Asymmetric Cyanine Dyes Research Articles

Tetraalkylammonium derivatives as real-time PCR enhancers and stabilizers of the qPCR mixtures containing SYBR Green I

Tetraalkylammonium (TAA) derivatives have been reported to serve as stabilizers of asymmetrical cyanine dyes in aqueous solutions and to increase the yield and efficiency of polymerase chain reaction (PCR) detected by end-point analysis. In this study, we compared the ability of various TAA derivatives (with alkyl chain ranging from 1 to 5 carbons) and some other compounds to serve as enhancers of real-time PCR based on fluorescence detection from intercalating dye SYBR Green I (SGI). Our data indicate that TAA chlorides and some other TAA derivatives serve as potent enhancers of SGI-monitored real-time PCR. Optimal results were obtained with 10–16 mM tetrapropylammonium chloride. The effect of TAA compounds was dependent on the nature of counter ions present and composition of the reaction mixtures used. Based on measurements of SGI-generated fluorescence signal in the presence of PCR-amplified DNA fragments, oligonucleotide primers and/or various additives, we propose that TAA-derivatives reduce the binding of SGI to oligonucleotide primers and thus enhance primer–template interactions during annealing phase. Furthermore, these compounds serve as stabilizers of SGI-containing PCR mixtures. The combined data indicate that TAA derivatives might be a new class of additives contributing to robustness of real-time PCR monitored by asymmetrical cyanine dye SGI.

New unsymmetrical cyanine dyes for real-time thermal cycling

Asymmetric cyanine dyes bind to the minor groove of double stranded DNA (dsDNA) owing to their crescent configuration; therefore, these dyes are widely used as a dsDNA probes. BOXTO-MEE is derived from BOXTO by adding the polar methoxyethoxyethyl tail in order to increase solubility, dissociation rate kinetics, and stability. As a result, BOXTO-MEE showed significant reduction in nonspecific amplification (primer dimers) without significant effect on target sequence amplification, PCR efficiency, and standard curve correlation coefficient. BETIBO is another example of an asymmetric cyanine dye that can binds to dsDNA but is less efficient than BOXTO-MEE for use in real-time PCR. Statistical analysis of reproducibility results shows that BETIBO is not strong enough to be used for quantifying low nucleic acid quantities. Statistical analysis for BOXTO-MEE results shows that there is no significant difference between the efficiency and correlation coefficient achieved by BOXTO-MEE and SYBR Green I, but a significant difference in the dynamic range is observed because BOXTO-MEE has a wider dynamic range. BOXTO-MEE stock solution was stable at -20 degrees C for more than 1 year and 40 microM solution was stable for 45 days (at least) at 4 degrees C.

Cyanine dye conjugates as probes for live cell imaging

A series of fluorescent compounds suitable for live cell imaging is described. Functionalized forms of four different asymmetric cyanine dyes are reported that are amenable to peptide conjugation. The photophysical properties of the modified dyes and conjugates and the use of the compounds as cellular imaging agents are described. The results obtained indicate that these spectrally versatile compounds, which have absorption and emission profiles spanning the visible spectrum, are useful probes for cellular imaging.

New cyanine dyes as base surrogates in PNA: Forced intercalation probes (FIT-probes) for homogeneous SNP detection

Forced intercalation probes (FIT-probes) are nucleic acid probes, in which an intercalator cyanine dye such as thiazole orange (TO) serves as a replacement of a canonical nucleobase. These probes signal hybridization by showing strong increases of fluorescence. TO in FIT-probes responds to adjacent base mismatches by attenuation of fluorescence intensities at conditions where both matched and mismatched target DNA are bound. The interesting features of TO labeled FIT-probes posed the question whether the forced intercalation concept can be extended to other cyanine dyes of the thiazole orange family. Herein, we present the synthesis of three asymmetrical cyanine dyes and their incorporation into PNA-conjugates by means of both divergent and linear solid-phase synthesis. Melting analysis revealed that the DNA affinity of PNA probes remained high irrespective of the replacement of a nucleobase by the cyanines YO (oxazole yellow), MO or JO. Of the three new tested dye–PNA-conjugates, the YO-containing PNA has properties useful for homogeneous SNP detection. YO–PNA is demonstrated to signal the presence of fully complementary DNA by up to 20-fold enhancement of fluorescence. In addition, YO emission discriminates against single base mismatches by attenuation of fluorescence. Oxazole yellow (YO) as a base surrogate in PNA may prove useful in the multiplex detection of single base mutations at non-stringent conditions.

