Cyanine dye labeling reagents--carboxymethylindocyanine succinimidyl esters

Ten carboxymethylindocyanine dyes which form the basis of a new series of fluorescent probes have been synthesized and converted into succinimidyl active esters for fluorescent labeling of proteins or other amino-containing substances. Fluorescence emission maxima for members of the series range from 575 to 780 nm. Hydrophilic, water-soluble reagents have been obtained which yield labeled antibodies with little tendency to form precipitates. The fluorescence intensities achieved are higher than those produced by labeling with the cyanine isothiocyanates described previously (Mujumdar et al.: Cytometry 10:11-19, 1989). The utility of these reagents has been demonstrated in antibody labeling for two-color immunofluorescent imaging of internal structures in a mammalian cell and for two-color flow-cytometry experiments. The use of values of chromophore-equivalent weight (W/Ceq), calculated from quantitative absorption data on dye samples, is proposed as an aid in formulating labeling procedures.     

Interaction of cyanine dyes with nucleic acids. Part 19: new method for the covalent labeling of oligonucleotides with pyrylium cyanine dyes

New chemistry for the fluorescent labeling of oligonucleotides with cyanine dyes is proposed. It is based on the use of pyrylium salts as amine-specific reagents. Monomethyne pyrylium cyanine dye 1 was covalently linked to 5'-aminoalkyl modified oligonucleotide, with simultaneous conversion of the non-fluorescent dye 1 into fluorescent pyridinium cyanine structure 2.     

Long-wavelength absorbing and fluorescent chameleon labels for proteins, peptides, and amines

Long-wavelength absorbing labels that change their color and fluorescence upon conjugation to proteins and other biomolecules provide two critical advantages over the wealth of conventional amine-reactive labels. At first, the progress of the labeling reaction can be monitored continuously either visually or by spectrometry without prior purification. Then, the labeled biomolecule can be investigated with red or near-infrared light, which minimizes background interference in biological samples. These unique characteristics are met by a group of long-wavelength absorbing cyanine dyes carrying a reactive chloro substituent for nucleophilic substitution with primary amines, which is accompanied by a color change from green to blue. In addition to this so-called chameleon effect, the dyes display an increase in fluorescence during the labeling reaction. Despite their structural similarity, the reactivity of the dyes differs strongly. The fastest labeling kinetics is observed with dye S 0378 as its five-membered ring affords a stabilizing effect on the intermediate carbocation during an S(N)1-type of nucleophilic substitution. The reaction mechanism of the amine-reactive cyanine dyes provides a blueprint for the design of future long-wavelength absorbing chameleon dyes.     

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.     

Heterogeneity of protein labeling with a fluorogenic reagent, 3-(2-furoyl)quinoline-2-carboxaldehyde

Fluorogenic reagents are used for protein labeling when high-sensitivity fluorescence detection is required. Similar to traditional labeling with activated fluorescent dyes, such as fluorescein isothiocyanate, a fluorogenic reaction is expected to change the physical-chemical properties of proteins. Knowledge of these changes may be essential for efficient separation and identification of labeled proteins. Here we studied the effect of labeling of myoglobin with a fluorogenic reagent on the acid-base properties of the protein. The fluorogenic reagent used was 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ). In slab-gel isoelectric focusing, we found that the labeling reaction generated at least six species with pI values lower than that of non-labeled myoglobin. These species can be identified as products of progressive labeling of myoglobin with one to six FQ molecules. The same series of FQ-labeled species were observed when the reaction products were analyzed by capillary zone electrophoresis. The comparison of experimental and theoretical pI values allowed us to elucidate the labeling pattern--the number of FQ molecules corresponding to each labeled product detected by isoelectric focusing.     

Syntheses and DNA-binding studies of a series of unsymmetrical cyanine dyes: structural influence on the degree of minor groove binding to natural DNA

Twelve crescent-shaped unsymmetrical dyes have been synthesized and their interactions with DNA have been investigated by spectroscopic methods. A new facile synthetic route to this type of cyanine dyes has been developed, involving the preparation of 6-substituted 2-thiomethyl-benzothiazoles in good yields. The new dyes are analogues to the 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 6-methylbenzothiazole substituent. Herein we further investigate the role of the extending benzazole heterocycle, as well as of the pyridine or quinoline moiety of the cyanine chromophore, for the binding mode of these crescent-shaped dyes to calf thymus DNA. Flow LD and CD studies of the 12 dyes show that the extent of minor groove binding to mixed sequence DNA varies significantly between the dyes. We find that hydrophobicity and size are the crucial parameters for recognition of the minor groove. The relatively high fluorescence quantum yield of many of these cyanines bound to DNA, combined with their absorption at long wavelengths, may render them useful in biological applications. In particular, two of the benzoxazole containing dyes BOXTO and 2-BOXTO show a high degree of minor groove binding and quantum yields of 0.52 and 0.32, respectively, when bound to DNA.     

