The nanoscale spatial organization of transmembrane tumor necrosis factor (TNF) receptors has been implicated in the regulation of cellular fate. Accordingly, molecular tools that can induce specific arrangements of these receptors on cell surfaces would give us an opportunity to study these effects in detail. To achieve this, we introduce DNA origami nanostructures that precisely scaffold the patterning of TNF-related apoptosis-inducing ligand-mimicking peptides at nanoscale level. Stimulating human breast cancer cells with these patterns, we find that around 5 nm is the critical interligand distance of hexagonally patterned peptides to induce death receptor clustering and a resulting apoptosis. We thus offer a strategy to reverse the non-efficacy of current ligand- and antibody-based methods for TNF superfamily activation.

As DNA origami applications in biomedicine are expanding, more knowledge is needed to assess these structures’ interaction with biological systems. Here, uptake and penetration in cell and cell spheroid tissue models (CSTMs) are studied to elucidate whether differences in internal structure can be a factor in the efficacy of DNA‐origami‐based delivery. Two structures bearing largely similar features in terms of both geometry and molecular weight, but with different internal designs—being either compact, lattice‐based origami or following an open, wireframe design—are designed. In CSTMs, wireframe rods are able to penetrate deeper than close‐packed rods. Moreover, doxorubicin‐loaded wireframe rods show a higher cytotoxicity in CSTMs. These results can be explained by differences in structural mechanics, local deformability, local material density, and accessibility to cell receptors between these two DNA origami design paradigms. In particular, it is suggested that the main reason for the difference in penetration dynamic arises from differences in interaction with scavenger receptors where lattice‐based structures appear to be internalized to a higher degree than polygonal structures of the same size and shape. It is thus argued that the choice of structural design method constitutes a crucial parameter for the application of DNA origami in drug delivery.

The nanoscale spatial organization of transmembrane tumor necrosis factor (TNF) receptors has been implied as a regulator of cellular fate. Accordingly, molecular tools that can induce specific arrangements of these receptors on cell surfaces would give us an opportunity to study these effects in detail. To achieve this, we introduce DNA origami nanostructures, that precisely scaffold the patterning of TNF-related apoptosis-inducing ligand (TRAIL)-mimicking peptides at nanoscale level. Stimulating human breast cancer cells with these patterns, we find that around 5 nm is the critical inter-ligand distance of hexagonally patterned peptides to induce death receptor clustering and a resulting apoptosis. We thus offer a strategy to reverse the non-efficacy of current ligand- and antibody-based methods for TNF superfamily (TNFRSF) activation.

By pairing adjacent molecules in-situ and then mapping these pairs, DNA microscopy could significantly reduce workload in spatial omics methods by directly inferring geometry from sequencing data alone. However, experimental artefacts can lead to errors in the adjacency data which distort the spatial reconstruction. Here, we describe a method to correct two such errors: spurious crosslinks formed between any two nodes, and fused nodes that are formed out of multiple molecules. We build on the principle that spatially close molecules should be connected and show that these errors violate this principle, allowing for their detection and correction. Our method corrects errors in simulated data, even in the presence of up to 20% errors, and proves to be more efficient at removing errors from experimental data than a read count filter. Integrating this method in DNA microscopy will significantly improve the accuracy of spatial reconstructions with lower data loss.