Double FIT hybridization probes - towards enhancing brightness, turn-on and specificity of RNA detection

Efficient fluorogenic hybridization probes combine high brightness and specificity of fluorescence signaling with large turn-on of fluorescence. Herein, we present an approach to enhance signaling by combining two identical fluorescence base surrogates in FIT² probes. Provided there is a suitable positioning of dyes, target-bound FIT² probes emit brighter than mono dye probes, while dye-dye contact in the single stranded state provides opportunities for decreasing background fluorescence. The probes were used to explore the single nucleotide-specific detection of a C → U edited RNA of the glycine receptor (GlyR). We observed strong self-quenching upon single base mismatched hybridization of FIT² probes, which helped in distinguishing edited from unedited RNA target in cell lysates.