Synthesis and Optical Properties of Cyanine Dyes as Fluorescent DNA Base Substitutions for Live Cell Imaging

The chromophore of thiazole orange (TO) and its derivative TO3 were incorporated synthetically as base surrogates into oligonucleotides using automated phosphoramidite chemistry. In comparison to TO, the TO3 chromophore contains an extended carbomethine bridge that shifts the absorption and emission significantly to the red. (S)-1-Aminopropane-2,3-diol served as an acyclic linker between the phosphodiesters. This linker was attached either to the quinoline or the thiazole nitrogen of the TO and TO3 dyes. The optical properties of TO and TO3 were studied in different DNA base environments and with different opposite bases. Both dyes as fluorescent DNA base substitutions show a brightness that is sufficient for bioanalytic and imaging applications. Additionally the TO3 dyes were combined as interstrand dimers, but in contrast to TO dimers, a red-shifted fluorescence was not observed. However, TO and TO3 can be combined to an interstrand chromophore pair and a DNA hybridization-dependent energy transfer process can be obtained between the modifications. As a result, the emission is shifted from the TO-typical value of 530 nm to 670 nm. This concept can be applied for fluorescence imaging to monitor DNA delivery as well as processing inside living cells by confocal microscopy. In contrast to the non-covalently binding TO dyes, the TO- and TO3-modified oligonucleotides are cell-permeable,