Abstract : New fluorescent oligonucleotide probes for the specific recognition of double-stranded nucleic acid structures were obtained. These triple helix forming oligonucleotides were linked through their 5'-or/and 3'-ends to a series of cyanine monomethine labels, with intercalating properties, via polymethylene linkers. In all cases, the hybridization of the labelled oligonucleotides with their double-stranded targets induced a strong increase of the fluorescence emission. The fluorescence increase was dependent on both the cyanine and the parameters of its linkage to the oligonucleotides (linker size and positions of its attachment to both the label and the oligonucleotide). In the presence of the single-stranded target, the fluorescence increase was weak. The triplex structures were stabilized by the presence of the labels. The probes involving thiazole orange attached by the benzothiazole ring and an octamethylene link provided the best balanced properties in terms of triplex stabilization, fluorescence intensity and fluorescence enhancement upon hybridization with the double-stranded target.
With the aim to obtain new probes that can be used in cellular and in vivo experiments, squaraine dyes were synthesized and used, for the first time to our knowledge, for the labelling of oligonucleotides. These squaraine-oligonucleotide conjugates are detectable beyond 650 nm and possess good quantum yields. The photophysical properties of the conjugates at 37 °C and in moderate acidic conditions can be strongly improved by the incorporation of fluorine atoms on the squaraine dye.