LNA-enhanced DNA FIT-probes for multicolour RNA imaging

The simultaneous imaging of different RNA molecules in homogeneous solution is a challenge and requires optimisation to enable unambiguous staining of intracellular RNA targets. Our approach relies on single dye forced intercalation (FIT) probes, in which a visco-sensitive reporter of the thiazole orange (TO) family serves as a surrogate nucleobase and provides enhancements of fluorescence upon hybridisation. Previous FIT probes spanned the cyan and green emission range. Herein, we report for the first time chromophores for FIT probes that emit in the red range (above 600 nm). Such probes are valuable to overcome cellular auto-fluorescent background and enable multiplexed detection. In order to find suitable chromophores, we developed a submonomer approach that facilitated the rapid analysis of different TO family dyes in varied sequence positions. A carboxymethylated 4,4'-methine linked cyanine, which we named quinoline blue (QB), provided exceptional response characteristics at the 605 nm emission maximum. Exceeding previously reported base surrogates, the emission of the QB nucleotide intensified by up to 195-fold upon binding of complementary RNA. Owing to large extinction coefficients and quantum yields (up to ε = 129.000 L mol^-1 cm^-1 and Φ = 0.47, respectively) QB-FIT probes enable imaging of intracellular mRNA. A mixture of BO-, TO- and QB-containing FIT probes allowed the simultaneous detection of three different RNA targets in homogenous solution. TO- and QB-FIT probes were used to localize oskar mRNA and other polyadenylated mRNA molecules in developing oocytes from Drosphila melanogaster by means of wash-free fluorescent in situ hybridisation and super resolution microscopy (STED).