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.     

Proteomic analysis of redox- and ErbB2-dependent changes in mammary luminal epithelial cells using cysteine- and lysine-labelling two-dimensional difference gel electrophoresis

Differential protein expression analysis based on modification of selected amino acids with labelling reagents has become the major method of choice for quantitative proteomics. One such methodology, two-dimensional difference gel electrophoresis (2-D DIGE), uses a matched set of fluorescent N-hydroxysuccinimidyl (NHS) ester cyanine dyes to label lysine residues in different samples which can be run simultaneously on the same gels. Here we report the use of iodoacetylated cyanine (ICy) dyes (for labelling of cysteine thiols, for 2-D DIGE-based redox proteomics. Characterisation of ICy dye labelling in relation to its stoichiometry, sensitivity and specificity is described, as well as comparison of ICy dye with NHS-Cy dye labelling and several protein staining methods. We have optimised conditions for labelling of nonreduced, denatured samples and report increased sensitivity for a subset of thiol-containing proteins, allowing accurate monitoring of redox-dependent thiol modifications and expression changes. Cysteine labelling was then combined with lysine labelling in a multiplex 2-D DIGE proteomic study of redox-dependent and ErbB2-dependent changes in epithelial cells exposed to oxidative stress. This study identifies differentially modified proteins involved in cellular redox regulation, protein folding, proliferative suppression, glycolysis and cytoskeletal organisation, revealing the complexity of the response to oxidative stress and the impact that overexpression of ErbB2 has on this response.     

Evaluation of Commercial Fluorescein Isothiocyanates Used in Fluorescent Antibody Studies

Commercial preparations of fluorescein isothiocyanate (FITC) for immunofluorescence applications were obtained from 12 sources and examined for purity by quantitative infrared spectrophotometry and by labeling efficiency for bovine serum albumin (BSA). Quantitative photometric measurements were made of nonspecific staining (NSS) produced by conjugates prepared from the dyes. The purity of FITC from different sources was highly variable. The risk of NSS appears to increase as the purity of the dye decreases. In immunofluorescence applications it is desirable to use the purest FITC available in order to obtain conjugates with minimum NSS. It is recommended that 70% FITC, as determined by BSA labeling efficiency, be accepted as the minimum purity for immunofluorescence applications.     

DNA and chromatin imaging with super-resolution fluorescence microscopy based on single-molecule localization

With the expansion of super-resolution fluorescence microscopy methods, it is now possible to access the organization of cells and materials at the nanoscale by optical means. This review discusses recent progress in super-resolution imaging of isolated and cell DNA using single-molecule localization methods. A high labeling density of photoswitchable fluorophores is crucial for these techniques, which can be provided by sequence independent DNA stains in which photoblinking reactions can be induced. In particular, unsymmetrical cyanine intercalating dyes in combination with special buffers can be used to image isolated DNA with a spatial resolution of 30-40 nm. For super-resolution imaging of chromatin, cell permeant cyanine dyes that bind the minor groove of DNA have the potential to become a useful alternative to the labeling of histones and other DNA-associated proteins. Other recent developments that are interesting in this context such as high density labeling methods or new DNA probes with photoswitching functionalities are also surveyed. Progress in labeling, optics, and single-molecule localization algorithms is being rapid, and it is likely to provide real insight into DNA structuring in cells and materials.     

Asymmetrical,water-soluble phthalocyanine dyes for covalent labeling of oligonucleotides

Two new water-soluble, porphyrazine (Pz) dyes containing an isothiocyanate function for covalent linking have each been prepared by cross condensation of two different aromatic dinitriles, one containing carboxylates for solubilizing purposes and the other containing a nitro group for conversion into the labeling function. The initial mononitrotricarboxylato Pzs have been purified to homogeneity from the mixture of Pz congeners formed in the condensation reaction by anion exchange chromatography. The phthalocyanine dye 1 has an absorption maxima at 683 nm while the trinaphthoporphyrazine dye 2 has an absorption maxima at 755 nm, due to the increased size of the aromatic system. Both dyes were successfully conjugated to oligonucleotide primers, showing their potential for use in near-infrared-based DNA diagnostic applications.     

