{"doi":"10.1093/nar/gkaf231","title":"Mapping small molecule–RNA binding sites via Chem-CLIP synergized with capillary electrophoresis and nanopore sequencing","abstract":"Target validation and identification of binding sites are keys to the development of bioactive small molecules that target RNA. Herein, we describe optimized protocols to profile small molecule-RNA interactions and to define binding sites of the small molecules in RNAs using covalent chemistry. Various reactive modules appended to an RNA-binding small molecule were studied for cross-linking to the RNA target. Electrophilic modules, whether N-chloroethyl aniline or diazirine, have reactive profiles consistent with induced proximity; however, probes with N-chloroethyl aniline were more reactive and more specific than those with a diazirine cross-linking moiety. Depending upon the identity of the cross-linking module, covalent adducts with different nucleotides that are proximal to a small molecule's binding site were formed. The nucleotides where cross-linking occurred were elucidated by using two different platforms: (i) automated capillary electrophoresis that identified a binding site by impeding reverse transcriptase, or \"RT stops\"; and (ii) nanopore sequencing where the cross-link produces mutations in the corresponding complementary DNA formed by reverse transcriptase-polymerase chain reaction amplification of the cross-linked RNA. These approaches are broadly applicable to aid in the advancement of chemical probes targeting RNA, including identifying binding sites and using covalent chemistry to screen for RNA-binding molecules in a high throughput format.","journal":"Nucleic Acids Research","year":2025,"id":516178,"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":0.9641,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1377185,"name":"Jielei Wang","orcid":"0009-0007-0072-039X","position":1,"is_corresponding":false},{"id":244754,"name":"Noah A. Springer","orcid":"0000-0001-8868-0415","position":2,"is_corresponding":false},{"id":804221,"name":"Patrick R. A. Zanon","orcid":"0000-0002-8883-8275","position":3,"is_corresponding":false},{"id":1377186,"name":"Yilin Jia","orcid":"0000-0002-2284-4250","position":4,"is_corresponding":false},{"id":1223782,"name":"Xiaoxuan Su","orcid":"0000-0002-5141-2692","position":5,"is_corresponding":false},{"id":249561,"name":"Matthew D. Disney","orcid":"0000-0001-8486-1796","position":6,"is_corresponding":false},{"id":819992,"name":"Xueyi Yang","orcid":"0000-0002-0207-5538","position":0,"is_corresponding":true}],"reference_count":57,"raw_metadata":null,"created_at":"2026-07-19T02:48:44.647022Z","pmid":"40156856","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":[]}