Monomethine Cyanine Research Articles

Fluorogenic Monomethine Cyanine Dyes for Nucleolar RNA Imaging in Living Cells

Fluorogenic Monomethine Cyanine Dyes for Nucleolar RNA Imaging in Living Cells

Modifying the terminal phenyl group of monomethine cyanine dyes as a pathway to brighter nucleic acid probes

Three novel monomethine cyanine dyes were synthesized carrying electron donating groups to obtain even brighter nucleic acids probes.

Asymmetric Monomethine Cyanine Dyes with Hydrophobic Functionalities for Fluorescent Intercalator Displacement Assay.

A new green procedure has been applied for the synthesis and purification of asymmetric monomethine cyanine dyes. The photophysical properties of the newly synthesized compounds have been examined by combined application of spectroscopic and theoretical methods. The structural characteristics of the molecules and dimer formation were characterized by quantum chemical computation and juxtaposed to the aggregachromism in UV/Vis spectra. The applicability of the dyes as fluorogenic nucleic acid probes has been proven by fluorescence titration, and their binding constants have been calculated. The mode of ligand-dsDNA/RNA interaction was rationalized by means of CD spectroscopy, molecular docking analysis, and fluorescent intercalator displacement experiments.

Photonics of Some Monomethine Cyanine Dyes in Solutions and in Complexes with Biomolecules.

In search of new probes for biomolecules, the spectral fluorescent study of four monomethine cyanine dyes (MCD), both unsymmetrical and symmetrical, has been carried out in different organic solvents, in aqueous buffer solutions, and in the presence of DNA and HSA. The complexation of MCD with biomacromolecules leads to a steep growth of the fluorescence intensity. Complexes of MCD with dsDNA and HSA of various types were modeled in silico by molecular docking. Experiments on thermal dissociation of dsDNA in the presence of MCD showed the formation of intercalative complexes of MCD with DNA. Quenching of intrinsic fluorescence of HSA by MCD occurred with rate constants much higher than the diffusion limit, that is, in dye-HSA complexes. Effective constants of MCD complexation with the biomacromolecules were estimated. MCD 1 has the best characteristics as a possible fluorescent probe for dsDNA and can serve as a sensitive and selective probe for dsDNA in the presence of HSA. Photochemical properties of MCD complexed with DNA have been also studied. An increase in the quantum yield of the triplet states of MCD in complexes with DNA has been found, which may be important for using these dyes as potential candidates in photodynamic therapy.

Aggregation induced nucleic acids recognition by homodimeric asymmetric monomethyne cyanine fluorochromes in mesenchymal stem cells

In the light of recent retrovirus pandemics, the issue of discovering new and diverse RNA-specific fluorochromes for research and diagnostics became of acute importance. The great majority of nucleic acid-specific probes either do not stain RNA or cannot distinguish between DNA and RNA. The versatility of polymethine dyes makes them suitable as stains for visualization, analysis, and detection of nucleic acids, proteins, and other biomolecules. We synthesized the asymmetric dicationic homodimeric monomethine cyanine dyes 1,1′-(1,3-phenylenebis(methylene))bis(4-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)pyridin-1-ium) bromide (Т1) and 1,1′-(1,3-phenylenebis(methylene))bis(4-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)quinolin-1-ium) bromide (M1) and tested their binding specificity, spectral characteristics, membrane penetration in living and fixed cells, cellular toxicity, and stability of fluorescent emission. Mesenchymal cells have diverse phenotypes and extensive proliferation and differentiation properties. We found dyes T1 and M1 to show high photochemical stability in living mesenchymal stem cells from apical papilla (SCAP) with a strong fluorescent signal when bound to nucleic acids. We found M1 to perform better than control fluorochrome (Hoechst 33342) for in vivo DNA visualization. T1, on the other hand, stains granular cellular structures resembling ribosomes in living cells and after permeabilization of the nuclear membrane stains the nucleoli and not the chromatin in the nucleus. This makes T1 suitable for the visualization of structures rich in RNA in living and fixed cells.

