Thiazole orange and Cy3: Improvement of fluorescent DNA probes with use of short range electron transfer

Thiazole orange was synthetically incorporated into oligo-nucleotides by using the corresponding phosphoramidite as the building block for automated DNA synthesis. Due to the covalent fixation of the TO dye as a DNA base surrogate, the TO-modified oligonucleotides do not exhibit a significant increase of fluorescence upon hybridization with the counterstrand. However, if 5-nitroindole (NI) is present as a second artificial DNA base (two base pairs away from the TO dye) a fluorescence increase upon DNA hybridization can be observed. That suggests that a short-range photoin-duced electron transfer causes the fluorescence quenching in the single strand. The latter result represents a concept that can be transferred to the commercially available Cy3 label. It. enables the Cy3 fluorophore to display the DNA hybridization by a fluorescence increase that is normally not observed with this dye.