Designed thiazole orange nucleotides for the synthesis of single labelled oligonucleotides that fluoresce upon matched hybridization

Probe molecules that enable the detection of specific DNA sequences are used in diagnostic and basic research. Most methods rely on the specificity of hybridization reactions, which complicates the detection of single base mutations at low temperature. Significant efforts have been devoted to the development of oligonucleotides that allow discrimination of single base mutations at temperatures where both the match and the mismatch probe-target complexes coexist. Oligonucleotides that contain environmentally sensitive fluorescence dyes such as thiazole orange (TO) provide single nucleotide specific fluorescence. However, most previously reported dye-DNA conjugates showed only little if any difference between the fluorescence of the single and the double stranded state. Here, we introduce a TO-containing acyclic nucleotide, which is coupled during automated oligonucleotide synthesis and provides for the desired fluorescence-up properties. The study reveals the conjugation mode as the most important issue. We show a design that leads to low fluorescence of the unbound probe (background) yet permits TO to adopt fluorescent binding modes after the probe-target complex has formed. In these probes, TO replaces a canonical nucleobase. Of note, the fluorescence of the "TO-base" remains low when a base mismatch is positioned in immediate vicinity.