Symmetric cyanine dyes for detecting nucleic acids

A novel approach to the design of sensitive fluorescent probes for nucleic acids detection is proposed. Suitable modifications of tri- and pentamethine cyanine dyes in the polymethine chain and/or in the heterocyclic residues can result in a significant decrease in unbound dye fluorescence intensity and an increase in dye emission intensity in the presence of DNA compared to the unsubstituted dye. The sharp enhancement in the fluorescence intensity upon dye interaction with double-stranded DNA permits the application of the modified tri- and pentamethine dyes as fluorescent probes in double-stranded DNA detection in homogeneous assays.     

Interaction of cyanine dyes with nucleic acids. XXI. Arguments for half-intercalation model of interaction

The spectral luminescent properties of two groups of monomethine cyanine dyes were studied in the presence of DNA. The first group included five dyes with 5,6-methylenedioxy-[d]-benzo-1,3-thiazole heterocycle and their unsubstituted analogs. Five monomethine pyrylium cyanines and their N-methyl-pyridine analogs were included in the second group. In each pair the pyrylium and pyridine dyes had similar geometry but differed in charge density distribution. The results presented some evidence in favor of the half-intercalation interaction mode between the studied dyes and DNA. When the benzothiazole residue had the lowest electron donor ability between the two heterocycles in the dye molecule, its substitution with the bulky methylenedioxy group led to a significant decrease in fluorescence enhancement of the dye-DNA complex. On the contrary, when the substituents that create steric hindrance (e.g., methylenedioxy and methyl groups) were introduced into the heterocycle with the higher electron donor ability, the fluorescence enhancement value of the dye-DNA complex was virtually unchanged. The changes in the Stock's shift values upon the formation of the dye-DNA complexes were in agreement with the proposed half-intercalation model. Interestingly, in the dye-DNA complexes the pyrylium dyes probably resided in a place similar to the pyridine ones. It is possible that the benzothiazole (or benzooxazole) ring intercalated between the DNA bases and the pyrylium (or pyridine) residue was located in the DNA groove closer to the phosphate backbone.     

Photoinactivation of bacteriophage PM2 by cyanine dyes]

Photoinactivation of the lipid-containing bacteriophage PM2 by visible light and cyanine dyes (carbo- and dicarbocyanines), aluminum phtalocyanine tetrasulfonate and methylene blue was studied. It was concluded that cyanine dye aggregates adsorbed on phage particles and oxygen are essential for phage photoinactivation.     

Interaction of cyanine dyes with nucleic acids: XXXI. Using of polymethine cyanine dyes for the visualization of DNA in agarose gels

Fifteen polymethine cyanine dyes were studied as fluorescent stains for DNA in electrophoretic gels. Among studied cyanines, two dyes CPent V and CCyan 2-O most effectively visualized covalently closed and linear double-stranded DNA molecules in gels under standard conditions using UV-illumination, green filter and black-and-white photo film. Ethidium bromide was 1.2-1.6 times more effective as compared to cyanine dyes in staining of DNA in the concentration range of 8-18 ng, while studied cyanines were more sensitive to DNA quantity above 50 ng.     

Sequence-dependent fluorescence of cyanine dyes on microarrays

Cy3 and Cy5 are among the most commonly used oligonucleotide labeling molecules. Studies of nucleic acid structure and dynamics use these dyes, and they are ubiquitous in microarray experiments. They are sensitive to their environment and have higher quantum yield when bound to DNA. The fluorescent intensity of terminal cyanine dyes is also known to be significantly dependent on the base sequence of the oligonucleotide. We have developed a very precise and high-throughput method to evaluate the sequence dependence of oligonucleotide labeling dyes using microarrays and have applied the method to Cy3 and Cy5. We used light-directed in-situ synthesis of terminally-labeled microarrays to determine the fluorescence intensity of each dye on all 1024 possible 5'-labeled 5-mers. Their intensity is sensitive to all five bases. Their fluorescence is higher with 5' guanines, and adenines in subsequent positions. Cytosine suppresses fluorescence. Intensity falls by half over the range of all 5-mers for Cy3, and two-thirds for Cy5. Labeling with 5'-biotin-streptavidin-Cy3/-Cy5 gives a completely different sequence dependence and greatly reduces fluorescence compared with direct terminal labeling.     

Facile synthesis of symmetric, monofunctional cyanine dyes for imaging applications

Efficient syntheses of several members of a new class of symmetric, monocarboxylate-functionalized cyanine dyes have been developed. The synthesis is a simple two-step method, typically with greater than 60% yield and easy final product purification. The new monocarboxylate-functionalized cyanine dyes exhibit excellent water solubility and similar excitation and emission properties to those of Cy5 and Alexa Fluor 647. The application of the new dyes in cellular imaging has been demonstrated through direct conjugating of the dye with an antibody, then imaging of microtubules inside cells, visualized by near-infrared fluorescence microscopy.     

