Reviewing the various methods and effectivity to stabilize DNA origami in biological environments.

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.

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.

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.

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.

Polymer nanomaterials in various shapes and sizes can be engineered to present nucleic acids and function as immune adjuvant.