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. 2022 Aug 8;21(10):736–762. doi: 10.1038/s41573-022-00521-4

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Fig. 2 — a–c | Methods to identify small molecules that bind RNA. a | Automated ligand identification system (ALIS) is a liquid chromatography–mass spectrometry (LC-MS) method. An RNA target is incubated with a library of small molecules. Unbound ligands are removed by size-exclusion chromatography and then bound ligands are identified by LC-MS. b | Fluorescence-based assays rely on a change in fluorescence upon small-molecule binding to the RNA target. This could be achieved by: displacing a non-selective RNA-binding dye (top); changing the microenvironment of a fluorescent nucleotide analogue (middle); or disrupting donor–acceptor pairs in a fluorescence resonance energy transfer (FRET)-based assay (bottom). c | Microarray-based screening in which a panel of small molecules is pinned to an array surface and incubated with labelled RNA targets, followed by washing and imaging to identify target-binding compounds. d | Compounds functionalized with a cross-linking module (such as diazirine or chlorambucil) and a pull-down tag (such as alkyne or biotin) can be screened against labelled RNA targets by using Chem-CLIP (chemical cross-linking and isolation by pull-down) to identify RNA binders and to map the binding sites. e | Identification of RNA-binding small molecules from a DNA-encoded library (DEL). The library is synthesized on beads, and each building block added during the synthesis is encoded with a DNA tag. The DEL is screened simultaneously for binding to the target of interest and a related RNA to which binding is undesired. The two RNAs are labelled with different fluorophores, and selective binders from the DEL can be identified and isolated by flow cytometry. f,g | Methods to design small molecules that bind RNA. f | Inforna is a lead identification strategy in which the structures present in a cellular RNA are compared with a database of experimentally determined RNA–small molecule interactions. Overlap affords lead targets and lead small molecules. g | Structure-based design of small molecules relies on a model of the structure of the RNA or of the RNA–ligand complex. Both can be used in docking studies while the latter can be used to guide modifications that improve interactions between the RNA and the small molecule. 2-AP, 2-aminopurine; RFU, relative fluorescence units.