{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,26]],"date-time":"2026-08-26T10:27:36Z","timestamp":1787740056643,"version":"build-2784847793"},"reference-count":47,"publisher":"Wiley","issue":"8","license":[{"start":{"date-parts":[[2026,8,20]],"date-time":"2026-08-20T00:00:00Z","timestamp":1787184000000},"content-version":"vor","delay-in-days":19,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,8,1]],"date-time":"2026-08-01T00:00:00Z","timestamp":1785542400000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"funder":[{"DOI":"10.13039\/100011769","name":"Scripps Research","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100011769","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Current Protocols"],"published-print":{"date-parts":[[2026,8]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>\n                    Targeting structured RNA elements with small molecules has emerged as a promising yet technically challenging strategy for antiviral drug discovery. Here, we present a comprehensive and experimentally validated workflow for the integrative\n                    <jats:italic>in silico<\/jats:italic>\n                    and\n                    <jats:italic>in vitro<\/jats:italic>\n                    screening of RNA\u2010binding small molecules. The approach is exemplified using conserved RNA elements from the SARS\u2010CoV\u20102 genome, including the 5\u2019\u2010terminal stem\u2010loop 1 and the programmed\u00a0\u20131 ribosomal frameshift pseudoknot, but is broadly applicable to other structured RNAs. The workflow integrates high\u2010resolution RNA structural ensemble generation with virtual screening (VS) and nuclear magnetic resonance (NMR)\u2010based experimental validation. Conformational ensembles generated by fragment\u2010assembly approaches serve as targets for docking chemically diverse fragments and lead\u2010like libraries. Top\u2010ranked compounds are prioritized through consensus scoring and evaluated using ligand\u2010 and RNA\u2010observed NMR experiments to confirm binding, characterize interaction modes, and assess specificity. Such ranking allows for NMR\u2010guided fragment optimization, which enables systematic improvement of solubility, affinity, and selectivity through iterative medicinal chemistry in the pharmaceutical pipeline. Detailed procedures are provided for library preparation, ensemble\u2010based VS, hit validation, data interpretation, and progression toward functional assays, together with practical considerations, quality\u2010control parameters, and troubleshooting guidance to ensure reproducibility. By combining computational and experimental strategies that select for high\u2010specificity ligands within a unified framework, this set of protocols accelerates the discovery and optimization of RNA\u2010targeting small molecules and provides a scalable platform for RNA\u2010focused drug discovery. \u00a9 2026 The Author(s).\n                    <jats:italic>Current Protocols<\/jats:italic>\n                    published by Wiley Periodicals LLC.\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Basic Protocol 1<\/jats:bold>\n                    : Target and library preparation for virtual screening\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Basic Protocol 2<\/jats:bold>\n                    : Ensemble\u2010based virtual screening of low\u2010molecular\u2010weight compounds against RNA targets\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Basic Protocol 3<\/jats:bold>\n                    : Comparative hit prioritization and selectivity filtering for RNA\u2010binding small molecules\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Basic Protocol 4<\/jats:bold>\n                    : Preparation of RNA samples for\n                    <jats:italic>in vitro<\/jats:italic>\n                    validation of small\u2010molecule binding\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Basic Protocol 5<\/jats:bold>\n                    : NMR\u2010based\n                    <jats:italic>in vitro<\/jats:italic>\n                    screening of RNA\u2013small molecule interactions\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Support Protocol<\/jats:bold>\n                    : Preparation of ligand stocks and NMR\u2010based quality control\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Basic Protocol 6<\/jats:bold>\n                    : Characterization and prioritization of validated RNA\u2010binding hits\n                  <\/jats:p>","DOI":"10.1002\/cpz1.70431","type":"journal-article","created":{"date-parts":[[2026,8,20]],"date-time":"2026-08-20T15:22:31Z","timestamp":1787239351000},"source":"Crossref","is-referenced-by-count":0,"title":["Integrative\n                    <i>in silico<\/i>\n                    and\n                    <i>in vitro<\/i>\n                    screening of small molecules targeting RNA"],"prefix":"10.1002","volume":"6","author":[{"ORCID":"https:\/\/orcid.org\/0009-0008-5501-6276","authenticated-orcid":false,"given":"Sabrina","family":"Toews","sequence":"first","affiliation":[{"name":"Institute for Organic Chemistry and Chemical Biology Goethe