Discovery 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 (22) exhibited improved affinity towards the yeast U4/U6 snRNA (K_D = 3.9 ± 2.2 μM). The specific interaction between 22 and the K-turn region was studied experimentally using deltaSHAPE and in silico with MD simulations. Moreover, this molecule was found to inhibit the binding of the U4 to Snu13 in biochemical assays (IC₅₀ = 3.2 ± 0.4 μM). This work reports new ligands for the U4 snRNA and reveals that a multiplexed, structure-based approach can be used to identify small molecules that bind to specific regions of complex RNAs.