{"doi":"10.1101/2021.12.28.474375","title":"Introducing a New Bond-Forming Activity in an Archaeal DNA Polymerase by Structure-Guided Enzyme Redesign","abstract":"ABSTRACT DNA polymerases have evolved to feature a highly conserved activity across the tree of life: formation of, without exception, phosphodiester linkages that create the repeating sugarphosphate backbone of DNA. Can this linkage selectivity observed in nature be overcome by design to produce non-natural nucleic acids? Here, we report that structure-guided redesign of an archaeal DNA polymerase (9°N) enables a new polymerase activity that is undetectable in the wild type enzyme: catalyzing the formation of N3’→P5’ phosphoramidate linkages in the presence of 3’-amino-2’,3’-dideoxynucleoside 5’-triphosphate (3’-NH 2 -ddNTP) building blocks. Replacing a highly conserved metal-binding aspartate in the 9°N active site (Asp-404) with asparagine was key to the emergence of this unnatural enzyme activity. Molecular dynamics simulations provided insights into how a single substitution could enhance the productive positioning of the 3’-amino nucleophile in the active site. Further remodeling of the protein-nucleic acid interface with substitutions in the finger subdomain led to a quadruple-mutant variant (9°N-NRQS) that incorporated 3’-NH 2 -ddNTPs into a 3’-amino-primer on various DNA templates. This work presents the first example of an active-site substitution of a metal-binding residue that leads to a novel activity in a DNA polymerase, and sheds light on the molecular basis of substrate fidelity and latent promiscuity in enzymes.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2021,"id":222258,"datarank":0.11594413407588787,"base_score":0.6931471805599453,"endowment":0.6931471805599453,"self_citation_contribution":0.10397207708399181,"citation_network_contribution":0.011972056991896065,"self_endowment_contribution":0.10397207708399181,"citer_contribution":0.011972056991896065,"corpus_percentile":null,"corpus_rank":null,"citation_count":1,"citer_count":1,"citers_with_citation_signal":1,"citers_with_endowment":1,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9427,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2021-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":705833,"name":"William A. Hansen","orcid":null,"position":1,"is_corresponding":false},{"id":823681,"name":"Jonathan Hong","orcid":null,"position":2,"is_corresponding":false},{"id":345740,"name":"Abir Ganguly","orcid":"0000-0002-0630-1109","position":3,"is_corresponding":false},{"id":61660,"name":"Darrin M. York","orcid":"0000-0002-9193-7055","position":4,"is_corresponding":false},{"id":524205,"name":"Sagar D. Khare","orcid":"0000-0002-2255-0543","position":5,"is_corresponding":false},{"id":366273,"name":"Enver Çagrı Izgü","orcid":"0000-0001-6673-3635","position":6,"is_corresponding":false},{"id":823413,"name":"Tushar Aggarwal","orcid":"0000-0002-6641-0964","position":0,"is_corresponding":true}],"reference_count":43,"raw_metadata":{"citation_network_status":"fetched"},"created_at":"2026-07-18T23:54:03.549854Z","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":[]}