{"doi":"10.1093/nar/gkae695","title":"Chemical synthesis of 2″OMeNAD+ and its deployment as an RNA 2′-phosphotransferase (Tpt1) ‘poison’ that traps the enzyme on its abortive RNA-2′-PO4-(ADP-2″OMe-ribose) reaction intermediate","abstract":"RNA 2'-phosphotransferase Tpt1 catalyzes the removal of an internal RNA 2'-PO4 via a two-step mechanism in which: (i) the 2'-PO4 attacks NAD+ C1″ to form an RNA-2'-phospho-(ADP-ribose) intermediate and nicotinamide; and (ii) transesterification of the ADP-ribose O2″ to the RNA 2'-phosphodiester yields 2'-OH RNA and ADP-ribose-1″,2″-cyclic phosphate. Although Tpt1 enzymes are prevalent in bacteria, archaea, and eukarya, Tpt1 is uniquely essential in fungi and plants, where it erases the 2'-PO4 mark installed by tRNA ligases during tRNA splicing. To identify a Tpt1 'poison' that arrests the reaction after step 1, we developed a chemical synthesis of 2″OMeNAD+, an analog that cannot, in principle, support step 2 transesterification. We report that 2″OMeNAD+ is an effective step 1 substrate for Runella slithyformis Tpt1 (RslTpt1) in a reaction that generates the normally undetectable RNA-2'-phospho-(ADP-ribose) intermediate in amounts stoichiometric to Tpt1. EMSA assays demonstrate that RslTpt1 remains trapped in a stable complex with the abortive RNA-2'-phospho-(ADP-2″OMe-ribose) intermediate. Although 2″OMeNAD+ establishes the feasibility of poisoning and trapping a Tpt1 enzyme, its application is limited insofar as Tpt1 enzymes from fungal pathogens are unable to utilize this analog for step 1 catalysis. Analogs with smaller 2″-substitutions may prove advantageous in targeting the fungal enzymes.","journal":"Nucleic Acids Research","year":2024,"id":481120,"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":0.9537,"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":411733,"name":"Shreya Ghosh","orcid":"0000-0002-1709-8011","position":1,"is_corresponding":false},{"id":989260,"name":"Renata Kasprzyk","orcid":"0000-0001-6163-2130","position":2,"is_corresponding":false},{"id":1321226,"name":"Marcel Brakonier","orcid":null,"position":3,"is_corresponding":false},{"id":1321227,"name":"M. G. Hanna","orcid":null,"position":4,"is_corresponding":false},{"id":1288726,"name":"Andreas Marx","orcid":"0000-0002-6471-3689","position":5,"is_corresponding":false},{"id":409034,"name":"Stewart Shuman","orcid":"0000-0001-5034-6438","position":6,"is_corresponding":false},{"id":1321225,"name":"Jakob Arnold","orcid":null,"position":0,"is_corresponding":true}],"reference_count":33,"raw_metadata":null,"created_at":"2026-07-19T02:07:06.225103Z","pmid":"39162230","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":[]}