Study of cis-trans equilibrium of oxacarbocyanine dyes in solution and in a complex with DNA

Cis-trans equilibrium for a number of meso-substituted oxacarbocyanine dyes, 3,3′-diethyloxacarbocyanine iodide (K1), 3,3′-diethyl-9-methyloxacarbocyanine iodide (K2), 3,3′-dimethyl-9-ethyloxacarbocyanine iodide (K3), 3,3′,9-triethyl-6,6′-dimethoxyoxacarbocyanine iodide (K4), and 3,3′,9-triethyl-5,5′-dimethyloxacarbocyanine iodide (K5), has been studied in solutions and in a complex with DNA by spectral and fluorescent methods. A shift of the cis-trans isomer equilibrium toward the formation of the trans-isomer was observed in the presence of DNA, which determined in many respects the spectral effects observed upon the complexation of the oxacarbocyanine dyes. A steep rise of fluorescence (due to binding of the trans-isomer) in a complex with DNA is favorable for using oxacarbocyanine dyes to determine DNA.     

Spectral and fluorescent study of the interaction of cyanine dyes Cyan 2 and Cyan 45 with DNA

The noncovalent interaction of the cyanine dyes Cyan 2 and Cyan 45 with DNA (in a pH 7 phosphate buffer) was studied by spectral and fluorescent methods. Upon titration of dye solutions with a concentrated DNA solution, a drop in the absorption band of the initial dye, a long-wavelength shift, and a rise of the absorption of the dye bound to DNA occur. Simultaneously, a growth of dye fluorescence is observed. Mathematical modeling of the fluorescence growth has been performed on the basis of the formation of one or two types of dye complexes with DNA. The experimental results are adequately described by the model involving one type of the dye-DNA complex. It is shown that Cyan 2 binds to DNA probably as the cis-isomer, whereas Cyan 45 as the trans-isomer.     

Verification and Biophysical Characterization of a New Three‐Color Förster Resonance‐Energy‐Transfer (FRET) System in DNA

We report on a new three‐color FRET system consisting of three fluorescent dyes, i.e., of a carbostyril (=quinolin‐2(1H)‐one)‐derived donor D, a (bathophenanthroline)ruthenium complex as a relay chromophore A₁, and a Cy dye as A₂ (FRET=Förster resonance‐energy‐transfer) (cf. Fig. 1). With their widely matching spectroscopic properties (cf. Fig. 2), the combination of these dyes yielded excellent FRET efficiencies. Furthermore, fluorescence lifetime measurements revealed that the long fluorescence lifetime of the Ru complex was transferred to the Cy dye offering the possibility to measure the whole system in a time‐resolved mode. The FRET system was established on double‐stranded DNA (cf. Fig. 3) but it should also be generally applicable to other biomolecules.     

New indolium and quinolinium dyes sensitive to aqueous halide ions at physiological concentrations

Twelve new highly fluorescent dyes have been produced by the reaction of two heterocyclic nitrogen bases with 8-bromooctanoic acid, 11-bromoundecanoic acid and 15-bromopentadecanoic acid. The bromide counter ions of the first six dyes have been replaced with the tetraphenylborate ion. Unlike the bases themselves, the quaternary salts are water soluble and have fluorescence characteristics independent of pH in the pH range 7–11. Both the fluorescence intensity and fluorescence lifetime of dyes 1–12 are reduced in the presence of aqueous halide ions allowing halide concentrations to be determined accurately at physiological levels. All the dyes have been characterised in terms of steady state fluorescence spectra and steady-state Stern-Volmer analysis.     

Assessing The Key Photophysical Properties of Triangulenium Dyes for DNA Binding by Alteration of the Fluorescent Core

Four-stranded G-quadruplex (G4) DNA is a non-canonical DNA topology that has been proposed to form in cells and play key roles in how the genome is read and used by the cellular machinery. Previously, a fluorescent triangulenium probe ( DAOTA-M2 ) was used to visualise G4s in cellulo, thanks to its distinct fluorescence lifetimes when bound to different DNA topologies. Herein, the library of available triangulenium probes is expanded to explore how modifications to the fluorescent core of the molecule affect its photophysical characteristics, interaction with DNA and cellular localisation. The benzo-bridged and isopropyl-bridged diazatriangulenium dyes, BDATA-M2 and CDATA-M2 respectively, featuring ethyl-morpholino substituents, were synthesised and characterised. The interactions of these molecules with different DNA topologies were studied to determine their binding affinity, fluorescence enhancement and fluorescence lifetime response. Finally, the cellular uptake and localisation of these optical probes were investigated. Whilst structural modifications to the triangulenium core only slightly alter the binding affinity to DNA, BDATA-M2 and CDATA-M2 cannot distinguish between DNA topologies through their fluorescence lifetime. It is argued theoretically and experimentally that this is due to reduced effectiveness of photoinduced electron transfer (PET) quenching. This work presents valuable new evidence into the critical role of PET quenching when using the fluorescence lifetime of triangulenium dyes to discriminate G4 DNA from duplex DNA, highlighting the importance of fine tuning redox and spectral properties when developing new triangulenium-based G4 probes.     

