biostudies-literatureUnknownYang MIntramural NIH HHSNCI NIH HHSThe spliceosome is a large ribonucleoprotein complex that regulates pre-mRNA splicing and has been an intriguing target for drug discovery. Essential to the assembly of the spliceosome are the small nuclear RNAs (snRNAs), which form RNA-RNA and RNA-protein interactions in the intact spliceosome and during its assembly. Here, we study the yeast U4/U6 snRNA assembly and report the rapid discovery of small molecule K-turn ligands via parallel small molecule microarray (SMM) screening of multiple related RNA constructs. For hit validation, biophysical analyses were conducted to confirm the binding and effects on thermodynamic stability and of RNA structure. One analog of the hit molecule (<b>22</b>) exhibited improved affinity towards the yeast U4/U6 snRNA (K_D = 3.9 ± 2.2 μM). The sbioRxiv : the preprint server for biology2025.10.10.681657https://www.ebi.ac.uk/biostudies/studies/S-EPMC12632352biostudies-literatureDiscovery of small molecule probes targeting the 5' stem-loop in the yeast U4/U6 snRNA.PMC1263235275N91019D00024ZIA BC011585Parmar SBalaratnam SBume DDPrestwood PRSchneekloth JSYang MKasprzak WKfalseDiscovery of small molecule probes targeting the 5' stem-loop in the yeast U4/U6 snRNA.The spliceosome is a large ribonucleoprotein complex that regulates pre-mRNA splicing and has been an intriguing target for drug discovery. Essential to the assembly of the spliceosome are the small nuclear RNAs (snRNAs), which form RNA-RNA and RNA-protein interactions in the intact spliceosome and during its assembly. Here, we study the yeast U4/U6 snRNA assembly and report the rapid discovery of small molecule K-turn ligands via parallel small molecule microarray (SMM) screening of multiple related RNA constructs. For hit validation, biophysical analyses were conducted to confirm the binding and effects on thermodynamic stability and of RNA structure. One analog of the hit molecule (<b>22</b>) exhibited improved affinity towards the yeast U4/U6 snRNA (K_D = 3.9 ± 2.2 μM). The s2025-01-01T00:00:00Z2025 Oct2026-05-31T03:11:43.026Z2026-05-31T03:09:49.104ZS-EPMC126323524127961510.1101/2025.10.10.681657