Asymmetric cyanine dyes as fluorescence probes and visible‐light photoinitiators of free‐radical polymerization processes

AbstractThis article introduces asymmetric cyanine dyes employed as visible‐light photoinitiators of vinyl monomer polymerization and as fluorescence probes monitoring the progress of polymerization. A degree of polymer cure from the measurement of the changes in the probe emission intensity and position shifts during the thermally initiated polymerization of monoacrylate was obtained. A distinct increase in the intensity of the probe fluorescence was observed during polymerization when the degree of monomer conversion was gradually increasing. This effect was accompanied by a blue‐shift of the probe emission maxima. The second part of this work is focused on the possibility of an application of the tested dyes, in combination with borate anions, as photoinitiating systems. The kinetics of polymerization of trimethylolpropane triacrylate, with cyanine borates as photoinitiators, was studied by a microcalorimetric method. Asymmetric cyanine borates were found to be effective photoinitiators, and both the initiator and coinitiator concentration as well as the light intensity strongly affected the progress of photopolymerization, leading, for example, to an increase in the polymerization rate. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci 99: 207–217, 2006

Thermodynamic characterization of the dimerization equilibrium of an asymmetric dye by spectral titration and chemometric analysis

The monomer–dimer equilibrium of an asymmetric cyanine dye has been investigated by means of UV-Vis spectroscopy. The data have been processed by a recently developed chemometric method for quantitative analysis of undefined mixtures, that is based on simultaneous resolution of the overlapping bands in the whole set of absorption. In this work the dimerization constant of 1-carboxydecyl-4-{3-[3-methyl-3H-benzothiazol-2-ylidene]-propenyl}-quinolinium (TO-3) has been determined by studying the dependence of absorption spectrum on temperature in the range 25–72.5 °C at different total concentrations of dye (8.5×10 −6 to 2.87×10 −5 M). Utilizing the van’t Hoff relation, which describes the dependence of the equilibrium constant on temperature, as constraint we determine the spectral responses of the monomer and dimer species as well as the enthalpy and entropy of the dimerization equilibrium.

Mesodomain separation in amalgamated J aggregate formation of cyanine dyes at a mica/solution interface

Aqueous mixed solutions of pseudoisocyanine (PIC) and its structural analogous asymmetric cyanine (AC) dye formed self-assembled amalgamated J aggregates (mixed J aggregates with a single excitonic band) at a mica/solution interface, and the formation mechanism of the aggregates was clarified by determining the optical, structural, and morphological properties of such aggregates. In this study, mesodomain separation in amalgamated J aggregate formation of the cyanine dyes at a mica/solution interface was revealed for the first time. Fluorescence microscopy showed that the emissive amalgamated J aggregates possessed mesoscopic island domain morphology. The aggregates covered the interface completely when the AC mole fraction was small (below 0.25). With an increase in the AC mole fraction, the emissive J aggregate islands decreased in size and number and were discretely distributed, although atomic force microscopy (AFM) showed that the interface was still completely covered by adsorbed molecules. Our results show that dye adsorption from the PIC/AC mixture solutions generated two types of domain formation at the mica/solution interface (mesodomain separation): emissive amalgamated J aggregate island (PIC-rich domain) and non-emissive island in the absence of J aggregates (AC-rich domain). On the basis of the anisotropic growth behavior of the islands, we proposed the internal structures of the emissive and non-emissive domains at the interface. Fluorescence microspectroscopy also revealed that each J aggregate mesodomain exhibited a different emission maximum.