Interaction of Crown Ether‐Annelated Styryl Dyes with Double‐Stranded DNA

DNA‐binding properties of 15‐crown‐5‐derived mono‐ and bis‐styryl dyes were investigated in the presence of calf thymus DNA. To access the factors that influence the DNA association in the series of these ligands, the structure of the molecules was varied by either changing size of the heterocyclic moiety or altering the position of the styryl substituents. The major binding mode for the monostyryl dyes is intercalation. Notably, binding of the dyes to the nucleic acids leads to a fluorescence enhancement by a factor of up to 54. Therefore, these cationic styryl derivatives may be applied as fluorescent “light‐up” probes for DNA detection.     

Evaluation of Antifungal Activity and Potential Application as Fluorescent Probes of Indolenine and Benzo[e]Indole-Based Squarylium Dyes

The antifungal performance and the possible use as fluorescent probes of a series of squarylium dyes derived from indolenine and benzo[e]indole previously synthesized was evaluated. Some photophysical properties were performed in ethanol and phosphate buffer, and the type of aggregates form in phosphate buffer was analyzed. Using the 1,3-diphenylisobenzofuran assay, a qualitative assessment of the capacity of dyes to produce singlet oxygen after irradiation was performed. Regarding the antifungal activity, this was studied through a broth microdilution assay using Saccharomyces cerevisiae PYCC 4072 as a biological model. The effect of irradiation of the dyes, with an appropriate light emitting diode system, on the antifungal activity was also evaluated, and it was verified that some of the dyes improve their activity after irradiation. Using fluorescence microscopy techniques, the colocalization of dyes in S. cerevisae cells was investigated and it was possible to verify that some of the squarylium dyes with a barbituric moiety in the four-membered central ring stained and accumulated preferentially in the mitochondrial web and perinuclear membrane of the cells. The possible use as a fluorescent probe for the detection of HSA was also evaluated for one of the dyes of the series, demonstrating a linear variation in the fluorescence intensity accompanied by the increase in the protein concentration.     

Electronic excitation energy transfer between molecules of carbocyanine dyes in complexes with DNA

Electronic excitation energy transfer has been carried out between molecules of carbocyanine dyes bound noncovalently to DNA. 3,3′,9-Triethyl-5,5′-dimethyloxacarbocyanine iodide was used as an energy donor and 3,3′-diethylthiacarbocyanine iodide as an acceptor dye. In this process, the band belonging to the donor is observed in the fluorescence excitation spectrum of the acceptor. Donor fluorescence quenching by the acceptor in the presence of DNA was studied. The results of the experiments are discussed in terms of the Dye-DNA stoichiometric complex formation and with respect to concentrating the dyes in the microphase (pseudophase) of the biopolymer.     

Label-free detection of nucleic acids by turn-on and turn-off G-quadruplex-mediated fluorescence

In this study we have used two fluorescent probes, tetrakis(diisopropylguanidino)-zinc-phthalocyanine (Zn-DIGP) and N-methylmesoporphyrin IX (NMM), to monitor the reassembly of “split” G-quadruplex probes on hybridization with an arbitrary “target” DNA. According to this approach, each split probe is designed to contain half of a G-quadruplex-forming sequence fused to a variable sequence that is complementary to the target DNA. Upon mixing the individual components, both base-pairing interactions and G-quadruplex fragment reassembly result in a duplex–quadruplex three-way junction that can bind to fluorescent dyes in a G-quadruplex-specific way. The overall fluorescence intensities of the resulting complexes were dependent on the formation of proper base-pairing interactions in the duplex regions, and on the exact identity of the fluorescent probe. Compared with samples lacking any “target” DNA, the fluorescence intensities of Zn-DIGP-containing samples were lower, and the fluorescence intensities of NMM-containing samples were higher on addition of the target DNA. The resulting biosensors based on Zn-DIGP are therefore termed “turn-off” whereas the biosensors containing NMM are defined as “turn-on”. Both of these biosensors can detect target DNAs with a limit of detection in the nanomolar range, and can discriminate mismatched from perfectly matched target DNAs. In contrast with previous biosensors based on the peroxidase activity of heme-bound split G-quadruplex probes, the use of fluorescent dyes eliminates the need for unstable sensing components (H₂O₂, hemin, and ABTS). Our approach is direct, easy to conduct, and fully compatible with the detection of specific DNA sequences in biological fluids. Having two different types of probe was highly valuable in the context of applied studies, because Zn-DIGP was found to be compatible with samples containing both serum and urine whereas NMM was compatible with urine, but not with serum-containing samples.     