Fluorescent Probes for Biomacromolecules Based on Monomethine Cyanine Dyes

Monomethine cyanine dyes (MCDs) are widely applied as biomolecular probes and stains in biochemical and biomedical research. This is based on the ability of MCDs to associate with biomolecules (mostly nucleic acids) with significant fluorescent growth. The present review considers the works devoted to the properties of MCDs and the influence of noncovalent interactions with biomacromolecules on their properties, as well as their use as noncovalent probes and stains for various biomacromolecules. The synthesis and photonics (photophysics and photochemistry; in particular, the generation of the triplet state) of MCDs are also considered. Areas and prospects of the practical applications of MCDs in biochemistry and biomedicine are discussed.

Selenium-Substituted Monomethine Cyanine Dyes as Selective G-Quadruplex Spectroscopic Probes with Theranostic Potential.

The binding interactions of six ligands, neutral and monocationic asymmetric monomethine cyanine dyes comprising benzoselenazolyl moiety with duplex DNA and RNA and G-quadruplex structures were evaluated using fluorescence, UV/Vis (thermal melting) and circular dichroism (CD) spectroscopy. The main objective was to assess the impact of different substituents (methyl vs. sulfopropyl vs. thiopropyl/thioethyl) on the nitrogen atom of the benzothiazolyl chromophore on various nucleic acid structures. The monomethine cyanine dyes with methyl substituents showed a 100-fold selectivity for G-quadruplex versus duplex DNA. Study results indicate that cyanines bind with G-quadruplex via end π-π stacking interactions and possible additional interactions with nucleobases/phosphate backbone of grooves or loop bases. Cyanine with thioethyl substituent distinguishes duplex DNA and RNA and G-quadruplex structures by distinctly varying ICD signals. Furthermore, cell viability assay reveals the submicromolar activity of cyanines with methyl substituents against all tested human cancer cell lines. Confocal microscopy analysis shows preferential accumulation of cyanines with sulfopropyl and thioethyl substituents in mitochondria and indicates localization of cyanines with methyl in nucleus, particularly nucleolus. This confirms the potential of examined cyanines as theranostic agents, possessing both fluorescent properties and cell viability inhibitory effect.

An Insight into Symmetrical Cyanine Dyes as Promising Selective Antiproliferative Agents in Caco-2 Colorectal Cancer Cells.

Cancer remains one of the diseases with the highest worldwide incidence. Several cytotoxic approaches have been used over the years to overcome this public health threat, such as chemotherapy, radiotherapy, and photodynamic therapy (PDT). Cyanine dyes are a class of compounds that have been extensively studied as PDT sensitisers; nevertheless, their antiproliferative potential in the absence of a light source has been scarcely explored. Herein, the synthesis of eighteen symmetric mono-, tri-, and heptamethine cyanine dyes and their evaluation as potential anticancer agents is described. The influences of the heterocyclic nature, counterion, and methine chain length on the antiproliferative effects and selectivities were analysed, and relevant structure–activity relationship data were gathered. The impact of light on the cytotoxic activity of the most promising dye was also assessed and discussed. Most of the monomethine and trimethine cyanine dyes under study demonstrated a high antiproliferative effect on human tumour cell lines of colorectal (Caco-2), breast (MCF-7), and prostate (PC-3) cancer at the initial screening (10 µM). However, concentration–viability curves showed higher potency and selectivity for the Caco-2 cell line. A monomethine cyanine dye derived from benzoxazole was the most promising compound (IC50 for Caco-2 = 0.67 µM and a selectivity index of 20.9 for Caco-2 versus normal human dermal fibroblasts (NHDF)) and led to Caco-2 cell cycle arrest at the G0/G1 phase. Complementary in silico studies predicted good intestinal absorption and oral bioavailability for this cyanine dye.

