carbocyanines and 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.

Topics: Benzothiazoles; Binding Sites; Carbocyanines; Coumarins; DNA; Fluorescent Dyes; Nucleic Acid Conformation; Thermodynamics

Hydrogen sulfide (H2S) is an important gaseous signaling agent mediated by many physiological processes and diseases. In order to explore its role in biological signaling, much effort has been focused on developing organic fluorescent probes to image H2S. However, these downconversion H2S probes are impractical for bio-imaging beyond a certain depth because of the short tissue penetration of UV/visible light (as an excitation source). In most circumstance, these probes are also not suitable for long-term assay due to photo-bleaching. Herein, a new design to detect H2S based on the coumarin-hemicyanine (CHC1)-modified upconversion nanophosphors is reported. This inorganic-organic integrated nanoprobe is demonstrated to display a fast response time with a large ratiometric upconversion luminescence (UCL) enhancement, and extraordinary photo-stability. CHC1-UCNPs not only can be used for ratiometric UCL monitoring of pseudo-enzymatic H2S production in living cells, but can also be used to identify the risk of endotoxic shock through ratiometric UCL imaging of tissue and measurement of endogenous H2S levels in plasma. The first ratiometric UCL H2S nanoprobe reported here may be further developed as the next-generation diagnostic tool for the detection of inflammatory-related diseases.

Topics: Animals; Carbocyanines; Coumarins; Disease Models, Animal; Fluorescent Dyes; HeLa Cells; Humans; Hydrogen Sulfide; Inflammation; Lipopolysaccharides; Luminescence; Magnetic Resonance Spectroscopy; Mice; Mice, Nude; Microscopy, Electron, Transmission; Nanoparticles; Nanostructures; Nanotechnology; Shock, Septic; Signal Transduction; Spectrophotometry, Ultraviolet; Spectroscopy, Near-Infrared

Cyanine dyes with indolium-coumarin linkages exhibit selective fluorescence enhancement for cyanide anions (CN(-)) via the nucleophilic interaction of CN(-) with their indolium carbon atoms. This facilitates rapid (detection time: 1 min) and sensitive (detection limit: 0.4 μM) sensing of CN(-) in aqueous media.

Topics: Anions; Carbocyanines; Coumarins; Fluorescent Dyes; Indoles; Nitriles; Quantum Theory; Spectrometry, Fluorescence; Water

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.

Topics: Base Pairing; Carbocyanines; Cell Cycle; Coumarins; DNA; Flow Cytometry; Fluorescence; Fluorescent Dyes; HEK293 Cells; HeLa Cells; Humans; Molecular Imaging; Thiazoles

Visualizing biomolecules by fluorescent tagging is a powerful method for studying their behaviour and function inside cells. We prepared and genetically encoded an unnatural amino acid (UAA) that features a bicyclononyne moiety. This UAA offered exceptional reactivity in strain-promoted azide-alkyne cycloadditions. Kinetic measurements revealed that the UAA reacted also remarkably fast in the inverse-electron-demand Diels-Alder cycloaddition with tetrazine-conjugated dyes. Genetic encoding of the new UAA inside mammalian cells and its subsequent selective labeling at low dye concentrations demonstrate the usefulness of the new amino acid for future imaging studies.

Topics: Alkynes; Azides; Bridged Bicyclo Compounds; Carbocyanines; Click Chemistry; Coumarins; Cycloaddition Reaction; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; HeLa Cells; Humans; Lysine; Microscopy, Fluorescence; Protein Engineering; Proteins; RNA, Transfer