{"doi":"10.1101/2020.03.10.985739","title":"Single-chain lanthanide luminescence biosensors for cell-based imaging and screening of protein-protein interactions","abstract":"ABSTRACT Research tools that enable imaging or analysis of protein-protein interactions (PPIs) directly within living cells provide unique and valuable biological insights and can also aid drug discovery efforts. Here, we present lanthanide-based, Förster resonance energy transfer (lanthanide-based FRET, or LRET) biosensors for time-gated luminescence (TGL) imaging or multiwell plate analysis of PPIs. Polypeptide chains comprised of an alpha helical linker flanked by a Tb(III) complex, GFP and two binding domains exhibit large differences in long-lifetime, Tb(III)-to-GFP LRET-sensitized emission between open (unbound) and closed (bound) states. We used TGL microscopy to image ca. 500% increases in Tb(III)-to-GFP LRET following rapamycin addition to NIH 3T3 cells that expressed biosensors bearing FKBP12 and the rapamycin binding domain of m-Tor (FRB) at each terminus. Much larger signal changes, up to ca. 2500%, were observed when cells were grown in 96-well or 384-well plates and analyzed using a TGL plate reader. We also measured the interaction of p53 and HDM2 and its inhibition within intact HeLa cells grown in 96-well plates and estimated a z’-factor of 0.5 for the assay. The modular design and high dynamic range of Tb(III)-based LRET biosensors will facilitate versatile imaging and cell-based screening of PPIs.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2020,"id":126370,"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":1,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9531,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2020-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":226326,"name":"Hà Phạm","orcid":"0000-0001-8484-470X","position":1,"is_corresponding":false},{"id":421465,"name":"Ali Mohamadi","orcid":null,"position":2,"is_corresponding":false},{"id":420747,"name":"Lawrence W. Miller","orcid":"0000-0003-4501-9357","position":3,"is_corresponding":false},{"id":420746,"name":"Ting Chen","orcid":"0000-0002-3124-4337","position":0,"is_corresponding":true}],"reference_count":41,"raw_metadata":null,"created_at":"2026-07-18T23:15:23.509897Z","pmid":null,"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":[]}