UCSF

This thesis details my work, both published and ongoing projects, on assembling proteins with modular DNA scaffolds in order to develop new strategies for engineering antibodies and probing cellular signaling complexes. The organization of cell surface receptors on a nanometer length scale can influence and regulate a cell’s functional response to signals. My goal was to systematically explore the effect of receptor complex composition, valency, and geometry on cell signaling. Towards this aim, I used modular DNA-based scaffolds to combinatorially control the position of attached proteins with low nanometer resolution. This strategy required a simple and modular technique for site-specifically conjugating synthetic oligonucleotides to proteins. My approach uses the aldehyde tag, which can be genetically incorporated into proteins at either protein terminus or in an internal loop, which provides the potential for orientational control of proteins on DNA scaffolds. We designed and optimized four different reactions that generate DNA−protein conjugates to provide flexibility in linker chemistry. The protein-DNA conjugates can be efficiently assembled into structures with greater valency and complexity. We are expanding our DNA scaffold libraries to generate collections of macromolecular assemblies varying in valency and architecture. These protein-bearing DNA-scaffolded multivalent probes may have novel activities as antibodies with unique specificities and biological activities, such as the ability to deliver protein-based effectors, increasing avidity, or modulating specificity. In one application, we are using this strategy to prepare and screen multivalent DNA-scaffolded biparatopic antibodies for inhibiting urokinase plasminogen activator receptor in triple negative breast cancer lines. Additionally, we are using these modular DNA scaffolds to investigate the effect of nanoscale organization on the signaling profiles of the Epidermal Growth Factor Receptor (EGFR) family by systematically guiding the assembly of signaling complexes on cells. By dimerizing EGFR in the absence of ligand using our DNA scaffolds, we discovered that although dimerization of EGFR is sufficient for autophosphorylation and recruitment of adaptor proteins Grb2 and SOS, it is not sufficient for Ras activation, activation of the AKT or MAPK signaling pathways, nor for the concentration of the receptor into clathrin coated pits and endocytosis.