A Thiazole Coumarin (TC) turn-on fluorescence probe for AT-base pair detection and multipurpose applications in different biological systems

Sequence-specific recognition of DNA by small turn-on fluorescence probes is a promising tool for bioimaging, bioanalytical and biomedical applications. Here, the authors report a novel cell-permeable and red fluorescent hemicyanine-based Thiazole Coumarin (TC) probe for DNA recognition, nuclear staining and cell cycle analysis. TC exhibited strong fluorescence enhancement in the presence of DNA containing AT-base pairs, but did not fluoresce with GC sequences, single-stranded DNA, RNA and proteins. The fluorescence staining of HeLa S3 and HEK 293 cells by TC followed by DNase and RNase digestion studies depicted the selective staining of DNA in the nucleus over the cytoplasmic region. Fluorescence-Activated Cell Sorting (FACS) analysis by flow cytometry demonstrated the potential application of TC in cell cycle analysis in HEK 293 cells. Metaphase chromosome and malaria parasite DNA imaging studies further confirmed the in vivo diagnostic and therapeutic applications of probe TC. Probe TC may find multiple applications in fluorescence spectroscopy, diagnostics, bioimaging and molecular and cell biology

Dual DNA binding mode of a turn-on red fluorescent probe thiazole coumarin.

Turn-on fluorescent probes show enhanced emission upon DNA binding, advocating their importance in imaging cellular DNA. We have probed the DNA binding mode of thiazole-coumarin (TC) conjugate, a recently reported hemicyanine-based turn-on red fluorescent probe, using a number of biophysical techniques and a series of short oligonucleotides. TC exhibited increased fluorescence anisotropy and decreased absorbance (~50%) at low [DNA]/[TC] ratio. Although the observed hypochromicity and the saturating value of [DNA base pair]:[TC] ratio is consistent with a previous study that suggested intercalation to be the DNA binding mode of TC, a distinctly different and previously unreported binding mode was observed at higher ratios of [DNA]:[TC]. With further addition of DNA, only oligonucleotides containing AnTn or (AT)n stretches showed further change-decreased hypochromicity, red shifted absorption peaks and concomitant fluorescence enhancement, saturating at about 1:1 [DNA]: [TC]. 1H-NMR chemical shift perturbation patterns and H1'-H6/H8 NOE cross-peaks of the 1:1 complex indicated minor groove binding by TC. ITC showed the 1:1 DNA binding event to be endothermic (ΔH° ~ 2 kcal/mol) and entropy driven (ΔS° ~ 32 cal/mol/K). Taken together, the experimental data suggest a dual DNA binding mode by TC. At low [DNA]/[TC] ratio, the dominant mode is intercalation. This switches to minor groove binding at higher [DNA]/[TC], only for sequences containing AnTn or (AT)n stretches. Turn-on fluorescence results only in the previously unreported minor groove bound state. Our results allow a better understanding of DNA-ligand interaction for the newly reported turn-on probe TC

A molecular beacon-based DNA switch for reversible pH sensing in vesicles and live cells

In this Communication, a molecular beacon-based DNA switch (LMB) is developed as an efficient and reversible pH sensing probe. Remarkably, LMB exhibited reversible structural transition between the closed (molecular beacon) and open (A-motif) states very efficiently in synthetic vesicles and live cells without the need for any transfection agents