Pontentiometric cyanine dyes are sensitive probes for mitochondria in intact plant cells : kinetin enhances mitochondrial fluorescence

Selected fluorescent dyes were tested for uptake by mitochrondria in intact cells of barley, maize, and onion. The cationic cyanine dye 3,3′-diheptyloxacarbocyanine iodide [DiOC₇(3)] accumulated in mitochondria within 15 to 30 minutes without appreciable staining of other protoplasmic constituents. The number, shape, and movement of the fluorescent mitochondria could be seen readily, and the fluorescence intensity of the mitochondria could be monitored with a microscope photometer. Fluorescence was eliminated in 1 to 5 minutes by the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) indicating that maintenance of dye concentration was dependent on the inside-negative transmembrane potential maintained by functional mitochondria. Fluorescence of prestained mitochondria was enhanced within 5 to 10 minutes after addition of 0.1 millimolar kinetin to cells. The fluorescence in kinetintreated cells was dissipated by CCCP. These results suggest that kinetin interacted with respiratory processes resulting in higher potential across the mitochondrial membrane.     

Groove-binding unsymmetrical cyanine dyes for staining of DNA: syntheses and characterization of the DNA-binding

Two new crescent-shaped unsymmetrical cyanine dyes have been synthesised and their interactions with DNA have been investigated by different spectroscopic methods. These dyes are analogues to a minor groove binding unsymmetrical cyanine dye, BEBO, recently reported by us. In this dye, the structure of the known intercalating cyanine dye BO was extended with a benzothiazole substituent. To investigate how the identity of the extending heterocycle affects the binding to DNA, the new dyes BETO and BOXTO have a benzothiazole group and a benzoxazole moiety, respectively. Whereas BEBO showed a heterogeneous binding to calf thymus DNA, linear and circular dichroism studies of BOXTO indicate a high preference for minor groove binding. BETO also binds in the minor groove to mixed sequence DNA but has a contribution of non-ordered and non-emissive species present. A non-intercalative binding mode of the new dyes, as well as for BEBO, is further supported by electrophoresis unwinding assays. These dyes, having comparable spectral properties as the intercalating cyanine dyes, but bind in the minor groove instead, might be useful complements for staining of DNA. In particular, the benzoxazole substituted dye BOXTO has attractive fluorescence properties with a quantum yield of 0.52 when bound to mixed sequence DNA and a 300-fold increase in fluorescence intensity upon binding.     

Specific fluorescent detection of fibrillar alpha-synuclein using mono- and trimethine cyanine dyes

With the aim of searching of novel amyloid-specific fluorescent probes the ability of series of mono- and trimethine cyanines based on benzothiazole, pyridine and quinoline heterocycle end groups to recognize fibrillar formations of alpha-synuclein (ASN) was studied. For the first time it was revealed that monomethine cyanines can specifically increase their fluorescence in aggregated ASN presence. Dialkylamino-substituted monomethine cyanine T-284 and meso-ethyl-substituted trimethine cyanine SH-516 demonstrated the higher emission intensity and selectivity to aggregated ASN than classic amyloid stain Thioflavin T, and could be proposed as novel efficient fluorescent probes for fibrillar ASN detection. Studies of structure-function dependences have shown that incorporation of amino- or diethylamino- substituents into the 6-position of the benzothiazole heterocycle yields in a appearance of a selective fluorescent response to fibrillar alpha-synuclein presence. Performed calculations of molecular dimensions of studied cyanine dyes gave us the possibility to presume, that dyes bind with their long axes parallel to the fibril axis via insertion into the neat rows (so called 'channels') running along fibril.     

Trimethine cyanine dyes as fluorescent probes for amyloid fibrils: The effect of N,N′-substituents

The effect of various N,N′-substituents in the molecule of benzothiazole trimethine cyanine dye on its ability to sense the amyloid aggregates of protein was studied. The dyes are low fluorescent when free and in the presence of monomeric proteins, but their emission intensity sharply increases in complexes with aggregated insulin and lysozyme, with the fluorescence quantum yield reaching up to 0.42.     

Novel cyanine dyes with vinylsulfone group for labeling biomolecules

Novel vinylsulfone cyanine dyes (em. 550-850 nm) were designed and synthesized for fluorescent labeling of biomolecules via 1,2-addition reaction in aqueous conditions. Due to the virtue of chemical structures of both fluorophore and reactive group, these dyes could be significantly stable and reactive in various aqueous/organic conditions. A wide variety of pH, temperature, buffer concentration, and protein were tested for the optimal labeling condition.     

