SYTO dyes and histoproteins—Myriad of applications

SINCE the first scientific publication (1) the vital nucleic acid stains for DNA and RNA of the SYTO family have gained substantial interest. More than 170 papers were published (source: ISI Web of Knowledge). This is a substantial number, since for comparison, the vital DNA dye HOECHST 33342, that is in use in cell sciences since the 1970s (2) has a publication record of 1,400 and the vital dye DRAQ5 that was first presented in 1999 (3) has a record of 24. This shows a substantial interest in this novel class of nucleic acid dyes. SYTO belongs to the class of substituted unsymmetrical cyanine dyes. This interesting class of molecules is thought to have multiple binding modes, including intercalation and charge interactions with the phosphate backbone, as well as potential for binding to the groove of the DNA double helix. One can think of thiazole orange (4) as a progenitor of the family – a dye with interesting properties that were modulated in order to develop improved versions with different properties. These dyes have structures that are flexible in aqueous solutions, and thus have very low fluorescence when unbound or free in solution, but become rigid when bound to nucleic acids, and as a result, exhibit very large increases in fluorescence in the bound complex. Although most of these dyes preferentially label double-stranded DNA, some of them have prevalence for single-stranded DNA and RNA. Their affinities can also be modulated, and many have binding values that are in the nanomolar range. Their permeability to live cells is mediated by modifying a number of the side groups of the molecules, and that is why there is variability in the behavior of the SYTO dye family, depending on structure. Today’s SYTO family consists of nearly 40 different dyes with absorption maxima ranging from violet (419 nm) to dark red (654 nm). The most popular dyes are SYTO13 (5), having a record of 67 publications, followed by SYTO16 (1) with 37. Most of the other family members in the blue (SYTO40-45), orange (SYTO80-86) and red have been so far seldom published. One reason may be the lack of appropriate excitation light sources. Still, the broad range of colors provided by the SYTO family could make them a very versatile tool for high-content analysis as the right color may be chosen for polychromatic and cytomic analysis (6). So, there is a major playground for research to determine the best fields of application for all SYTO dyes. These UVor visible light–excitable dyes can be used to stain RNA and DNA in both live and dead eukaryotic cells, as well as in Gram-positive and Gram-negative bacteria. SYTO dyes share several important characteristics. They exhibit permeability to virtually all cell membranes, including mammalian cells and bacteria and they have high molar absorptivity (extinction coefficients > 50,000 cmM at visible absorption maxima), very low intrinsic fluorescence (quantum yields typically < 0.01 when not bound to nucleic acids) and increased quantum yields (typically >0.4) when bound to nucleic acids. For comparison, quantum yield of HOECHST 33342 in water is 0.034 and 10 when bound to double-stranded DNA (7), that of DRAQ5 is 0.003 with and without binding to DNA (8). SYTO dyes differ from each other in one or more characteristics, including cell permeability, fluorescence enhancement upon binding nucleic acids, excitation and emission spectra, DNA/RNA selectivity and binding affinity. This makes them versatile tools for cell biologists. SYTO14 has been used to visualize the trafficking of endogenous RNA found in polyribosome complexes in living neurons (9). Other applications of SYTO include staining of plant chromosomes (10,11) or bacteria (12). In fact, nearly 50% of all SYTO related publications are on prokaryotic cells or yeast including applications as a substitute for Gram stain-