A new minor groove binding asymmetric cyanine reporter dye for real-time PCR

The minor groove binding asymmetric cyanine dye 4-[(3-methyl-6-(benzothiazol-2-yl)-2,3-dihydro- (benzo-1,3-thiazole)-2-methylidene)]-1-methyl-pyridin ium iodide (BEBO) is tested as sequence non- specific label in real-time PCR. The fluorescence intensity of BEBO increases upon binding to double-stranded DNA allowing emission to be measured at the end of the elongation phase in the PCR cycle. BEBO concentrations between 0.1 and 0.4 micro M generated sufficient fluorescence signal without inhibiting the PCR. A comparison with the commonly used reporter dye SYBR Green I shows that the two dyes behave similarly in all important aspects.

Synthesis and DNA binding studies of a new asymmetric cyanine dye binding in the minor groove of [poly(dA-dT)] 2

A new asymmetric cyanine dye has been synthesised and its interaction with different DNA has been investigated. In this dye, BEBO, the structure of the known intercalating cyanine dye BO has been extended with a benzothiazole substituent. The resulting crescent-shape of the molecule is similar to that of the well-known minor groove binder Hoechst 33258. Indeed, comparative studies of BO illustrate a considerable change in binding mode induced by this structural modification. Linear and circular dichroism studies indicate that BEBO binds in the minor groove to [poly (dA-dT)] 2, but that the binding to calf thymus DNA is heterogeneous, although still with a significant contribution of minor groove binding. Similar to other DNA binding asymmetric cyanine dyes, BEBO has a large increase in fluorescence intensity upon binding and a relatively large quantum yield when bound. The minor groove binding of BEBO to [poly (dA-dT)] 2 affords roughly a 180-fold increase in intensity, which is larger than to that of the commonly used minor groove binding probes DAPI and Hoechst 33258.

Solid-phase synthesis of asymmetric cyanine dyes

An efficient solid-phase synthesis of asymmetric cyanine dyes is described. Combinatorial synthesis and spectroscopic analysis of four dyes was carried out. The yields are quantitative after 20 h, but the reaction can be halted earlier, since it is shown that the starting material does not disturb the fluorescence measurements in these binding studies.

Light-Up Probes: Thiazole Orange-Conjugated Peptide Nucleic Acid for Detection of Target Nucleic Acid in Homogeneous Solution

We have constructed light-up probes for nucleic acid detection. The light-up probe is a peptide nucleic acid (PNA) oligonucleotide to which the asymmetric cyanine dye thiazole orange (TO) is tethered. It combines the excellent hybridization properties of PNA and the large fluorescence enhancement of TO upon binding to DNA. When the PNA hybridizes to target DNA, the dye binds and becomes fluorescent. Free probes have low fluorescence, which may increase almost 50-fold upon hybridization to complementary nucleic acid. This makes the light-up probes particularly suitable for homogeneous hybridization assays, where separation of the bound and free probe is not necessary. We find that the fluorescence enhancement upon hybridization varies among different probes, which is mainly due to variations in free probe fluorescence. For eight probes studied the fluorescence quantum yield at 25°C in the unbound state ranged from 0.0015 to 0.08 and seemed to depend mainly on the PNA sequence. The binding of the light-up probes to target DNA is highly sequence specific and a single mismatch in a 10-mer target sequence was readily identified.

Fischer's base as an electron donor for new penta- and heptamethines

Asymmetric cyanine dyes (penta- and heptamethines) prepared by coupling Fischer's base or Fischer's aldehyde with malononitrile dimer or analogous compounds are described. Dye characteristics are given and structure-color correlations are discussed.

