FluoTag-EMSA: a fast and accessible quantitative method to assess RNA-binding specificity using 3′-tagged hybrid duplexes

Electrophoretic mobility shift assay (EMSA) is widely used to study RNA-protein interactions but remains limited by safety, cost, and time constraints associated with radioactive or covalent fluorescent labeling. Here, a novel method termed FluoTag-EMSA overcomes these hurdles by providing the RNA 3'ends with short sequence tags that can be hybridized to specific complementary fluorescent DNA probes, eliminating the need for chemical or enzymatic labeling steps. Specifically, two independent sequence tags are described that do not disrupt RNA folding and allow efficient annealing to complementary oligonucleotides carrying far-red/near-infrared dyes (700 nm and 800 nm), enabling direct in-gel fluorescence detection. Both tag/probe duplexes exhibit identical thermodynamic properties and can be used interchangeably. FluoTag-EMSA is a streamlined and reproducible non-radioactive alternative method for studying RNA-protein interactions without the need for specialized equipment.