Understanding the roles of avidity in biological systems

Abstract

Understanding avidity is essential for elucidating complex molecular interactions and designing effective therapeutic strategies. This thesis aims to explore strategies and tools to understand how avidity influences receptor-ligand binding kinetics, cellular uptake mechanisms, intracellular trafficking pathways, and modulation of immune responses.

In Paper I, we developed a method called NanoDeep that utilizes DNA sequencing to decipher the nanoscale spatial distribution of membrane proteins. NanoDeep can characterize differences in protein nanoenvironments in different cellular contexts and provides insights into the roles of the composition and spatial organization of protein nanoenvironments. NanoDeep has the potential to be a tool for simultaneous analysis of the spatial distribution of many proteins at the membrane without microscopy measurements, providing a means to understand the importance of membrane protein assemblies in cellular function.

In Paper II, we used DNA origami nanotechnology to precisely control insulin multivalency and spatial arrangement at the nanoscale. This methodology enabled us to study the binding and signaling characteristics of insulin nanoclusters, revealing that the arrangement and number of insulin molecules significantly influence their interaction with insulin receptors and subsequent activation of the insulin receptor signaling cascade. These insights offer guidance for designing and refining insulin-based nanotherapeutics aimed at treating diabetes.

In Paper III, we constructed and characterized a novel platform that consists of self-assembling virus-like particles displaying single-chain Fc molecules on their surface. This engineered platform facilitated the investigation of Fc valency on Fcγ receptor clustering and activation. The findings revealed that Fc valency influences the binding of single-chain Fc virus-like particles to various FcγR-expressing immune cells. Interestingly, single-chain Fc virus-like particles showed limited internalization and receptor clustering, hindering phagocytosis and FcγR activation. These insights shed light on the potential of engineered Fc multimers for developing innovative therapies targeting autoimmune diseases by suppressing FcγR activation and immune complex-mediated functions such as antibody-dependent cellular phagocytosis.

In Paper IV, we used DNA origami nanostructures presenting aptamers that bind to transferrin receptors to study the relationship between avidity and receptor-mediated endocytosis. We found that the binding, uptake, and sorting of the DNA nanostructures were specific to the transferrin receptor, aptamer-mediated, and modulated by aptamer valency. These findings provide insights into the role of avidity in cellular uptake and suggest potential strategies for modulating the intracellular behaviour of DNA nanosheets for drug delivery.