Synthesis, photophysical, electrochemical, tumor-imaging, and phototherapeutic properties of purpurinimide-N-substituted cyanine dyes joined with variable lengths of linkers

Purpurinimide methyl esters, bearing variable lengths of N-substitutions, were conjugated individually to a cyanine dye with a carboxylic acid functionality. The results obtained from in vitro and in vivo studies showed a significant impact of the linkers joining the phototherapeutic and fluorescence imaging moieties. The photosensitizer-fluorophore conjugate with a PEG linker showed the highest uptake in the liver, whereas the conjugate linked with two carbon units showed excellent tumor-imaging and PDT efficacy at 24 h postinjection. Whole body imaging and biodistribution studies at variable time points portrayed enhanced fluorescent uptake of the conjugates in the tumor compared to that in the skin. Interestingly, the conjugate with the shortest linker and the one joining with two carbon units showed faster clearance from normal organs, e.g., the liver, kidney, spleen, and lung, compared to that in tumors. Both imaging and PDT efficacy of the conjugates were performed in BALB/c mice bearing Colon26 tumors. Compared to the others, the short linker conjugate showed poor tumor fluorescent properties and as a corollary does not exhibit the dual functionality of the photosensitizer-fluorophore conjugate. For this reason, it was not evaluated for in vivo PDT efficacy. However, in Colon26 tumor cells (in vitro), the short linker was highly effective. Among the conjugates with variable linkers, the rate of energy transfer from the purpurinimide moiety to the cyanine moiety increased with deceasing linker length, as examined by femtosecond laser flash photolysis measurements. No electron transfer from the purpurinimide moiety to the singlet excited state of the cyanine moiety or from the singlet excited state of the cyanine moiety to the purpurinimide moiety occurred as indicated by a comparison of transient absorption spectra with spectra of the one-electron oxidized and one-electron reduced species of the conjugate obtained by spectroelectrochemical measurements.     

Rational approach to select small peptide molecular probes labeled with fluorescent cyanine dyes for in vivo optical imaging

We demonstrate that the structure of carbocyanine dyes, which are commonly used to label small peptides for molecular imaging and not the bound peptide, controls the rate of extravasation from blood vessels to tissue. By examining several near-infrared (NIR) carbocyanine fluorophores, we demonstrate a quantitative correlation between the binding of a dye to albumin, a model plasma protein, and the rate of extravasation of the probe into tissue. Binding of the dyes was measured by fluorescence quenching of the tryptophans in albumin and was found to be inversely proportional to the rate of extravasation. The rate of extravasation, determined by kurtosis from longitudinal imaging studies using rodent ear models, provided a basis for quantitative measurements. Structure-activity studies aimed at evaluating a representative library of NIR fluorescent cyanine probes showed that hydrophilic dyes with binding constants several orders of magnitude lower than their hydrophobic counterparts have much faster extravasation rate, establishing a foundation for rational probe design. The correlation provides a guideline for dye selection in optical imaging and a method to verify if a certain dye is optimal for a specific molecular imaging application.     

Red-fluorescent argininamide-type NPY Y1 receptor antagonists as pharmacological tools

Fluorescently labelled NPY Y(1) receptor (Y(1)R) ligands were synthesized by connecting pyrylium and cyanine dyes with the argininamide-type Y(1)R antagonist core structure by linkers, covering a wide variety in length and chemical nature, attached to the guanidine group. The most promising fluorescent probes had Y(1)R affinities (radioligand binding) and antagonistic activities (calcium assay) in the one- to two-digit nanomolar range. These compounds turned out to be stable under assay conditions and to be appropriate for the detection of Y(1)Rs by confocal microscopy in live cells. To improve the signal-to-noise ratio by shifting the emission into the near infrared, a new benzothiazolium-type fluorescent cyanine dye (UR-DE99) was synthesized and attached to the parent antagonist via a carbamoyl linker yielding UR-MK131, a highly potent fluorescent Y(1)R probe, which was also successfully applied in flow cytometry.     

Synthesis of some novel dimethine,bis-dimethine cyanine dyes and octacosamethine cyanine dyes endowed with promising biological potency against (HepG2), (Hela), (MCF-7), (MIA), (SN12C) and (H358) cell lines

Successfully, one step two component synthesis of dimethine cyanine dyes, bis-dimethine cyanine dyes and icosamethine cyanine dyes 2–10via reaction of pyridinium salt 1 with some different aldehydes hope to obtain these compounds with enhanced biological potency as antitumor agents against spontaneous liver (HepG2), cervical (Hela), breast (MCF-7), pancreas (MIA), kidney (SN12C) and lung (H358). The impact of substituted drugs on the tumor cells was reflected by means of structure activity relationship (SAR). Among these dyes, icosamethine cyanine dye 8 recorded an excellent activity toward all the tested cell lines. The newly destined drugs were identified and emphasized by spectroscopy and elemental analyses.     

