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 FIT(2) probes. Provided there is a suitable positioning of dyes, target-bound FIT(2) 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 FIT(2) probes, which helped in distinguishing edited from unedited RNA target in cell lysates.