{"doi":"10.1093/nar/gkac1108","title":"G-rich motifs within phosphorothioate-based antisense oligonucleotides (ASOs) drive activation of <i>FXN</i> expression through indirect effects","abstract":"Friedreich's ataxia is an incurable disease caused by frataxin (FXN) protein deficiency, which is mostly induced by GAA repeat expansion in intron 1 of the FXN gene. Here, we identified antisense oligonucleotides (ASOs), complementary to two regions within the first intron of FXN pre-mRNA, which could increase FXN mRNA by ∼2-fold in patient fibroblasts. The increase in FXN mRNA was confirmed by the identification of multiple overlapping FXN-activating ASOs at each region, two independent RNA quantification assays, and normalization by multiple housekeeping genes. Experiments on cells with the ASO-binding sites deleted indicate that the ASO-induced FXN activation was driven by indirect effects. RNA sequencing analyses showed that the two ASOs induced similar transcriptome-wide changes, which did not resemble the transcriptome of wild-type cells. This RNA-seq analysis did not identify directly base-paired off-target genes shared across ASOs. Mismatch studies identified two guanosine-rich motifs (CCGG and G4) within the ASOs that were required for FXN activation. The phosphorodiamidate morpholino oligomer analogs of our ASOs did not activate FXN, pointing to a PS-backbone-mediated effect. Our study demonstrates the importance of multiple, detailed control experiments and target validation in oligonucleotide studies employing novel mechanisms such as gene activation.","journal":"Nucleic Acids Research","year":2022,"id":266263,"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":10,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9565,"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":575100,"name":"Ezequiel Calvo-Roitberg","orcid":"0000-0002-2431-515X","position":1,"is_corresponding":false},{"id":806203,"name":"Julia M Rembetsy-Brown","orcid":null,"position":2,"is_corresponding":false},{"id":925947,"name":"Minggang Fang","orcid":"0000-0002-8267-6738","position":3,"is_corresponding":false},{"id":302205,"name":"Jacquelyn Sousa","orcid":"0009-0008-2592-9613","position":4,"is_corresponding":false},{"id":925948,"name":"Zachary J. Kartje","orcid":"0000-0001-9798-8382","position":5,"is_corresponding":false},{"id":650493,"name":"Pranathi M. Krishnamurthy","orcid":null,"position":6,"is_corresponding":false},{"id":925949,"name":"Jonathan Lee","orcid":"0000-0002-7941-5684","position":7,"is_corresponding":false},{"id":645803,"name":"Michael R. Green","orcid":"0000-0003-3017-3298","position":8,"is_corresponding":false},{"id":65303,"name":"Athma A. Pai","orcid":"0000-0002-7995-9948","position":9,"is_corresponding":false},{"id":348431,"name":"Jonathan K. Watts","orcid":"0000-0001-5706-1734","position":10,"is_corresponding":false},{"id":575969,"name":"Feng Wang","orcid":"0000-0002-3383-2992","position":0,"is_corresponding":true}],"reference_count":65,"raw_metadata":null,"created_at":"2026-07-19T00:26:54.081997Z","pmid":"36511872","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":[]}