Monitoring DNA structures by dual fluorescence of pyrene derivatives

We have developed a nucleotide modified by a pyrene derivative with dual fluorescence. The dual fluorescence of the fluorophore, which was incorporated into DNA, was effectively controlled at ambient temperature according to DNA structural status. Our nucleoside with dual fluorescence is effective as a conceptually new probe for monitoring DNA hybridization by the color change without multilabeling with fluorescent dyes.     

Asymmetric Distyrylpyridinium Dyes as Red‐Emitting Fluorescent Probes for Quadruplex DNA

The interactions of three cationic distyryl dyes, namely 2,4‐bis(4‐dimethylaminostyryl)‐1‐methylpyridinium ( 1 a ), its derivative with a quaternary aminoalkyl chain ( 1 b ), and the symmetric 2,6‐bis(4‐dimethylaminostyryl)‐1‐methylpyridinium ( 2 a ), with several quadruplex and duplex nucleic acids were studied with the aim to establish the influence of the geometry of the dyes on their DNA‐binding and DNA‐probing properties. The results from spectrofluorimetric titrations and thermal denaturation experiments provide evidence that asymmetric (2,4‐disubstituted) dyes 1 a and 1 b bind to quadruplex DNA structures with a near‐micromolar affinity and a fair selectivity with respect to double‐stranded (ds) DNA [K_a(G4)/K_a(ds)=2.5–8.4]. At the same time, the fluorescence of both dyes is selectively increased in the presence of quadruplex DNAs (more than 80–100‐fold in the case of human telomeric quadruplex), even in the presence of an excess of competing double‐stranded DNA. This optical selectivity allows these dyes to be used as quadruplex‐DNA‐selective probes in solution and stains in polyacrylamide gels. In contrast, the symmetric analogue 2 a displays a strong binding preference for double‐stranded DNA [K_a(ds)/K_a(G4)=40–100), presumably due to binding in the minor groove. In addition, 2 a is not able to discriminate between quadruplex and duplex DNA, as its fluorescence is increased equally well (20–50‐fold) in the presence of both structures. This study emphasizes and rationalizes the strong impact of subtle structural variations on both DNA‐recognition properties and fluorimetric response of organic dyes.     

Probing the charge-transfer dynamics in DNA at the single-molecule level

Photoinduced charge-transfer fluorescence quenching of a fluorescent dye produces the nonemissive charge-separated state, and subsequent charge recombination makes the reaction reversible. While the information available from the photoinduced charge-transfer process provides the basis for monitoring the microenvironment around the fluorescent dyes and such monitoring is particularly important in live-cell imaging and DNA diagnosis, the information obtainable from the charge recombination process is usually overlooked. When looking at fluorescence emitted from each single fluorescent dye, photoinduced charge-transfer, charge-migration, and charge recombination cause a "blinking" of the fluorescence, in which the charge-recombination rate or the lifetime of the charge-separated state (τ) is supposed to be reflected in the duration of the off time during the single-molecule-level fluorescence measurement. Herein, based on our recently developed method for the direct observation of charge migration in DNA, we utilized DNA as a platform for spectroscopic investigations of charge-recombination dynamics for several fluorescent dyes: TAMRA, ATTO 655, and Alexa 532, which are used in single-molecule fluorescence measurements. Charge recombination dynamics were observed by transient absorption measurements, demonstrating that these fluorescent dyes can be used to monitor the charge-separation and charge-recombination events. Fluorescence correlation spectroscopy (FCS) of ATTO 655 modified DNA allowed the successful measurement of the charge-recombination dynamics in DNA at the single-molecule level. Utilizing the injected charge just like a pulse of sound, such as a "ping" in active sonar systems, information about the DNA sequence surrounding the fluorescent dye was read out by measuring the time it takes for the charge to return.     

