engleski

CHARACTERIZATION OF NOVEL MONOMETHINE CYANINE DYES AS INTERCALATING AGENTS AND FLUORESCENT PROBES

The novel asymmetric cyanine dyes were synthesized, their spectral characteristics and interaction with double stranded (ds)DNA have been investigated. Used as sensitizers for colour photography and recently as fluorescent labels for biomolecules and nucleic acids, cyanines are characterized by their high molar absorptivity and relatively high affinity for nucleic acids. Upon binding to double helical DNA, dyes exhibit large fluorescence emission intensity enhancements. Additionally, cyanine dyes are used as molecular probes in detection of specific DNA sequences important for genetic screening, clinical diagnostics and microchip analysis of gene expression, whereas fluorescence is the most common form of detection. In this work, we have analysed fluorimetric, spectrophotometric, thermal melting, circular dichroism measurements in order to elucidate the mode of binding and the specific affinity for different polynucleotide secondary structure motifs. Our further research was focused on elucidating the biological activity of novel cyanines with potential antitumor activity. We have screened our own designed and synthesized asymmetric dicationic monomethine cyanine dyes (AK-A1, AK-A2, AK-A3, AK-A4 and AK-A7) for antiproliferative activity (MTT assay) and showed negligible activity on human carcinogenic lung (H460) and breast cancer (MCF-7) cells. AK-A7 dye showed weak antiproliferative activity (IC50 = 7, 74 x 10-4 M) toward H460 cells. Considering that the spectroscopic measurements indicated intercalative mode of binding with the DNA, low antiproliferation activity was a surprising result. Having two positive charges, safe assumption was that the dyes do not pass the semi permeable membrane of mammalian cells. Therefore, dyes AK-A1 (abs/em=483/516 nm) and AK-A4 (abs/em=510/533 nm) have been further checked by confocal microscopy for subcellular localization and the results suggested their mitochondrial localization in human cells. Commercial dyes such as Mitotracker Deep Red and DiOC6(3) were used for colocalization experiments. Another colocalization strategy involved transient transfection of human cancerogenic cells with pMito-ECFP vector. After adding specific dyes (AK-A1 and AK-A4) and their incubation, colocalization of cyan fluorescent protein produced in mitochondria and dyes was successfully observed. The correlation of the fluorophores colocalization was calculated using Pearson correlation coefficient (PCC). Image J (Wayne Rasband, NIH, Bethesda, USA) and LAS X Leica Microsystems software packages have been used for overlaying of images and calculation of PCC. To conclude, tested cyanine dyes are excellent imaging agents with low cytotoxicity and no photobleaching effects observed.

cyanine dyes; DNA binding intercalator; fluorescence; cells; bioimaging

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