{"doi":"10.1101/2025.01.06.631531","title":"High-throughput protein binder discovery by rapid in vivo selection","abstract":"Abstract Proteins that selectively bind to a target of interest are foundational components of research pipelines 1,2 , diagnostics 3 , and therapeutics 4 . Current immunization-based 5,6 , display-based 7–14 , and computational approaches 15–17,18 for discovering binders are laborious and time-consuming – taking months or more, suffer from high false positives – necessitating extensive secondary screening, and have a high failure rate, especially for disordered proteins and other challenging target classes. Here we establish Phage-Assisted Non-Continuous Selection of Protein Binders (PANCS-binders), an in vivo selection platform that links the life cycle of M13 phage to target protein binding though customized proximity-dependent split RNA polymerase biosensors, allowing for complete and comprehensive high-throughput screening of billion-plus member protein variant libraries with high signal-to-noise. We showcase the utility of PANCS-Binders by screening multiple protein libraries each against a panel of 95 separate therapeutically relevant targets, thereby individually assessing over 10 11 protein-protein interaction pairs, completed in two days. These selections yielded large, high-quality datasets and hundreds of novel binders, which we showed can be affinity matured or directly used in mammalian cells to inhibit or degrade targets. PANCS-Binders dramatically accelerates and simplifies the binder discovery process, the democratization of which will help unlock new creative potential in proteome-targeting with engineered binder-based biotechnologies.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2025,"id":555056,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":4,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9513,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1374677,"name":"Joshua A. Pixley","orcid":"0009-0000-4564-5345","position":1,"is_corresponding":false},{"id":1221735,"name":"Tongyao Wei","orcid":"0000-0002-0602-6979","position":2,"is_corresponding":false},{"id":1374678,"name":"Christopher Basile","orcid":"0009-0009-9764-4745","position":3,"is_corresponding":false},{"id":528746,"name":"Shannon S. Lu","orcid":"0000-0003-1556-7520","position":4,"is_corresponding":false},{"id":551852,"name":"Bryan C. Dickinson","orcid":"0000-0002-9616-1911","position":5,"is_corresponding":false},{"id":506946,"name":"Matthew J. Styles","orcid":"0000-0003-3236-0425","position":0,"is_corresponding":true}],"reference_count":43,"raw_metadata":null,"created_at":"2026-07-19T02:54:54.542303Z","pmid":"39829796","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}