiomyocytes, as well as their therapeutic potential toward ischemic myocardial injury, are not clear. EV content beyond miRNA that may contribute to cardioprotection has not been fully illuminated. This study characterized the protein cargo of human vascular endothelial EVs (EEVs) to identify lead cardioactive proteins and assessed the effect of EEVs on human laminar cardiac tissues (hlCTs) exposed to IRI. We mapped the protein content of human vascular EEVs and identified proteins that were previously associated with cellular metabolism, redox state, and calcium handling, among other processes. Analysis of the protein landscape of human cardiomyocytes revealed corresponding modifications induced by EEV treatment. To assess their human-specific cardioprotection in vitro, we developed a human heart-on-a-chip IRI assay using human stem cell-derived, engineered cardiac tissues. We found that EEVs alleviated cardiac cell death as well as the loss in contractile capacity during and after simulated IRI in an uptake- and dose-dependent manner. Moreover, we found that EEVs increased the respiratory capacity of normoxic cardiomyocytes. These results suggest that vascular EEVs rescue hlCTs exposed to IRI possibly by supplementing injured myocytes with cargo that supports multiple metabolic and salvage pathways and therefore may serve as a multitargeted therapy for IRI.

Nucleic acids presenting polymer nanomaterials as vaccine adjuvants.

Most vaccines developed today include only the antigens that best stimulate the immune system rather than the entire virus or microbe, which makes vaccine production and use safer and easier, though they lack potency to induce acceptable immunity and long-term protection. The incorporation of additional immune stimulating components, named adjuvants, is required to generate a strong protective immune response. Nucleic acids (DNA and RNA) and their synthetic analogs are promising candidates as vaccine adjuvants activating Toll-like receptors (TLRs). Additionally, in the last few years several nanocarriers have emerged as platforms for targeted co-delivery of antigens and adjuvants. In this review, we focus on the recent developments in polymer nanomaterials presenting nucleic acids as vaccine adjuvants. We aim to compare the effectiveness of the various classes of polymers in immune modulating materials (nanoparticles, dendrimers, single-chain particles, nanogels, polymersomes and DNA-based architectures). In particular, we address the critical role of parameters such as size, shape, complexation and release of TLR ligands, cellular uptake, stability, toxicity and potential importance of spatial control in ligand presentation.

Engineering a stable future for DNA-origami as a biomaterial.

DNA as a biomaterial has evoked great interest as a potential platform for therapeutics and diagnostics and as hydrogel scaffolds due to the relative ease of programming its robust and uniform shape, site-specific functionality and controlled responsive behavior. However, for a stable self-assembled product, a relatively high cation concentration is required to prevent denaturation. Physiological and cell-culture conditions do not match these concentrations and present additional nucleases that cause a serious threat to the integrity of DNA-based materials. For the translation of this promising technology towards bioengineering challenges, stability needs to be guaranteed. Over the past years, various methods have been developed addressing the stability-related weaknesses of DNA-origami. This mini-review explains the common stability issues and compares the stabilization strategies recently developed. We present a detailed overview of each method in order to ease the selection process on which method to use for future users of DNA-origami as a biomaterial.

Quantifying Guest-Host Dynamics in Supramolecular Assemblies to Analyze Their Robustness.

The most basic function of synthetic microenvironments for tissue engineering is to act as a physical substrate for cell attachment, migration, and proliferation, similar to the natural cell environment. Functionalization of supramolecular materials with guest compounds that display the same recognition moieties is a common strategy to introduce biofunctionality. However, besides a robust interaction with the material, a certain level of dynamics needs to be conserved for an adaptive interface toward the living environment. A balance between robust material functionalization and dynamic cell interaction needs to be met. The detailed analysis hereof using a ureido-pyrimidinone (UPy) poly(ethylene glycol) system in dilute and transient network regime is demonstrated. Monovalent and bivalent UPy-functionalized guest molecules are designed and their interaction with UPy-host fibers is evaluated. Analysis of guest interaction in the dilute state by microfluidics, and in the gel state, by fluorescence recovery after photobleaching and fluorescence resonance energy transfer is proven to be suitable to quantify the local and ensemble guest mobility. The results demonstrate that the interaction of bioactive moieties through supramolecular host-guest chemistry yields a dynamic system, which is stronger for divalent guests but risks unintended leakage in the case of functional monomeric units.

Modulation of the Cellular Uptake of DNA Origami through Control over Mass and Shape.

Designer nanoparticles with controlled shapes and sizes are increasingly popular vehicles for therapeutic delivery due to their enhanced cell-delivery performance. However, our ability to fashion nanoparticles has offered only limited control over these parameters. Structural DNA nanotechnology has an unparalleled ability to self-assemble three-dimensional nanostructures with near-atomic resolution features, and thus, it offers an attractive platform for the systematic exploration of the parameter space relevant to nanoparticle uptake by living cells. In this study, we examined the cell uptake of a panel of 11 distinct DNA-origami shapes, with the largest dimension ranging from 50-400 nm, in 3 different cell lines. We found that larger particles with a greater compactness were preferentially internalized compared with elongated, high-aspect-ratio particles. Uptake kinetics were also found to be more cell-type-dependent than shape-dependent, with specialized endocytosing dendritic cells failing to saturate over 12 h of study. The knowledge gained in the current study furthers our understanding of how particle shape affects cellular uptake and heralds the development of DNA nanotechnologies toward the improvement of current state-of-the-art cell-delivery vehicles.