{"doi":"10.1101/2022.09.17.508400","title":"Synthesis of Long RNA with a Site-Specific Modification by Enzymatic Splint Ligation","abstract":"ABSTRACT Synthesis of RNA molecules that contain an internal site-specific modification is important for RNA research and therapeutics. While solid-state synthesis is attainable for such RNA in the range of 100 nucleotides (nts), it is currently impossible with kilobase (kb)-long RNA. Instead, long RNA with an internal modification is usually assembled in an enzymatic 3-part splint ligation to join a short RNA oligonucleotide, containing the site-specific modification, with both a left-arm and a right-arm long RNA that are synthesized by in vitro transcription. However, long RNAs have structural heterogeneity and those synthesized by in vitro transcription have 3’-end sequence heterogeneity, which together substantially reduce the yield of 3-part splint ligation. Here we describe a method of 3-part splint ligation with an enhanced efficiency utilizing a ribozyme cleavage reaction to address the 3’-end sequence heterogeneity and involving DNA disruptors proximal to the ligation sites to address the structural heterogeneity. The yields of the synthesized kb-long RNA are sufficiently high to afford purification to homogeneity for practical RNA research. We also verify the sequence accuracy at each ligation junction by nanopore sequencing.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2022,"id":298837,"datarank":0.26876392038420827,"base_score":1.791759469228055,"endowment":1.791759469228055,"self_citation_contribution":0.26876392038420827,"citation_network_contribution":0.0,"self_endowment_contribution":0.26876392038420827,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":5,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9545,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2022-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":807911,"name":"Caroline A. McCormick","orcid":"0009-0000-0670-6707","position":1,"is_corresponding":false},{"id":807910,"name":"Sepideh Tavakoli","orcid":"0000-0002-0802-7051","position":2,"is_corresponding":false},{"id":572586,"name":"Meni Wanunu","orcid":"0000-0002-9837-0004","position":3,"is_corresponding":false},{"id":807912,"name":"Sara H. Rouhanifard","orcid":"0000-0002-6991-4877","position":4,"is_corresponding":false},{"id":17829,"name":"Ya-Ming Hou","orcid":"0000-0001-6546-2597","position":5,"is_corresponding":false},{"id":488621,"name":"Howard Gamper","orcid":"0000-0001-9007-8546","position":0,"is_corresponding":true}],"reference_count":64,"raw_metadata":{"citation_network_status":"fetched"},"created_at":"2026-07-19T00:31:40.528568Z","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":[]}