Unlocking High Affinity Binders: A Novel DAmFRET-Based High-Throughput Screening Platform

Abstract

Protein interactions are fundamental to life, and the capability to engineer such interactions has immense potential for therapeutics and synthetic biology. Established methods for screening protein binders, such as yeast and phage display, are limited by the need first to purify the target protein and by the requirement to express binders extracellularly. These requirements limit throughput, preclude screening against complex or insoluble targets, and require hit binders to be subsequently validated for intracellular activity before they can be used in synthetic biology applications. To overcome these limitations, we are developing the Intracellular Binder–Antigen Screening System (iBASS), a high-throughput screening platform that operates intracellularly and can screen for high-affinity binders to an antigen of interest in a pooled fashion. iBASS exploits a prion-like domain to amplify a fluorescent signal triggered by a stable interaction of antigen with binder. For this purpose, we have designed a simple diblock amyloid-forming sequence whose saturating concentration and nucleation barrier can be independently tuned, allowing for its intracellular supersaturation and nucleation upon recruitment to a multivalent seed. This sequence is fused to the binder pool and mEos3.1, a photoconvertible fluorescent protein that reports on the protein’s self-association using a flow cytometry technique, DAmFRET (Distributed Amphofluoric FRET). The antigen is expressed in trans as the multivalent seed by fusing it to a homo-oligomeric domain. The interaction of the binder with the antigen increases its local concentration and triggers amyloid nucleation, which then drives the entire cellular pool of binder fusion essentially into a high-density aggregate, resulting in a massive digital gain in ratiometric FRET (AmFRET). We use fluorescence-activated cell sorting (FACS) to isolate FRET-positive cells, followed by targeted sequencing to identify successful binders. Using the well-characterized NbALFA/ALFA pair, we validated and optimized iBASS, demonstrating its ability to perform pooled, intracellular binder screening that is faster, easier and cheaper to use than other binder screening platforms. Following validation, We adapted the iBASS pipeline to screen a binder pool designed against the intrinsically disordered C-terminus of BFSP1 (Mus musculus) to isolate and identify novel high-affinity binders.