Exploring the interplay of molecular pattern and valency for selective interactions at the bio-interface

Cell-cell communication is fundamental for immune balance. Multivalent interactions of surface receptors at immune interfaces drive specific communication, with stimulatory and inhibitory signals guiding the immune outcome. Notably, the valency, affinity, cooperativity, and spatial distribution of the interacting signals were naturally evolved to selectively induce immune activation or suppression. The complex receptor network on the cell surface poses significant challenges in addressing nanoscale spatial organization and functional implications in cellular processes. While other available materials have been limited in their selective abilities, DNA represents an ideal material for constructing platforms with nano-controlled spatial arrangements of multivalent ligands allowing for selective targeting at cell interfaces. In this thesis, we explored relevant spatial organizational parameters leading to selective multivalent interactions and their significance in immune cell activity using DNA origami.

In chapter 2, we started with a bio-interface model to study the design principles guiding selective multivalent interactions using a rigid-disk shaped DNA origami structure. We showed that the ligand's intrinsic affinity defines the threshold for multivalency to overcome individual binding events and that six ligands were required for stable interaction. Furthermore, we revealed that pattern-controlled multivalent presentation of ligands can result in a super-selective binding behavior that we defined as multivalent pattern recognition.

In chapter 3, we turned to a biologically relevant system and explored the role of spatial organization of the immune regulator PD-1 known to inhibit T cell activity. Using DNA-PAINT imaging, we resolved the natural organization of its binding partner, PD-L1, in dendritic cells (DCs). We then designed DNA disks to mimic these features, presenting PD-1 at defined valencies, nano-spacings, and geometric patterns. We show that the spatial arrangement of PD-1 has profound effects on its interaction to DCs and that valency modulates the effectiveness in restoring T cell function.

In chapter 4, we used a similar approach to develop sensitive tools for the detection of pMHC complexes, which would ultimately enable the investigation of their nanoscale organization in DCs. The natural TCR clustering found on T cells was used as inspiration to create multivalent TCR displays on DNA disks. As the intrinsic pMHC-TCR affinity is one of the weakest observed in nature, we found that high TCR valency was necessary to achieve strong binding to pMHC complexes for sensitive detection.

In chapter 5, we interrogated the spatial organization of multiple relevant stimulatory and inhibitory receptors involved in T cell function. Their nanoscale arrangement was first resolved in DCs versus cancer cells by DNA-PAINT imaging, and we further investigated the significance of nanoscale differences observed in MHC and PD-L1 arrangement. By ma