Extended Data Fig. 1: To-Pro-1 dye displacement assay validation and newly identified small molecules that bind RNA are chemically dissimilar from previously known RNA binders.
From: Programming inactive RNA-binding small molecules into bioactive degraders
a, To-Pro assay optimization and validation. Optimization of the signal to noise ratio of To-Pro-1 dye displacement from an RNA 3×3 internal loop library (3×3 ILL) by altering the concentration of the dye and the RNA (left; n = 3 independent replicates). Validation using Hoechst 33258 as a positive control to displace To-Pro-1 (middle; n = 3 independent replicates). Z-factor analysis of the screening conditions using 10 and 100 µM Hoechst 33258 (right). For each concentration, n = 690 in a single independent experiment. Data are reported as the mean ± SD. b, AbsorbArray screening with a 5’-32P-labelled 3×3 ILL. Compounds were delivered to the array surface in dose response (200 nL of 10 – 0.625 mM, log2 dilutions). c, Heat map of the Tanimoto analysis of 404 molecules found within Inforna show that C1–C20 are chemically dissimilar to known RNA-binding matter. d, Average chemical similarity of C1–C20 to compounds within the R-BIND database (n = 104 unique compounds). e, Comparison of the physicochemical properties of C1–C20 and molecules housed within Inforna. The compounds identified herein are chemically distinct, having ~35-fold higher CLogP, an ~20% reduction in polar surface area (TPSA), and an ~15% reduction in the number of both H-bond donors and acceptors15,57,58. f, Unique chemical patterns indicate chemically similar compounds within the azolium scaffold. Compounds C7–C10 have a Tanimoto scores ranging from 0.7 to >0.995 and cluster together. Compounds C12–C15 are nearly chemically identical as they share the cholesterol-derived azolium core, differing only by the other N-substituent. However, when compared to the remaining compounds (C1–C11 and C16–C20), their Tanimoto score ranges from 0.29–0.32, indicating they are unique among the hits obtained. C1 and C11 exhibit high similarity (Tanimoto coefficient = 0.82), although they appear very different structurally. This could be due to their similar spatial orientations, as both have alkyl substituted benzenes on their azolium cores. Compounds C4, C5, C6, and C20 are structurally unique compared to all other hits.
