Non-coding RNAs (ncRNAs) are a novel class of biomolecules implicated in many important biological processes and are often misregulated in various diseases. Currently, few rational approaches exist to develop chemical probes for RNA, which hinders the study of disease-driving ncRNAs. One successful approach is the identification of RNA binding preferences of small molecule scaffolds; however, most of these studies evaluate a subset of secondary structures, which leads to an incomplete binding profile of the small molecule. In order to fundamentally understand RNA:small molecule recognition, there is a need to more generally and rapidly evaluate RNA:small molecule interactions. We herein describe a TO-PRO-1 displacement assay as an appropriate screening tool along with current efforts to correlate binding preferences to small molecule properties. Patented TO-PRO-1 was synthesized via an optimized, three-step synthetic route. To investigate several assay parameters, a truncated sequence of the HIV-1 Transactivation Response Element (TAR) was used as a model system. Using TAR, the dissociation constant of TO-PRO-1, signal-to-background ratio, and effective RNA concentration for displacement were assessed. To confirm these optimized conditions, small molecules known to bind TAR were used to evaluate the displacement of TO-PRO-1 and binding curves were obtained. Currently, the TO-PRO-1 displacement assay is being utilized with various RNA libraries to screen diverse, RNA-biased small molecules and identify binding preferences for secondary structures of differing motif and size. At the same time, the physicochemical properties of small molecules are being analyzed and compared to elucidate guiding principles that bias recognition. Small molecules that are identified to bind a particular secondary structure are further investigated for sequence specificity and unique interactions are characterized by NMR techniques. At the conclusion of this work, the RNA structural preferences of hundreds of RNA-biased small molecules will be defined and the physicochemical properties that lead to specific interactions will be determined. Furthermore, by expanding the diversity of RNA motifs, small molecules, and methods used to identify and analyze these interactions, rational approaches for targeting RNA will be developed. Such approaches can result in selective small molecule probes to study the role of RNA in disease and the discovery of therapeutic leads.

Fluorescent indicator displacement assay to identify and characterize secondary structure-specific RNA: Small molecule interactions

254th National Meeting (2017)

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