Light-Harvesting FIT Probes for mRNA Detection in Live T Cells

Fluorogenic hybridization probes allow the detection and localization of specific mRNA molecules in living cells without the need for genetic engineering. However, with the fluorescence properties of currently available oligonucleotide probes, detection in live cells is difficult when the mRNA target is expressed at medium levels, let alone at low copy numbers. Here, we report the development of RNA hybridization probes (RNA FIT probes) that use a light harvesting principle to enhance the brightness of fluorescence signaling. The probes contain a fluorescent base surrogate, such as quinoline blue (QB) or thiazole orange (TO), which is weakly fluorescent in the single-stranded state. In this state, the QB/TO nucleotides quench the fluorescence of auxiliary dyes. Upon target recognition, QB/TO stack with adjacent base pairs, resulting in enhanced fluorescence quantum yields. The light harvesting auxiliary 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 and enabled the detection of T cell receptor mRNA in live T cells by flow cytometry. We show that one T cell clone can be distinguished from another based on TCR mRNA expression using a single light-harvesting FIT probe.