A Novel Method to Quantify RNA-Protein Interactions In Situ Using FMTRIP and Proximity Ligation

RNA binding proteins (RBP) and small RNAs regulate the editing, localization, stabilization, translation, and degradation of ribonucleic acids (RNAs) through their interactions with specific cis-acting elements within target RNAs. Here, we describe a novel method to detect protein-mRNA interactions, which combines FLAG-peptide modified, multiply-labeled tetravalent RNA imaging probes (FMTRIPs) with proximity ligation (PLA), and rolling circle amplification (RCA). This assay detects native RNA in a sequence specific and single RNA sensitive manner, and PLA allows for the quantification and localization of protein-mRNA interactions with single-interaction sensitivity.