Formation of triplex rA/dA-containing nucleic acid helices induced by new thiazole orange analogues and their biological evaluation. H-aggregate formation conditioned by rA sequence

A series of 3 dicationic monomethine cyanine dyes was synthesized in moderate yields, applying structural alternations with respect to both the peripheral carrier of additional possitive charge - cyclic amine (pyrrolidine or pyridine) and the introduction of a halogen atom (fluorine or chlorine) at position 6 of the benzothiazole moiety. Structural elucidation of the target fluorophores was achieved employing a combination of 1D, 2D-NMR spectroscopy and ESI mass spectrometry in both positive and negative mode.One of the main goals of current investigation was to examine the impact of the introduced steric and electronic properties on interactions with various DNA and RNA. The cyanine dyes induced the triplex formation of consecutive rA/dA-containing nucleic acid helices. In particular, the chlorinated cyanine 3 stabilized all three triplex helices (dAdTdT, rAdTdT, rArUrU) much stronger than corresponding duplex nucleic acid forms (dAdT, rAdT, rArU). The same derivative, showed a strong tendency for H-aggregate formation in the presence of rA chain in single-stranded, double-stranded, or triple-stranded forms. The three monomethines exerted a significant effect on all tumour cell lines' viability, especially towards HeLa and A549 cell lines (5–6 μM). Co-localization studies revealed that fluorophores 2 and 3 preferentially accumulate in mitochondria, which, in addition to their proven influence on cell viability, makes them promising therapeutic leads.

Molecular Docking of Monomethine Cyanine Dyes to Lysozyme Amyloid Fibrils

Protein aggregation into highly ordered supramolecular aggregates is the hallmark of many degenerative diseases including the neurological disorders (Parkinson’s, Alzheimer’s, and Huntington’s diseases), type II diabetes, systemic amyloidosis, spongiform encephalopathies, etc. One of the simplest and effective methods for the identification and characterization of amyloid fibrils in vitro and the visualization of amyloid inclusions in vivo is based on the use of probes sensitive to the beta-pleated motifs. In the attempt to design new amyloid-sensing dyes or to optimization the existing molecules, it is crucial to have the sufficient knowledge of the molecular and atomic levels interactions in the binding sites. Among the especially useful methods available to provide the atomic-level insights into the mechanisms of various types of biomolecular interactions is molecular docking technique. In the present study, the molecular docking tool has been employed to investigate the interactions between the monomethine cyanine dyes and the lysozyme amyloid fibrils constructed from the K-peptide of lysozyme, GILQINSRW (residues 54–62 of the wild-type protein). Using the AutoDOCK and the protein-ligand interaction profiler PLIP it was found: i) monomethines interact with the fibril surface (with the aromatic residues on the top of β-sheet or with the edges of the β-sheet); ii) the dye binding is governed by the hydrophobic interactions, salt bridges and the hydrogen bonds between the aliphatic substituents on the nitrogen atom of benzothiazole part of dye molecules and the lysozyme amyloid fibril; iii) the variations in the cyanine structure and in the lysozyme amiloid twisting didn’t insert significant effect on the binding mode of cyanines.

Bright and Light-Up Sensing of Benzo[c,d]indole-oxazolopyridine Cyanine Dye for RNA and Its Application to Highly Sensitive Imaging of Nucleolar RNA in Living Cells.

Small molecular weight probes that can show a fluorescence signaling response upon binding to RNAs are promising for RNA imaging in living cells. Live-cell RNA imaging probes that can achieve a large light-up ability (>100-fold) and high Φbound value for RNA (>0.50) have been rarely reported to date. Here, benzo[c,d]indole-oxazolopyridine (BIOP), an unsymmetrical monomethine cyanine analogue, was newly developed as a bright and large light-up probe for imaging of nucleolar RNA in living cells. BIOP served as a yellow-emissive probe (λem = 570 nm) and exhibited a significant light-up response upon RNA binding (770-fold) with a high Φbound value (0.52). We demonstrated the advantages of BIOP over a commercially available RNA-staining probe, SYTO RNA select, for robust and sensitive RNA sensing by a systematic comparison of fluorescent properties for RNA. In addition, BIOP was found to possess high membrane permeability and low cytotoxicity in living cells. The examination of live-cell imaging revealed that BIOP exhibited emission in the nucleolus upon binding to nucleolar RNA much stronger than that of SYTO RNA select. Furthermore, BIOP facilitated the highly sensitive imaging of nucleolar RNA, in which 50 nM BIOP can stain nucleolar RNA in living cells with a 20 min incubation.

