2-amino-1,3-benzothiazole-6-carboxamide Preferentially Binds the Tandem Mismatch Motif r(UY:GA)

Abstract

RNA helices are often punctuated with non-Watson-Crick features that can be the target of chemical compounds, but progress towards identifying small molecules specific for non-canonical elements has been slow. We have used a tandem UU:GA mismatch motif (5'-UG-3':5'-AU-3') embedded within the helix of an RNA hairpin as a model to identify compounds that bind the motif specifically. The three-dimensional structure of the RNA hairpin and its interaction with a small molecule compound identified through a virtual screen are presented. The G-A of the mismatch forms a sheared pair upon which the U-U base pair stacks. The hydrogen bond configuration of the U-U pair involves the O2 of the U adjacent to the G and the O4 of the U adjacent to the A. The G-A and U-U pairs are flanked by A-U and G-C base pairs, respectively, and the mismatch exhibits greater stability than when the motif is within the context of other flanking base pairs or when the 5'-3' orientation of the G-A and U-U is swapped.Residual dipolar coupling constants were used to generate an ensemble of structures against which a virtual screen of 64,480 small molecules was performed to identify candidate compounds that the motif specifically binds. The tandem mismatch was found to be specific for one compound, 2-amino-1,3-benzothiazole-6-carboxamide, which binds with moderate affinity but extends the motif to include the flanking A-U and G-C base pairs. The finding that affinity for the UU:GA mismatch is flanking sequence dependent emphasizes the importance of motif context and potentially increases the number of small non-canonical features within RNA that can be specifically targeted by small molecules.

An overview of structural approaches to study therapeutic RNAs

“…A campaign of high-throughput screening against ~100,000 drug-like small molecules (Ganser et al, 2018) has been performed on this target, revealing again the superiority of RDC-mediated ensembles that enrich libraries with true hits. Chang et al (2020) resolved with the help of RDCs the structure of a microRNA (miRNA) that contains the tandem UU:GA base pair mismatch and used it for a drug-discovery purpose. This mismatch motif occurs in the helices of large rRNA subunits of several organisms, including human pathogens like HIV (Bilbille et al, 2009), making it an optimal target for pharmaceutical purposes.…”