{"doi":"10.1002/ange.202316428","title":"Enhanced Directed Evolution in Mammalian Cells Yields a Hyperefficient Pyrrolysyl tRNA for Noncanonical Amino Acid Mutagenesis","abstract":"Abstract Heterologous tRNAs used for noncanonical amino acid (ncAA) mutagenesis in mammalian cells typically show poor activity. We recently introduced a virus‐assisted directed evolution strategy (VADER) that can enrich improved tRNA mutants from naïve libraries in mammalian cells. However, VADER was limited to processing only a few thousand mutants; the inability to screen a larger sequence space precluded the identification of highly active variants with distal synergistic mutations. Here, we report VADER2.0, which can process significantly larger mutant libraries. It also employs a novel library design, which maintains base‐pairing between distant residues in the stem regions, allowing us to pack a higher density of functional mutants within a fixed sequence space. VADER2.0 enabled simultaneous engineering of the entire acceptor stem of M. mazei pyrrolysyl tRNA (tRNA Pyl ), leading to a remarkably improved variant, which facilitates more efficient incorporation of a wider range of ncAAs, and enables facile development of viral vectors and stable cell‐lines for ncAA mutagenesis.","journal":"Angewandte Chemie","year":2024,"id":461873,"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":2,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9498,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2024-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":992119,"name":"Rachel E. Kelemen","orcid":null,"position":1,"is_corresponding":false},{"id":991759,"name":"Rachel L. Huang","orcid":"0000-0003-4664-5069","position":2,"is_corresponding":false},{"id":331272,"name":"Zeyu Zhu","orcid":"0000-0002-9218-9351","position":3,"is_corresponding":false},{"id":254149,"name":"Bharathi Sundaresh","orcid":"0000-0002-1924-6612","position":4,"is_corresponding":false},{"id":1234742,"name":"Kaitlin Malley","orcid":null,"position":5,"is_corresponding":false},{"id":1079898,"name":"Quan Pham","orcid":"0009-0001-9559-7772","position":6,"is_corresponding":false},{"id":987774,"name":"Conor Loynd","orcid":null,"position":7,"is_corresponding":false},{"id":986523,"name":"Zeyi Huang","orcid":"0009-0009-3221-2597","position":8,"is_corresponding":false},{"id":228745,"name":"Tim van Opijnen","orcid":"0000-0001-6895-6795","position":9,"is_corresponding":false},{"id":117143,"name":"Abhishek Chatterjee","orcid":"0000-0002-6231-5302","position":10,"is_corresponding":false},{"id":987775,"name":"Delilah Jewel","orcid":null,"position":0,"is_corresponding":true}],"reference_count":44,"raw_metadata":null,"created_at":"2026-07-19T02:04:16.410510Z","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":[]}