Ultrafast excited-state dynamics of DNA fluorescent intercalators: new insight into the fluorescence enhancement mechanism

The excited-state dynamics of the DNA bisintercalator YOYO-1 and of two derivatives has been investigated using ultrafast fluorescence up-conversion and time-correlated single photon counting. The free dyes in water exist in two forms: nonaggregated dyes and intramolecular H-type aggregates, the latter form being only very weakly fluorescent because of excitonic interaction. The excited-state dynamics of the nonaggregated dyes is dominated by a nonradiative decay with a time constant of the order of 5 ps associated with large amplitude motion around the monomethine bridge of the cyanine chromophores. The strong fluorescence enhancement observed upon binding of the dyes to DNA is due to both the inhibition of this nonradiative deactivation of the nonaggregated dyes and the dissociation of the aggregates and thus to the disruption of the excitonic interaction. However, the interaction between the two chromophoric moieties in DNA is sufficient to enable ultrafast hopping of the excitation energy as revealed by the decay of the fluorescence anisotropy. Finally, these dyes act as solvation probes since a dynamic fluorescence Stokes shift was observed both in bulk water and in DNA. Very similar time scales were found in bulk water and in DNA.     

Amphiphilic Fluorescence Resonance Energy-Transfer Dyes: Synthesis,Fluorescence, and Aggregation Behavior in Water

Amphiphilic pyrene/perylene bis-chromophore dyes were synthesized from unsymmetrically substituted perylene bisimide dyes, which were obtained through three synthetic methods. The optical and aggregation behaviors of these functional dyes were studied by means of UV/Vis absorption and fluorescence spectroscopy, dynamic light scattering, and TEM. These dyes are highly fluorescent and cover the whole visible-light region. A donor/acceptor dye displays intramolecular fluorescence resonance energy transfer (FRET), with a high efficiency of up to 96.4 % from pyrene to perylene bisimide chromophores, which leads to a high fluorescence color sensitivity to environmental polarity. Under a λ=365 nm UV lamp, the light-emitting colors of the donor/acceptor dye change from green to yellow with increasing solvent polarity, which demonstrates application potential as a new class of FERT probes. The donor/acceptor dye in water was assembled into hollow vesicles with a narrow size distribution. The bilayer structure of the vesicular wall was directly observed by means of TEM. These vesicular aggregates in water are fluorescent at λ=650–850 nm within the near-infrared region.     

Structure-fluorescence contrast relationship in cyanine DNA intercalators: toward rational dye design

The fluorescence enhancement mechanisms of a series of DNA stains of the oxazole yellow (YO) family have been investigated in detail using steady-state and ultrafast time-resolved fluorescence spectroscopy. The strong increase in the fluorescence quantum yield of these dyes upon DNA binding is shown to originate from the inhibition of two distinct processes: 1) isomerisation through large-amplitude motion that non-radiatively deactivates the excited state within a few picoseconds and 2) formation of weakly emitting H-dimers. As the H-dimers are not totally non-fluorescent, their formation is less efficient than isomerisation as a fluorescent contrast mechanism. The propensity of the dyes to form H-dimers and thus to reduce their fluorescence contrast upon DNA binding is shown to depend on several of their structural parameters, such as their monomeric (YO) or homodimeric (YOYO) nature, their substitution and their electric charge. Moreover, these parameters also have a substantial influence on the affinity of the dyes for DNA and on the ensuing sensitivity for DNA detection. The results give new insight into the development and optimisation of fluorescent DNA probes with the highest contrast.     

Excitonic interaction: another photophysical process for fluorescence‐controlled nucleic acid sensing

An excitonic interaction caused by the H‐aggregation of fluorescent dyes is a new type of useful photophysical process for fluorescence‐controlled nucleic acid sensing. We designed a fluorescence‐labeled nucleotide in which two thiazole orange dyes were linked covalently. A DNA strand containing this fluorescence‐labeled nucleotide showed absorption at 480 nm before hybridization, whereas an absorption band at 510 nm became predominant when the DNA was hybridized with the complementary strand. The shift in the absorption bands shows the existence of an excitonic interaction between dyes in the nucleotide, and as a result, emission from the doubly thiazole orange‐labeled DNA was well controlled. This clear change in fluorescence intensity depending on hybridization is applicable to multicolor RNA imaging in living cells. © 2010 The Japan Chemical Journal Forum and Wiley Periodicals, Inc. Chem Rec 10: 188–196; 2010: Published online in Wiley InterScience ( www.interscience.wiley.com ) DOI 10.1002/tcr.201000003     

Fabrication of Novel Polymer Nanoparticle-Based Fluorescence Resonance Energy Transfer Systems and their Tunable Fluorescence Properties