Small S0–S1 energy gaps for certain twisted conformations of unsymmetric polymethine dyes: quantum chemical treatment and spectroscopic manifestations

The AM1–CI (Austin model 1 configuration interaction) method has been used to calculate energy gaps between the ground state (S0) and the lowest biradicaloid (BR) excited state as a measure for roughly estimating the probability of reaching an S0–BR conical intersection by bond twisting of one of the bonds along the polymethine chain of unsymmetric cyanine dyes. Invoking qualitative concepts for interpreting the computational data, a pronounced alternation of successive bonds is shown and explained. The magnitude of the S0–BR energy gap was found to be governed by the position of the corresponding bond with respect to the polymethine chain ends and by the degree of electronic asymmetry of the chain, i.e. by the difference in electron donor abilities of its end groups. These results are compared to experimental photophysical data for a series of differently bridged asymmetric cyanine dyes and suggest that the magnitude of the energy gap for a given twisted bond can be modulated by bridge-induced perturbations. Nonreactive low-lying minima caused by the accessibility of several twisted excited-state conformers have to be invoked in order to explain the experimentally observed ‘‘ inverse-loose-bolt’’ effect of the nonradiative decay rates and should be taken into account in the treatment of possible pathways of fluorescence quenching and in the design of high-quantum-yield fluorescent dyes.

Structure-color correlations of penta- and heptamethines: Syntheses with nitriles XCIV

The synthesis of asymmetric cyanine dyes (penta- and heptamethines), prepared by coupling Fischer's base with indandione, benzo[b]thiophene and pyrazole derivatives via a C-1 unit, is described. Three different synthetic strategies for substituted polymethines are presented. Dye characteristics are given and the correlation between the chemical structures and the dye properties are discussed by means of semiempirical PPP calculations.

Stable fluorescent complexes of double-stranded DNA with bis-intercalating asymmetric cyanine dyes: properties and applications.

The synthesis, proof of structure, and the absorption and fluorescence properties of two new unsymmetrical cyanine dyes, thiazole orange dimer (TOTO; 1,1'-(4,4,7,7-tetramethyl-4,7- diazaundecamethylene)-bis-4-[3-methyl-2,3-dihydro-(benzo-1,3-thiaz ole)-2- methylidene]-quinolinium tetraiodide) and oxazole yellow dimer (YOYO; an analogue of TOTO with a benzo-1,3-oxazole in place of the benzo-1,3-thiazole) are reported. TOTO and YOYO are virtually non-fluorescent in solution, but form highly fluorescent complexes with double-stranded DNA (dsDNA), up to a maximum dye to DNA bp ratio of 1:4, with greater than 1000-fold fluorescence enhancement. The dsDNA-TOTO (lambda max 513 nm; lambda maxF 532 nm) and dsDNA-YOYO (lambda max 489 nm; lambda maxF 509 nm) complexes are completely stable to electrophoresis on agarose and acrylamide gels. Mixtures of restriction fragments pre-labeled with ethidium dimer (EthD; lambda maxF 616 nm) and those pre-labeled with either TOTO or YOYO were separated by electrophoresis. Laser excitation at 488 nm and simultaneous confocal fluorescence detection at 620-750 nm (dsDNA-EthD emission) and 500-565 nm (dsDNA-TOTO or dsDNA-YOYO emission) allowed sensitive detection, quantitation, and accurate sizing of restriction fragments ranging from 600 to 24,000 bp. The limit of detection of dsDNA-TOTO and YOYO complexes with a laser-excited confocal fluorescence gel scanner for a band 5-mm wide on a 1-mm thick agarose gel was 4 picograms, about 500-fold lower than attainable by conventional staining with ethidium bromide.

ChemInform Abstract: Synthesis, Spectral Behaviour and Application of Oxazolo/Imidazolo Pyrimidine Cyanines.

AbstractThe asymmetric cyanine dyes (III) and (V) are synthesized from the fused pyrimidines (I) and the pyridinium or quinolinium salts (II).

The basicity and color of asymmetric cyanine dyes

The basicity and color of asymmetric cyanine dyes