Interaction of Novel Monomethine Cyanine Dyes with Proteins in Native and Amyloid States

Molecular interactions between novel monomethine cyanine dyes and non-fibrilar and fibrilar proteins were assessed using fluorescence spectroscopy and molecular docking techniques. To this end, the fluorescence spectral properties of dyes have been explored in the buffer solution and in the presence of insulin and lysozyme in the native and amyloid states. It was observed that association of monomethines with the native and fibrillar proteins was accompanied with a significant enhancement of the fluorophore fluorescence, being more pronounced in the presence of aggregated insulin and lysozyme. The quantitative information about the dye-protein binding was obtained through approximating the experimental dependencies of the fluorescence intensity increase vs protein concentration by the Langmuir model. Analysis of the spectral properties and the binding characteristics of monomethines in the presence of the fibrillar insulin and lysozyme showed that the introduction of chloro- and fluorine-substitutients to the oxazole yellow derivatives, as well as the long aliphatic substitution on the nitrogen atom of the benzazole chromophore of YO-dyes had a negative impact on the dye amyloid specificity. Molecular docking studies showed that monomethines tend to form the most stable complexes with the B-chain residues Val 17, Leu17, Ala 14, Phe1, Gln 4 and Leu 6 and the A-chain residue Leu 13, Tyr 14, Glu 17 of non-fibrilar insulin and interact with the deep cleft of native lysozyme lined with both hydrophobic (Ile98, Ile 58, Thr108, Thr 62 and Thr 63 residues) and negatively (Asp101, Asp 107) charged residues. The wet surface groove Gln15_Glu17 and groove G2-L4/S8-W10 were found as the most energetically favorable binding sites for examined monomethine dyes in the presence of the insulin and lysozyme fibrils, respectively.

Theoretical Modeling of Absorption and Fluorescent Characteristics of Cyanine Dyes

The rational design of cyanine dyes for the fine-tuning of their photophysical properties undoubtedly requires theoretical considerations for understanding and predicting their absorption and fluorescence characteristics. The present study aims to assess the applicability and accuracy of several DFT functionals for calculating the absorption and fluorescence maxima of monomethine cyanine dyes. Ten DFT functionals and different basis sets were examined to select the proper theoretical model for calculating the electronic transitions of eight representative molecules from this class of compounds. The self-aggregation of the dyes was also considered. The pure exchange functionals (M06L, HFS, HFB, B97D) combined with the triple-zeta basis set 6-311+G(2d,p) showed the best performance during the theoretical estimation of the absorption and fluorescent characteristics of cyanine dyes.

A new auspicious scaffold for small dyes and fluorophores

An unprecedented chromophore, based on a nitrogen fused tricyclic heterocycle, is presented. This prototype molecule can be considered a “rigidified” monomethine cyanine dye and the central core is isoelectronic to pyridine-based BODIPY analogues, such as boron difluoride dipyridylmethene. The chromophore was synthesized and its photophysical properties examined. The new molecule can be considered the starting point to develop dyes, fluorophores or dual-emission fluorescent probes with excitation window in the green region of the spectrum.

Tricationic asymmetric monomeric monomethine cyanine dyes with chlorine and trifluoromethyl functionality – Fluorogenic nucleic acids probes

The nucleic acids staining ability of a series asymmetric monomeric monomethine cyanine dyes prepared by simple and reliable synthetic procedure is demonstrated. The dyes show ratiometric red shift of their longer wavelength absorption maxima. It is in the range 514–522 nm neat in TE buffer, 516–528 nm in the presence of DNA, and 518–524 nm in the presence of RNA. Practically the target dyes are not fluorescent in water solutions or possess negligible fluorescence, but become strongly fluorescent after contacting with nucleic acids. A considerable increase of the fluorescence intensity upon binding to RNA compared to dsDNA is observed particularly in the group of chlorine substituted analogues 1a-1c. The intensive ICD signal of dye 1a in the presence of RNA unequivocally proves that finding. Molecular docking studies have been performed to investigate the nature of binding mode of the dyes with DNA. 1a-1c are groove binders and 2b-2c are interacting with DNA through partial intercalation mode. The nature of electronic transitions and their manifestation in the electronic spectra are revealed by DFT and TDDFT calculations.

