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.
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.
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.
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