In this study, two hydrophobic fluorescent dyes, nitrobenzoxadiazolyl (NBD) and 9-(diethylamino)benzo[a]phenoxazin-5-one (NR) with different doping ratios were incorporated into polymer nanoparticles to constitute novel polymer nanoparticle-based fluorescence resonance energy transfer (FRET) systems via a facile one-step mini-emulsion polymerization. Spectroscopic characteristics demonstrate that the two fluorophores have been successfully embedded into the nanoparticles, and the fluorescence emission intensity of the two hydrophobic dyes can be greatly enhanced in aqueous media. The as-prepared fluorescent nanoparticles also display a uniform small size (ca. 55 nm), high dye load, intense fluorescence, as well as controllable amount and ratio of the two dyes. The observed FRET efficiencies (16.0–75.2%), as well as the distance (r) between NBD (donor) and NR (acceptor), is closely correlated to the doping ratio of two dyes. Moreover, by varying the doping ratio of two dyes, the fluorescent nanoparticles would exhibit multicolor through FRET upon a single wavelength excitation, and the fluorescence emission signals of the dye-doped nanoparticles could be accurately tuned. These results indicate that the as-prepared uniform FRET-mediated nanoparticles are of high interest in multiplexed bioanalysis.     

Luminescence spectroscopic studies of trimethinecyanines substituted in polymethine chain with nucleic acids and proteins

The series of symmetrical beta-substituted and alpha,gamma-substituted trimethinecyanine dyes were studied for their absorption and fluorescent characteristics in unbound state and in the presence of nucleic acids and proteins. It was shown that beta-substituted and alpha,gamma-bridged trimethinecyanines containing extended heterocyclic systems or N-phenyl as well as N-cyclohexyl substituents demonstrate increased affinity to proteins. At the same time the presence of both N-phenyl and N-cyclohexyl substituents leads to the decrease of the dye fluorescence intensity in complexes with nucleic acids. For trimethinecyanines similarly to unsymmetrical monomethines the presence of N-omega-hydroxy alkyl substituents results in the increase of fluorescence intensity of dye-DNA complex and the emission decrease of dye-RNA complex.     

Ultrafast excited-state dynamics of oxazole yellow DNA intercalators

The excited-state dynamics of the DNA intercalator YO-PRO-1 and of three derivatives has been investigated in water and in DNA using ultrafast fluorescence spectroscopy. In the free form, the singly charged dyes exist both as monomers and as H-dimers, while the doubly charged dyes exist predominantly as monomers. Both forms are very weakly fluorescent: the monomers because of ultrafast nonradiative deactivation, with a time constant on the order of 3-4 ps, associated with large amplitude motion around the methine bridge, and the H-dimers because of excitonic interaction. Upon intercalation into DNA, large amplitude motion is inhibited, H-dimers are disrupted, and the molecules become highly fluorescent. The early fluorescence dynamics of these dyes in DNA exhibits substantial differences compared with that measured with their homodimeric YOYO analogues, which are ascribed to dissimilarities in their local environment. Finally, the decay of the fluorescence polarization anisotropy reveals ultrafast hopping of the excitation energy between the intercalated dyes. In one case, a marked change of the depolarization dynamics upon increasing the dye concentration is observed and explained in terms of a different binding mode.     

Effect of nucleic acid binding dyes on DNA extraction,amplification, and STR typing

We report on the effects of six dyes used in the detection of DNA on the process of DNA extraction, amplification, and detection of STR loci. While dyes can be used to detect the presence of DNA, their use is restricted if they adversely affect subsequent DNA typing processes. Diamond? Nucleic Acid Dye, GelGreen?, GelRed?, RedSafe?, SYBR^® Green I, and EvaGreen? were evaluated in this study. The percentage of dye removed during the extraction process was determined to be: 70.3% for SYBR^® Green I; 99.6% for RedSafe?; 99.4% for EvaGreen?; 52.7% for Diamond? Dye; 50.6% for GelRed?, and; could not be determined for GelGreen?. It was then assumed that the amount of dye in the fluorescent quantification assay had no effect on the DNA signal. The presence of all six dyes was then reviewed for their effect on DNA extraction. The t‐test showed no significant difference between the dyes and the control. These extracts were then STR profiled and all dyes and control produced full DNA profiles. STR loci in the presence of GelGreen^TM at 1X concentration showed increased amplification products in comparison to the control samples. Full STR profiles were detected in the presence of EvaGreen? (1X), although with reduced amplification products. RedSafe? (1X), Diamond? Dye (1X), and SYBR^® Green I (1X) all exhibited varying degrees of locus drop‐out with GelRed? generating no loci at all. We provide recommendations for the best dye to visualize the presence of DNA profile as a biological stain and its subsequent amplification and detection.