{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,20]],"date-time":"2026-05-20T04:39:09Z","timestamp":1779251949570,"version":"3.51.4"},"reference-count":50,"publisher":"Oxford University Press (OUP)","issue":"16","license":[{"start":{"date-parts":[[2025,8,30]],"date-time":"2025-08-30T00:00:00Z","timestamp":1756512000000},"content-version":"vor","delay-in-days":3,"URL":"https:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001717","name":"Leiden University","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100001717","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Leiden Institute of Chemistry"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2025,8,27]]},"abstract":"<jats:title>Abstract<\/jats:title>\n               <jats:p>There is an indisputable need for new screening methodologies to identify small molecules that target RNA tertiary structures, such as pseudoknots or G-quadruplexes. Here, we present a high-throughput competitive binding antisense assay\u00a0designed to identify ligands for complex RNA tertiary structures. In this assay, initially customized for the bacterial PreQ1-I riboswitch pseudoknot, ligands compete with a rationally designed quencher-labelled antisense oligonucleotide for binding to a fluorophore-labelled riboswitch. The method is validated for four PreQ1-I riboswitches, using the natural riboswitch ligand PreQ1 and various analogues. A commercial RNA-focused library consisting of \u223c15\u00a0000 compounds was then screened against the Fusobacterium nucleatum riboswitch, leading to the identification of a promising hit, 4494, which showed competitive binding activity to all PreQ1-I riboswitches and was able to inhibit translation of a riboswitch-regulated reporter gene. Although resynthesis of 4494 revealed that its activity originated from an \u223c1% guanine contamination, this result underscores the assay\u2019s exceptional sensitivity. To demonstrate its versatility, the assay was tailored for a SARS-CoV-2 G-quadruplex\u00a0structure and validated with several known G-quadruplex ligands. This work shows that the competitive binding antisense assay is a powerful addition to the RNA-targeting toolbox, facilitating the discovery of ligands for diverse RNA tertiary structures.<\/jats:p>","DOI":"10.1093\/nar\/gkaf819","type":"journal-article","created":{"date-parts":[[2025,8,30]],"date-time":"2025-08-30T10:32:42Z","timestamp":1756549962000},"source":"Crossref","is-referenced-by-count":2,"title":["High-throughput competitive binding assay for targeting RNA tertiary structures with small molecules: application to pseudoknots and G-quadruplexes"],"prefix":"10.1093","volume":"53","author":[{"ORCID":"https:\/\/orcid.org\/0009-0004-7955-6788","authenticated-orcid":false,"given":"Sophie E L","family":"Wintermans","sequence":"first","affiliation":[{"name":"Leiden Institute of Chemistry, Leiden University Department of Medical Biochemistry, , Einsteinweg 55, 2333 CC Leiden ,","place":["The 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55, 2333 CC\u00a0Leiden,","place":["The Netherlands"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hans","family":"van\u00a0den\u00a0Elst","sequence":"additional","affiliation":[{"name":"Leiden Institute of Chemistry, Leiden University Department of Bio-organic Synthesis, , Einsteinweg 55, 2333 CC Leiden,","place":["The Netherlands"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Bjorn\u00a0R","family":"van\u00a0Doodewaerd","sequence":"additional","affiliation":[{"name":"Division of Chemical Biology and Drug Discovery, Leiden University Medical Center Department of Cell and Chemical Biology, , Einthovenweg 20, 2333 ZC\u00a0Leiden,","place":["The Netherlands"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1849-1111","authenticated-orcid":false,"given":"Paul P","family":"Geurink","sequence":"additional","affiliation":[{"name":"Division of Chemical Biology and Drug Discovery, Leiden University Medical 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