Light harvesting FIT DNA hybridization probes for brightness-enhanced RNA detection

Abstract

Fluorogenic hybridization probes are essential tools in modern molecular biology techniques. They allow detection of specific nucleic acid molecules without the need to separate target-bound from unbound probes. To enable detection of targets at low concentration, fluorogenic probes should have high brightness. Here, we report the development of RNA hybridization probes (RNA FIT probes) that use smart quenching and a light harvesting principle to enhance the brightness of fluorescence signaling. The signaling mechanism is based on FRET between brightly emitting donor dyes and a fluorescent base surrogate, such as quinoline blue (QB) or thiazole orange (TO). In the single-stranded state, QB/TO nucleotides fluoresce weakly and quench the fluorescence of the donor dyes. Upon target recognition, QB/TO stack with adjacent base pairs, resulting in enhanced fluorescence quantum yields. The donor dyes are blue-shifted by only 5-20 nm relative to the QB/TO nucleotides, allowing simultaneous excitation of both dye groups with efficient energy transfer. The combined photon absorption results in exceptionally bright FIT probes. This feature facilitated the detection of RNA target in undiluted cell lysates. The present study examines the utilization of probes to detect mRNA targets in live T cells using flow cytometry.