University Frankfurt  Frankfurt am Main, Hesse Germany"},{"name":"Center of Biomolecular Magnetic Resonance (BMRZ) Goethe University Frankfurt  Frankfurt am Main, Hesse Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8336-9935","authenticated-orcid":false,"given":"Megan","family":"Ken","sequence":"additional","affiliation":[{"name":"Department of Integrative Structural and Computational Biology The Scripps Research Institute  La Jolla California"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5693-7909","authenticated-orcid":false,"given":"Harald","family":"Schwalbe","sequence":"additional","affiliation":[{"name":"Institute for Organic Chemistry and Chemical Biology Goethe University Frankfurt  Frankfurt am Main, Hesse Germany"},{"name":"Center of Biomolecular Magnetic Resonance (BMRZ) Goethe University Frankfurt  Frankfurt am Main, Hesse Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,8,20]]},"reference":[{"key":"e_1_2_13_2_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41589\u2010024\u201001801\u20103"},{"key":"e_1_2_13_3_1","doi-asserted-by":"publisher","DOI":"10.3791\/62262"},{"key":"e_1_2_13_4_1","doi-asserted-by":"publisher","DOI":"10.1002\/cbic.202000476"},{"key":"e_1_2_13_5_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.sbi.2024.102894"},{"key":"e_1_2_13_6_1","doi-asserted-by":"publisher","DOI":"10.1080\/00268978300100381"},{"key":"e_1_2_13_7_1","doi-asserted-by":"publisher","DOI":"10.1002\/mco2.70342"},{"key":"e_1_2_13_8_1","doi-asserted-by":"publisher","DOI":"10.1002\/anie.202417961"},{"key":"e_1_2_13_9_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41573\u2010022\u201000521\u20104"},{"key":"e_1_2_13_10_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.jmedchem.9b01927"},{"key":"e_1_2_13_11_1","doi-asserted-by":"publisher","DOI":"10.1023\/A:1008272928075"},{"key":"e_1_2_13_12_1","doi-asserted-by":"publisher","DOI":"10.1023\/A:1013302231549"},{"key":"e_1_2_13_13_1","doi-asserted-by":"publisher","DOI":"10.1021\/jacs.8b13419"},{"key":"e_1_2_13_14_1","doi-asserted-by":"publisher","DOI":"10.1039\/D0CS01261K"},{"key":"e_1_2_13_15_1","doi-asserted-by":"publisher","DOI":"10.1021\/ja00295a052"},{"key":"e_1_2_13_16_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41580\u2010019\u20100136\u20100"},{"key":"e_1_2_13_17_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41594\u2010018\u20100062\u20104"},{"key":"e_1_2_13_18_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0407141102"},{"key":"e_1_2_13_19_1","doi-asserted-by":"publisher","DOI":"10.1021\/ja9715962"},{"key":"e_1_2_13_20_1","doi-asserted-by":"publisher","DOI":"10.1002\/wrna.1373"},{"key":"e_1_2_13_21_1","doi-asserted-by":"publisher","DOI":"10.1002\/wrna.1718"},{"key":"e_1_2_13_22_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.jmedchem.6c00070"},{"key":"e_1_2_13_23_1","doi-asserted-by":"publisher","DOI":"10.1126\/science.aah3963"},{"key":"e_1_2_13_24_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.sbi.2023.102550"},{"key":"e_1_2_13_25_1","doi-asserted-by":"publisher","DOI":"10.1021\/ci2005934"},{"key":"e_1_2_13_26_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41467\u2010022\u201029135\u20105"},{"key":"e_1_2_13_27_1","doi-asserted-by":"publisher","DOI":"10.1039\/D0CS00560F"},{"key":"e_1_2_13_28_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jmr.2006.10.014"},{"key":"e_1_2_13_29_1","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkab119"},{"key":"e_1_2_13_30_1","doi-asserted-by":"publisher","DOI":"10.1007\/s12104\u2010021\u201010047\u20102"},{"key":"e_1_2_13_31_1","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkaf560"},{"key":"e_1_2_13_32_1","doi-asserted-by":"publisher","DOI":"10.1039\/D4CB00266K"},{"key":"e_1_2_13_33_1","doi-asserted-by":"publisher","DOI":"10.1042\/EBC20200011"},{"key":"e_1_2_13_34_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41467\u2010020\u201019371\u2010y"},{"key":"e_1_2_13_35_1","doi-asserted-by":"publisher","DOI":"10.1002\/anie.202103693"},{"key":"e_1_2_13_36_1","doi-asserted-by":"publisher","DOI":"10.1038\/nchembio.596"},{"key":"e_1_2_13_37_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2012217117"},{"key":"e_1_2_13_38_1","doi-asserted-by":"publisher","DOI":"10.1039\/D4CB00272E"},{"key":"e_1_2_13_39_1","doi-asserted-by":"publisher","DOI":"10.1042\/BSR20240139"},{"key":"e_1_2_13_40_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.ymeth.2019.05.015"},{"key":"e_1_2_13_41_1","doi-asserted-by":"publisher","DOI":"10.1002\/cbic.202500668"},{"key":"e_1_2_13_42_1","doi-asserted-by":"publisher","DOI":"10.1021\/jacs.5c05264"},{"key":"e_1_2_13_43_1","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkae477"},{"key":"e_1_2_13_44_1","doi-asserted-by":"publisher","DOI":"10.1101\/2025.11.05.686775"},{"key":"e_1_2_13_45_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2422346122"},{"key":"e_1_2_13_46_1","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkaa1013"},{"key":"e_1_2_13_47_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.chembiol.2021.03.003"},{"key":"e_1_2_13_48_1","doi-asserted-by":"publisher","DOI":"10.1002\/wcms.1571"}],"container-title":["Current 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