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

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.