Fluorescent intercalator displacement as a tool for RNA‐ligand discovery

Fluorescent intercalator displacement (FID) is a useful tool for identifying new nucleic‐acid‐binding ligands. The success of this method is based on the fact that it can be fashioned into a versatile high‐throughput assay, which can then be used to assess relative binding affinities of compounds to nucleic acids in a simple and efficient manner. FID is becoming an increasingly attractive method because it is a tag‐less approach; neither the nucleic acid nor the small molecule under investigation has to be modified. In this study, a modified FID method for screening RNA‐binding ligands was established, using 3‐methyl‐2‐((1‐(3‐(trimethylammonio)propyl)‐4‐quinolinylidene)methyl)benzothiazolium (TO‐PRO) as the fluorescent intercalator. Electrospray ionization mass spectrometry (ESI‐MS) was employed to investigate the FID mechanism. Our results provide molecular evidence that correlates a reduction in fluorescence intensity in the FID assay with displacement of the TO‐PRO dye molecule from structured RNA. The FID assay was then used to screen a variety of RNA‐binding ligands. With FID, it is possible to differentiate ligands that bind to a variety of RNA constructs from the ribosome and HIV (A‐site, h31, H69, and TAR) with moderate selectivity. This work is supported by the National Institute of Health (NIH)