Diversification of RNA-targeted scaffolds offers great promise in the search for selective ligands of therapeutically relevant RNA such as HIV-1 TAR. We herein report the establishment of amiloride as a novel RNA-binding scaffold along with synthetic routes for combinatorial C(5)- and C(6)-diversification. Iterative modifications at the C(5)- and C(6)- positions yielded derivative 24, which demonstrated a 100-fold increase in activity over the parent dimethylamiloride in peptide displacement assays. NMR chemical shift mapping was performed using the 2D SOFAST- [1H-13C] HMQC NMR method, which allowed for facile and rapid evaluation of binding modes for all library members. Cheminformatic analysis revealed distinct differences between selective and non-selective ligands. In this study, we evolved dimethylamiloride from a weak TAR ligand to one of the tightest binding selective TAR ligands reported to date through a novel combination of synthetic methods and analytical techniques. We expect these methods to allow for rapid library expansion and tuning of the amiloride scaffold for a range of RNA targets and for SOFAST NMR to allow unprecedented evaluation of small molecule:RNA interactions.

Three-dimensional RNA structures are notoriously difficult to determine, and the link between secondary structure and RNA conformation is only beginning to be understood. These challenges have hindered the identification of guiding principles for small molecule:RNA recognition. We herein demonstrate that the strong and differential binding ability of aminoglycosides to RNA structures can be used to classify five canonical RNA secondary structure motifs through principal component analysis (PCA). In these analyses, the aminoglycosides act as receptors, while RNA structures labeled with a benzofuranyluridine fluorophore act as analytes. Complete (100%) predictive ability for this RNA training set was achieved by incorporating two exhaustively guanidinylated aminoglycosides into the receptor library. The PCA was then externally validated using biologically relevant RNA constructs. In bulge-stem-loop constructs of HIV-1 transactivation response element (TAR) RNA, we achieved nucleotide-specific classification of two independent secondary structure motifs. Furthermore, examination of cheminformatic parameters and PCA loading factors revealed trends in aminoglycoside:RNA recognition, including the importance of shape-based discrimination, and suggested the potential for size and sequence discrimination within RNA structural motifs. These studies present a new approach to classifying RNA structure and provide direct evidence that RNA topology, in addition to sequence, is critical for the molecular recognition of RNA.

Using pattern recognition of RNA with small molecules (PRRSM) with fluorescent RNA chemosensors and aminoglycosides, we reveal the impact of changing environmental conditions on the differentiation of a range of RNA structures as well as the ability to predict different sequence/size compositions of five canonical RNA motifs.