Spatial control of membrane receptor function using ligand nanocalipers.

The spatial organization of membrane-bound ligands is thought to regulate receptor-mediated signaling. However, direct regulation of receptor function by nanoscale distribution of ligands has not yet been demonstrated, to our knowledge. We developed rationally designed DNA origami nanostructures modified with ligands at well-defined positions. Using these 'nanocalipers' to present ephrin ligands, we showed that the nanoscale spacing of ephrin-A5 directs the levels of EphA2 receptor activation in human breast cancer cells. Furthermore, we found that the nanoscale distribution of ephrin-A5 regulates the invasive properties of breast cancer cells. Our ligand nanocaliper approach has the potential to provide insight into the roles of ligand nanoscale spatial distribution in membrane receptor-mediated signaling.

Soluble and multivalent Jag1 DNA origami nanopatterns activate Notch without pulling force.

The Notch signaling pathway has fundamental roles in embryonic development and in the nervous system. The current model of receptor activation involves initiation via a force-induced conformational change. Here, we define conditions that reveal pulling force-independent Notch activation using soluble multivalent constructs. We treat neuroepithelial stem-like cells with molecularly precise ligand nanopatterns displayed from solution using DNA origami. Notch signaling follows with clusters of Jag1, and with chimeric structures where most Jag1 proteins are replaced by other binders not targeting Notch. Our data rule out several confounding factors and suggest a model where Jag1 activates Notch upon prolonged binding without appearing to need a pulling force. These findings reveal a distinct mode of activation of Notch and lay the foundation for the development of soluble agonists.

Clustering of Death Receptor for Apoptosis Using Nanoscale Patterns of Peptides.

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.

DNA Origami Penetration in Cell Spheroid Tissue Models is Enhanced by Wireframe Design.

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.

Specificity of linear motifs that bind to a common mitogen-activated protein kinase docking groove.

Mitogen-activated protein kinases (MAPKs) have a docking groove that interacts with linear "docking" motifs in binding partners. To determine the structural basis of binding specificity between MAPKs and docking motifs, we quantitatively analyzed the ability of 15 docking motifs from diverse MAPK partners to bind to c-Jun amino-terminal kinase 1 (JNK1), p38α, and extracellular signal-regulated kinase 2 (ERK2). Classical docking motifs mediated highly specific binding only to JNK1, and only those motifs with a sequence pattern distinct from the classical MAPK binding docking motif consensus differentiated between the topographically similar docking grooves of ERK and p38α. Crystal structures of four complexes of MAPKs with docking peptides, representing JNK-specific, ERK-specific, or ERK- and p38-selective binding modes, revealed that the regions located between consensus positions in the docking motifs showed conformational diversity. Although the consensus positions in the docking motifs served as anchor points that bound to common MAPK surface features and mostly contributed to docking in a nondiscriminatory fashion, the conformation of the intervening region between the anchor points mostly determined specificity. We designed peptides with tailored MAPK binding profiles by rationally changing the length and amino acid composition of intervening regions located between anchor points. These results suggest a coherent structural model for MAPK docking specificity that reveals how short linear motifs binding to a common kinase docking groove can mediate diverse interaction patterns and contribute to correct MAPK partner selection in signaling networks.