Staining with chromoxane cyanine R.

Since Pearse in 1957 introduced chromoxane cyanine R as a dual nuclear and cytoplasmic stain there have appeared numerous procedures for use of this dye. These have differed widely, sharing in common mainly the implication that each is best. A defendable procedure has been developed on an experimental basis and is reported here. Four stock solutions are needed: (1) a 0.2% solution of chromoxane cyanine R in 0.5% aqueous H2SO4 (v/v); boil this solution for 5 min, (2) 10% FeCl3 in 3% HCl, (3) 1% aqueous HN4OH, and (4) 1% HCl in 70% ethanol. The staining solution: 40 ml of dye solution, 2 ml of FeCl3 solution, 8 ml H2O. Dewax and hydrate sections and stain for 10 min. If a myelin sheath stain is desired differentiate for 1 min in solution (3). For a nuclear stain differentiate for 1 min in solution (4). The nuclear stain when counterstained with eosin closely resembles the routine hematoxylin and eosin. Histochemical tests show that the functional group for myelin staining contains nitrogen, and probably hydrogen bonding is involved. The nuclear stain involves a different functional group and possibly neither electrostatic nor hydrogen bonding.     

Simple one-pot preparation of water-soluble, cysteine-reactive cyanine and merocyanine dyes for biological imaging

A simple one-pot-procedure for preparation of protein-reactive, water-soluble merocyanine and cyanine dyes has been developed. The 1-(3-ammoniopropyl)-2,3,3-trimethyl-3H-indolium-5-sulfonate bromide (1) was used as a common starting intermediate. The method allows easy preparation of dyes with chloro- and iodoacetamide side chains for covalent attachment to cysteine. By placing a sulfonato group directly on the dye fluorophore system, dyes with high fluorescence quantum yields in water were generated. Both iodo- and chloroacetamido derivatives were shown to be useful in protein labeling. Less reactive chloroacetamides will be preferential for selective labeling of the most reactive cysteines.     

Fluorescent cyanine dyes for the quantification of low amounts of dsDNA

In this research six cyanine fluorophores for the quantification of dsDNA in the pg-ng range, without amplification, are compared under exactly identical conditions: EvaGreen, SYBR Green, PicoGreen, AccuClear, AccuBlue NextGen and YOYO-1. The fluorescence intensity as a function of the amount of dsDNA is measured at the optimal wavelengths for excitation and emission and for each dye the limit of detection and the response linearity at low levels of dsDNA are determined. No linear range was found for SYBR Green and YOYO-1 for pg-ng quantities of dsDNA. EvaGreen, PicoGreen, AccuClear and AccuBlue NextGen show good linearity in the pg-ng range. AccuClear exhibits the widest linear range of 3 pg–200 ng, whereas AccuBlue NextGen turned out to have the highest sensitivity of the tested dyes with a limit of detection of 50 pg.     

The use of fluorescent dyes to measure membrane potentials: a response

The use of fluorescent cyanine dyes to estimate membrane potential in cell suspensions has been considered. Several problems related tot he application of the dyes have been reviewed. These problems include: 1) alteration of the membrane potential (Em) and factors involved in establishing Em by the dyes themselves, 2) the effects of altered energy metabolism on the fluorescent response of the dyes and on Em, and 3) calibration of dye fluorescence. Recent reports that advocate the use of the fluorescent dyes are misleading.     

Studies of monomeric and homodimeric oxazolo[4,5-b]pyridinium cyanine dyes as fluorescent probes for nucleic acids visualization

The series of recently synthesized monomeric and homodimeric cyanine dyes based on monomethine cyanine chromophore with oxazolo[4,5-b]pyridinium and quinoline end groups [Vassilev A, Deligeorgiev T, Gadjev N, Drexhage K-H. Synthesis of novel monomeric and homodimeric cyanine dyes based on oxazolo[4,5-b]pyridinium and quinolinium end groups for nucleic acid detection, Dyes Pigm 2005;66:135-142] were studied as possible fluorescent probes for nucleic acids detection. Significant fluorescence enhancement and intensity level (quantum yield up to 0.75) was observed for all the dyes in the presence of DNA. The oxazolo[4,5-b]pyridinium cyanines demonstrated high sensitivity as fluorescent stains for post-electrophoretic visualization of nucleic acids in agarose gels upon both VIS and UV transillumination, and the visualized band contained 0.8 ng of dsDNA.