Cyanine dye labeling reagents for sulfhydryl groups

Cyanine and merocyanine dyes are introduced as new fluorescent reagents for covalently labeling proteins and other biomolecules. These dyes, which contain iodoacetamide functional groups, have high extinction coefficients and moderate quantum yields. A major advantage of these polymethine dyes is the easy manipulation of their spectral properties during synthesis. Cyanines containing reactive functional groups can be made with absorption maxima ranging from less than 500 nm to greater than 750 nm. This property opens additional regions of the spectrum for experiments involving the simultaneous multicolor analysis of different fluorescent probes. The cyanines, which are relatively insensitive to solvent property changes, are complemented by the merocyanines, which are keen indicators of solvent polarity.     

Multiple labeling of antibodies with dye/DNA conjugate for sensitivity improvement in fluorescence immunoassay

Fluorescence immunoassays are widely used in life science research, medical diagnostics, and environmental monitoring due to the intrinsically high specificity, simplicity, and versatility of immunoassays, as well as the availability of a large variety of fluorescent labeling molecules. However, the sensitivity needs to be improved to meet the ever-increasing demand in the new proteomics era. Here, we report a simple method of attaching multiple fluorescent labels on an antibody with a dye/DNA conjugate to increase the immunoassay sensitivity. In the work, mouse IgG adsorbed on the surface of a 96-well plate was detected by its immunoreaction with biotinylated goat anti-mouse antibody. A 30 base pair double-stranded oligonucleotide terminated with biotin was attached to the antibody through the biotin/streptavidin/biotin interaction. Multiple labeling of the antibody was achieved after a fluorescent DNA probe was added into the solution and bound to the oligonucleotide at high ratios. By comparison with fluorescein-labeled streptavidin, the assay with the dye/DNA label produced up to 10-fold increase in fluorescence intensity, and consequently about 10-fold lower detection limit. The multiple labeling method uses readily available reagents, and is simple to implement. Further sensitivity improvement can be obtained by using longer DNAs for antibody labeling, which can incorporate more fluorescent dyes on each DNA.     

Cyanine dye-protein interactions: looking for fluorescent probes for amyloid structures

We ascertained the ability to detect fibrillar beta-lactoglobulin (BLG) of a series of mono-, tri-, penta-, and heptamethinecyanines based on benzothiazole and benzimidazole heterocycles, and of benzothiazole squaraine. Fluorescence properties of these cyanine dyes were measured in the unbound state and in the presence of monomeric and fibrillar BLG and compared with those for the commercially available benzothiazole dye Thioflavin T. The correlation between the chemical nature of the dye molecules and the ability of dyes to bind aggregated proteins was established. We found that meso-substituted cyanines with amino substituents in heterocycle in contrast to the corresponding unsubstituted dyes have a binding preference to fibrillar BLG and a noticeable fluorescence response in the presence of the aggregated protein. For the squaraines and benzimidazole penthamethinecyanines studied, fluorescence emission increased both in the presence of native and fibrillar protein. The trimethinecyanines T-49 and SH-516 exhibit specifically increased fluorescence in the presence of fibrillar BLG. These dyes demonstrated the same or higher emission intensity and selectivity to aggregated BLG as Thioflavin T, and are proposed for application in selective fluorescent detection of aggregated proteins.     

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.     

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.     

Use of fluorescein hydrazide and fluorescein thiosemicarbazide reagents for the fluorometric determination of protein carbonyl groups and for the detection of oxidized protein on polyacrylamide gels

Highly fluorescent thiosemicarbazide and hydrazide prepared by reaction of fluorescein isothiocyanate with hydrazine or adipic acid dihydrazide have been used to monitor the presence of carbonyl groups in oxidatively modified proteins. After oxidation, proteins react with these reagents under anaerobic conditions in the dark to yield fluorescent protein conjugates (presumably thiosemicarbazones or hydrazones) which can be visualized as fluorescent bands following electrophoresis (0-4 degrees C) on lithium dodecyl sulfate-polyacrylamide gels. These reagents do not react with unoxidized proteins. The conjugates formed dissociate readily at room temperature but are fairly stable at pH 6-9, 0 degrees C. Current data suggest that these reagents will be useful in the detection and quantitation of oxidatively modified proteins in biological systems.     

Fluoro-Gold: An alternative viability stain for multicolor flow cytometric analysis.

