Project Summary
Protein-protein interactions (PPIs) drive countless processes in biology. The ability to block these interactions
with high specificity is crucial for probing the basic science of these processes, as well as for developing imaging
agents or novel therapeutics. However, most traditional molecules for blocking protein-protein interactions—like
small molecules, peptides, or antibodies—rely on the precise targeting of the crucial interface or binding pocket,
which can be difficult for some targets. Furthermore, none of these approaches can be easily tuned to match the
valency or size of the target, and binding to patches on the protein not directly involved in PPIs can fail to block
activity. Here, we propose to develop a nanoscale synthetic antibody (“nano-synbody”) consisting of a tunable
DNA nanostructure bearing 2-3 peptide/protein ligands that can bind to distinct surfaces of a target protein and
block its association with its partner through the steric bulk of the DNA structure. The individual peptide/protein
binding agents will be derived from either known molecules, or found independently through methods like phage
display. Critically, our method merges computational simulation—and in silico “evolution”—of these hybrid
protein-DNA nano-synbodies, creating a library of structures and probing their association with the target. We
aim to create a feedback loop, whereby the computational simulations yield candidate nano-synbodies that can
be experimentally tested, further informing the next round of simulations. We will first apply this pipeline to a
homo-trimeric nano-synbody against the SARS-CoV-2 spike protein trimer (Aim 1). This test bed will allow us to
optimize the process and find a high-affinity blocking structure. Then, we will apply our method to nano-synbodies
for blocking the assembly of fibrinogen into fibrin clots (Aim 2). The second Aim will involve phage display against
fibrin to find novel binding agents, and thereby convert them into high-affinity hetero-trivalent structures. In both
Aims, we will demonstrate the advantage of nano-structuring ligand presentation over simple oligomerization
with flexible linkers. Taken together, our work will generate a new computational-experimental paradigm for the
design of tunable, user-defined nanostructures that can present three or more peptides/proteins for binding to
any protein, and blocking its association with its target. Crucially, our approach does not require binding directly
to the interface, which should enable it to target a much larger range of proteins that may not be amenable to
traditional approaches, large protein complexes, or mutants/variants of the targets that might escape single
binding agents.