Simultaneous enumeration of CD34+ and CD45+ cells using EasyCounter image cytometer

Accurate counting of CD34-positive cells is important for successful hematopoietic stem cell transplantation that is applied to various diseases. The aim of this study was simultaneous counting of viable CD34+ (vCD34+) and CD45+ (vCD45+) cells in apheresis samples by automatic immunofluorescence counter – EasyCounter BC. CD34+ and CD45+ cells were counted using two conjugates anti-CD34 antibody – dR110 and anti-CD45 antibody – ATTO620, respectively. The conjugates were prepared by carbodiimide method. Dead nuclear cells were counted by using monomethine cyanine dye PO-TEDM 1. The linearity and reproducibility of EasyCounter BC for CD34+ cell counting were determined (R2 = 0.99; CV values for vCD34+ cells were 6.8 ÷ 8.5% and for vCD45+ cells 4.1 ÷ 7.2%). The obtained results by EasyCounter BC were compared with those by other two standard methods – flow cytometry (Guava easyCyte 8HT) and fluorescence microscopic method (Olympus BX51) with the same conjugates. Passing–Bablok regression was performed to determine the relationship between the results of the three methods, analyzing 43 apheresis samples. Correlation coefficients for vCD45+ and vCD34+ between EasyCounter BC and Olympus microscope were 0.987 and 0.982, respectively (P < 0.0001). Better results were obtained between EasyCounter BC and flow cytometer Guava, 0.998 for vCD45+ and 0.998 for vCD34+ (P < 0.0001).

Polycationic Monomeric and Homodimeric Asymmetric Monomethine Cyanine Dyes with Hydroxypropyl Functionality-Strong Affinity Nucleic Acids Binders.

New analogs of the commercial asymmetric monomethine cyanine dyes thiazole orange (TO) and thiazole orange homodimer (TOTO) with hydroxypropyl functionality were synthesized and their properties in the presence of different nucleic acids were studied. The novel compounds showed strong, micromolar and submicromolar affinities to all examined DNA ds-polynucleotides and poly rA–poly rU. The compounds studied showed selectivity towards GC-DNA base pairs over AT-DNA, which included both binding affinity and a strong fluorescence response. CD titrations showed aggregation along the polynucleotide with well-defined supramolecular chirality. The single dipyridinium-bridged dimer showed intercalation at low dye-DNA/RNA ratios. All new cyanine dyes showed potent micromolar antiproliferative activity against cancer cell lines, making them promising theranostic agents.

Monomethine cyanine probes for visualization of cellular RNA by fluorescence microscopy

We have studied spectral-luminescent properties of the monomethine cyanine dyes both in their free states and in the presence of either double-stranded deoxyribonucleic acids (dsDNAs) or single-stranded ribonucleic acids (RNAs). The dyes possess low fluorescence intensity in an unbound state, which is increased up to 479 times in the presence of the nucleic acids. In the presence of RNAs, the fluorescence intensity increase was stronger than that observed in the presence of dsDNA. Next, we have performed staining of live and fixed cells by all prepared dyes. The dyes proved to be cell and nuclear membrane permeant. They are photostable and brightly stain RNA-containing organelles in both live and fixed cells. The colocalization confirmed the specific nucleoli staining with anti-Ki-67 antibodies. The RNA digestion experiment has confirmed the selectivity of the dyes toward intracellular RNA. Based on the obtained results, we can conclude that the investigated monomethine cyanine dyes are useful fluorescent probes for the visualization of intracellular RNA and RNA-containing organelles such as nucleoli by using fluorescence microscopy.

Polymorphic Structures of Monolayer J-Aggregates of Two Monomethine Cyanine Dyes at the Meso- and Nanoscale

Polymorphic Structures of Monolayer J-Aggregates of Two Monomethine Cyanine Dyes at the Meso- and Nanoscale

Fluorogenic Indicator for Targeting HIV-1 TAR RNA: Evaluation for Binging and Signaling Functions of Monomethine Cyanine Dye Thiazole Orange and Its Application to FID Assay

Fluorogenic Indicator for Targeting HIV-1 TAR RNA: Evaluation for Binging and Signaling Functions of Monomethine Cyanine Dye Thiazole Orange and Its Application to FID Assay