A theoretical and experimental study of two thiazole orange derivatives with single- and double-stranded oligonucleotides, polydeoxyribonucleotides and DNA.

The effect of interaction with DNA and oligonucleotides on the photophysical properties of two thiazole orange (TO) derivatives, with different side chains (-(CH2)3-N+(CH3)3 and -(CH2)6-I)) linked to the nitrogen of the quinoline ring of the thiazole orange, is presented here. The first one called TO-PRO1 is a commercially available dye, whereas the second one called TO-MET has been specially synthesized for further covalent binding to oligonucleotides with the aim of being used for specific in situ detection of biomolecular interactions. Both photophysical measurements and molecular calculations have been done to assess their possible mode of interaction with DNA. When dissolved in buffered aqueous solutions both derivatives exhibit very low fluorescence quantum yields of 8 x 10(-5) and 2 x 10(-4), respectively. However, upon binding to double-stranded DNA, large spectroscopic changes result and the quantum yield of fluorescence is enhanced by four orders of magnitude, reaching values up to phi F = 0.2 and 0.3, respectively, as a result of an intercalation mechanism between DNA base pairs. A modulation of the quantum yield is observed as a function of the base sequence. The two derivatives also bind with single-stranded oligonucleotides, but the fluorescence quantum yield is not so great as that when bound to double-stranded samples. Typical fluorescence quantum yields of 7 x 10(-3) to 3 x 10(-2) are observed when the dyes interact with short oligonucleotides, whereas the fluorescence quantum yield remains below 10(-2) when interacting with single-stranded oligonucleotides. This slight but significant quantum-yield increase is interpreted as a folding of the single strand around the dye, which reduces the internal rotation of the two heterocycles around the central methine bridge that links the two moieties of the dye. From these properties, it is proposed to link monomer covalently to oligonucleotides for the subsequent detection of target sequences within cells.