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Abstract
Long-lived and photostable fluorescent probes for real-time monitoring of cell division, particularly for dynamic visualization of mitosis and interphase, are rare. We have developed a water soluble diazaoxatriangulenium cation based fluorescent probe, Nuc-DAOTA+, that meets these criteria. Nuc-DAOTA+ has a long fluorescence lifetime (~20 ns) and exclusively targets the nucleus for specific DNA binding in live cells, essential for real-time dynamic imaging of cell-division, including mitosis and interphase. Addition of dsDNA (0 – 50 μg/mL) to Nuc-DAOTA+, results in a red-shift (~10 nm) of the absorption and a blue shift (~5 nm) of the fluorescence maxima, along with an intensity increase in both. A very good linear correlation (R2 = 0.999) from plot between fluorescence intensity versus concentration of dsDNA (up to 30 μg/mL) resulted in a detection limit of 0.7 μg/mL. The Benesi – Hildebrand plot was used to calculate the binding constant between the Nuc-DAOTA+ and DNA, which was found to be 1.6 x 104 M-1 and mechanism of binding interactions was investigated using CD spectroscopy. The long fluorescence lifetime and excellent biocompatibility of Nuc-DAOTA+ enabled its use for real-time dynamic imaging of mitotic phases and interphase in live CHO (Chinese hamster ovary) cells by using confocal microscopy. In addition, the Nuc-DAOTA+ exhibited high photostability during photo-bleaching experiments and was successfully applied for fluorescence lifetime imaging microscopy (FLIM) and time gated imaging of mouse embryonic fibroblast 3T3 cell line.
Supplementary materials
Title
A Water Soluble, Long Fluorescent Lifetime DNA Probe for Real-Time Dynamic Visualization of Mitosis in Live Cells and Applicability for FLIM/Time-gated Imaging
Description
Here we are presenting a highly water soluble derivative Nuc-DAOTA+ of diazaoxatriangulenium dyes endowed with long fluorescence lifetime and high photostability, used for real-time dynamic visualization of complete cell-division, particularly mitosis. Nuc-DAOTA+ showed selective binding with DNA over RNA and BSA. CD spectroscopy data revealed the role of electrostatic, π-π stacking interactions and intercalations between the
dicationic Nuc-DAOTA+ probe and DNA molecules. Confocal microscopic studies showed the exclusive localization of the probe in the nucleus of live cells and dynamic monitoring of different mitotic phases during cell division by following in a single cell. The long fluorescence lifetime of the Nuc-DAOTA+ was also explored for the applications in the FLIM and time gated imaging.
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