Fluorescent indicator displacement assays to identify and characterize small molecule interactions with RNA

Fluorescent indicator displacement (FID) assays are an advantageous approach to convert receptors into optical sensors that can detect binding of various ligands. In particular, the identification of ligands that bind to RNA receptors has become of increasing interest as the roles of RNA in cellular processes and disease pathogenesis continue to be discovered. Small molecules have been validated as tools to elucidate unknown RNA functions, underscoring the critical need to rapidly identify and quantitatively characterize RNA:small molecule interactions for the development of chemical probes. The successful application of FID assays to evaluate interactions between diverse RNA receptors and small molecules has been facilitated by the characterization of distinct fluorescent indicators that reversibly bind RNA and modulate the fluorescence signal. The utility of RNA-based FID assays to both academia and industry has been demonstrated through numerous uses in high-throughput screening efforts, structure-activity relationship studies, and in vitro target engagement studies. Furthermore, the development, optimization, and validation of a variety of RNA-based FID assays has led to general guidelines that can be utilized for facile implementation of the method with new or underexplored RNA receptors. Altogether, the use of RNA-based FID assays as a general analysis tool has provided valuable insights into small molecule affinity and selectivity, furthering the fundamental understanding of RNA:small molecule recognition. In this review, we will summarize efforts to employ FID assays using RNA receptors and describe the significant contributions of the method towards the development of chemical probes to reveal unknown RNA functions.