BACKGROUND: The viability stains propidium iodide (PI) and 7-amino-actinomycin D (7-AAD) are excited at 488 nm, as are the commonly used antibody conjugates fluorescein isothiocyanate (FITC), phycoerythrin (PE), and cyanine 5 dye covalently coupled to R-phycoerythrin (RPE-Cy5). When excited by a single laser, spectral overlap in the emission of PI and 7-AAD with RPE-Cy5 precludes the use of these viability stains for three-color immunophenotyping, particularly when evaluating low levels of marker expression in viable target cells. The ultraviolet excitable dye hydroxystilbamidine methanesulfonate (Fluoro-Gold, or FG) binds to DNA at the A-T-rich regions of the minor groove in permeabilized or dead cells. We assessed the suitability of this dye as a viability stain. METHODS: The ability of FG to detect nonviable cells in fresh and cryopreserved human apheresed peripheral blood cells was compared with that of PI and 7-AAD. The stability of FG staining and the effects of dye and cell concentration on the discrimination of nonviable cells was determined by measuring changes in the median fluorescence of viable and nonviable cells. RESULTS: FG labeling at dye concentrations of 2-8 microM is stable for at least 3 h over a wide range of cell concentrations (4 x 10(5) to 4 x 10(7) cells/ml). Costaining studies and linear regression analysis show that cell viability as determined by FG is strongly correlated with estimates using PI (r = 0.9636) and 7-AAD (r = 0.9879). CONCLUSIONS: FG is a reliable, alternative viability stain that can be used in conjunction with fluorochromes including FITC, PE, and RPE-Cy5 for multicolor analysis using dual-laser instruments.     

NMR screening of new carbocyanine dyes as ligands for affinity chromatography

Four new carbocyanines containing symmetric and asymmetric heterocyclic moieties and N‐carboxyalkyl groups have been synthesized and characterized. The binding mechanism established between these cyanines and several proteins was evaluated using saturation transfer difference (STD) NMR. The results obtained for the different dyes revealed a specific interaction to the standard proteins lysozyme, α‐chymotrypsin, ribonuclease (RNase), bovine serum albumin (BSA), and gamma globulin. For instance, the two un‐substituted symmetrical dyes (cyanines 1 and 3) interacted preferentially through its benzopyrrole and dibenzopyrrole units with lysozyme, α‐chymotrypsin, and RNase, whereas the symmetric disulfocyanine dye (cyanine 2) bound BSA and gamma globulin through its carboxyalkyl chains. On the other hand, the asymmetric dye (cyanine 4) interacts with lysozyme and α‐chymotrypsin through benzothiazole moiety and with RNase through dibenzopyrrole unit. Thus, STD‐NMR technique was successfully used to screen cyanine–protein interactions and determine potential binding sites of the cyanines for posterior use as ligands in affinity chromatography. Copyright © 2014 John Wiley & Sons, Ltd.     

Symplast domains in extrastelar tissues of Egeria densa Planch.

A set of hydrophilic fluorescent dyes of known molecular weight has been used to determine the molecular exclusion limit and the extent of apical, epidermal and cortical symplasts in the root, stem and leaf of Egeria densa. These dyes are unable to pass the plasmalemma, so that any cell-to-cell movement of injected dye must occur via the symplast. The shoot-apex symplast has a high molecular exclusion limit, excluding dyes with a molecular weight of 749 dalton (fluorescein hexaglycine) and greater but allowing dyes of up to 665 dalton (fluorescein diglutamic acid) to pass. The leaf epidermal symplast is similar to that in the apex: fluorescein pentaglycine (674 dalton) moves to a limited extent, but fluorescein hexaglycine is immobile. Stem and root epidermal cells have a lower molecular exclusion limit, only the dye 6-carboxyfluorescein (376 dalton) is able to move from cell-to-cell. Cortical and epidermal tissues in both the stem and the root have similar symplast permeabilities. However, a barrier to dye (6-carboxyfluorescein) movement is found between the epidermis and the cortex in both organs. Barriers are also found at the nodes between expanded internodes. The stem barriers are not found in the unexpanded nodes near the shoot tip; apparently they are formed early during internode expansion. In the root tip, a barrier to the movement of dye is found between the root cap and the remainder of the root. Plasmodesmata are found linking all cell types studied, even cells where barriers to dye movement occur. Thus, the plant, far from being one uniform symplast, consists of a large number of symplast domains, which may or may not differ in molecular exclusion limit.Abbreviations F fluorescein isothiocyanate isomer I - Glu l-glutamic acid - (Glu)₂ l-glutamylglutamic acid - (Gly)₅ l-pentaglycine - (Gly)₆ l-hexaglycine