Transposon-Display of AI-designed binders enables manipulation of the proteome in human cells

ABSTRACT Transposon-Display is a highly scalable screening method that links proteins to their encoding DNA during expression in E. coli via a mutant transposase. Leveraging this system, we identified AI-designed binders targeting four intracellular human proteins, saturation-mutagenized top candidates, and charge-balanced variants to improve compatibility with the intracellular environment while preserving target binding. The resulting neutral binders can be fused to EGFP allowing antibody-free intracellular staining. Expressing neutral binders in human cells as fusions with functional domains can drive small molecule-controlled protein aggregation and trigger proteasomal degradation of endogenous target proteins in living cells. Manipulating the proteome of living cells using libraries of AI-designed binders may provide a new avenue to screen for disease-relevant protein functions and large-scale functional data may help to refine protein design algorithms. Competing Interest Statement A.H. and J.L.S.-B. are inventors on a patent application related to Transposon-Display filed by the University of Bonn. J.L.S.-B. is a co-founder and shareholder of LAMPseq Diagnostics GmbH and ions.bio GmbH. S.V.W. and P.E.G. are co-founders, shareholders and employees of ions.bio GmbH. M.H. reports travel expenses, honoraria for webinars and research support (consumables) from TME Pharma AG unrelated to this work. M.H. also reports honoraria and clinical advisory board membership from OncoMAGNETx Inc unrelated to this work.