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.

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.

Long noncoding RNAs (lncRNAs), defined as nontranslated transcripts greater than 200 nucleotides in length, are often differentially expressed throughout developmental stages, tissue types, and disease states. The identification, visualization, and suppression/overexpression of these sequences have revealed impacts on a wide range of biological processes, including epigenetic regulation. Biochemical investigations on select systems have revealed striking insight into the biological roles of lncRNAs and lncRNA:protein complexes, which in turn prompt even more unanswered questions. To begin, multiple protein- and RNA-centric technologies have been employed to isolate lncRNA:protein and lncRNA:chromatin complexes. LncRNA interactions with the multi-subunit protein complex PRC2, which acts as a transcriptional silencer, represent some of the few cases where the binding affinity, selectivity, and activity of a lncRNA:protein complex have been investigated. At the same time, recent reports of full-length lncRNA secondary structures suggest the formation of complex structures with multiple independent folding domains and pave the way for more detailed structural investigations and predictions of lncRNA three-dimensional structure. This review will provide an overview of the methods and progress made to date as well as highlight new methods that promise to further inform the molecular recognition, specificity, and function of lncRNAs.