Fluorescence properties and photophysics of the sulfoindocyanine Cy3 linked covalently to DNA

The sulfoindocyanine Cy3 is one of the most commonly used fluorescent dyes in the investigation of the structure and dynamics of nucleic acids by means of fluorescence methods. In this work, we report the fluorescence and photophysical properties of Cy3 attached covalently to single-stranded and duplex DNA. Steady-state and time-resolved fluorescence techniques were used to determine fluorescence quantum yields, emission lifetimes, and fluorescence anisotropy decays. The existence of a transient photoisomer was investigated by means of transient absorption techniques. The fluorescence quantum yield of Cy3 is highest when attached to the 5' terminus of single-stranded DNA (Cy3-5' ssDNA), and decreases by a factor of 2.4 when the complementary strand is annealed to form duplex DNA (Cy3-5' dsDNA). Substantial differences were also observed between the 5'-modified strands and strands modified through an internal amino-modified deoxy uridine. The fluorescence decay of Cy3 became multiexponential upon conjugation to DNA. The longest lifetime was observed for Cy3-5' ssDNA, where about 50% of the decay is dominated by a 2.0-ns lifetime. This value is more than 10 times larger than the fluorescence lifetime of the free dye in solution. These observations are interpreted in terms of a model where the molecule undergoes a trans-cis isomerization reaction from the first excited state. We observed that the activation energy for photoisomerization depends strongly on the microenvironment in which the dye is located. The unusually high activation energy measured for Cy3-5' ssDNA is an indication of dye-ssDNA interactions. In fact, the time-resolved fluorescence anisotropy decay of this sample is dominated by a 2.5-ns rotational correlation time, which evidences the lack of rotational freedom of the dye around the linker that separates it from the terminal 5' phosphate. The remarkable variations in the photophysical properties of Cy3-DNA constructs demonstrate that caution should be used when Cy3 is used in studies employing DNA conjugates.     

Homodimeric monomethine cyanine dyes as fluorescent probes of biopolymers

The fluorescence properties of newly synthesized homodimeric monomethine cyanine dyes in the presence of biopolymers are investigated. They do not fluoresce in TE buffer and bidistilled water but become strongly fluorescent (Q(F)=0.3-0.9) in the region 530-650 nm when bound to dsDNA and ssDNA. The detection limit of dsDNA is about 1.7 ng/ml. Some of dyes studied are able to distinguish between dsDNA and ssDNA, RNA, BSA in solution and gel electrophoresis. The influence of different factors (temperature, pH and viscosity of the medium, presence of histone) on the formation of the dye-biopolymer complexes is investigated. The results of steady-state and dynamic fluorescence measurements concerning the different types of binding between dyes and biopolymers show that the new dyes are applicable in molecular biology as highly sensitive fluorescence labels.     

The staining efficiency of cyanine dyes for single‐stranded DNA is enormously dependent on nucleotide composition

The staining of nucleic acids with fluorescent dyes is one of the most fundamental technologies in relevant areas of science. For reliable and quantitative analysis, the staining efficiency of the dyes should not be very dependent on the sequences of the specimens. However, this assumption has not necessarily been confirmed by experimental results, especially in the staining of ssDNA (and RNA). In this study, we found that both SYBR Green II and SYBR Gold did not stain either homopyrimidines or ssDNA composed of only adenine (A) and cytosine (C). However, these two dyes emit strong fluorescence when the ssDNA contains both guanine (G) and C (and/or both A and thymine (T)) and form potential Watson‐Crick base pairs. Interestingly, SYBR Gold, but not SYBR Green II, strongly stains ssDNA consisting of G and A (or G and T). Additionally, we found that the secondary structure of ssDNA may play an important role in DNA staining. To obtain reliable results for practical applications, sufficient care must be paid to the composition and sequence of ssDNA.     

Photochemical Characteristics of the mode-locking dyes 1,1′-diethyl-4,4′ carbocyanine iodide (cryptocayanine,DCI) and 1,1′-diethyl-2,2′ dicarb

The fluorescence quantum efficiencies, lifetimes and triplet—triplet absorption spectra of the cyanine dyes DCI     

Key Structural Elements of Unsymmetrical Cyanine Dyes for Highly Sensitive Fluorescence Turn‐On DNA Probes

Unsymmetrical cyanine dyes, such as thiazole orange, are useful for the detection of nucleic acids with fluorescence because they dramatically enhance the fluorescence upon binding to nucleic acids. Herein, we synthesized a series of unsymmetrical cyanine dyes and evaluated their fluorescence properties. A systematic structure–property relationship study has revealed that the dialkylamino group at the 2‐position of quinoline in a series of unsymmetrical cyanine dyes plays a critical role in the fluorescence enhancement. Four newly designed unsymmetrical cyanine dyes showed negligible intrinsic fluorescence in the free state and strong fluorescence upon binding to double‐stranded DNA (dsDNA) with a quantum yield of 0.53 to 0.90, which is 2 to 3 times higher than previous unsymmetrical cyanine dyes. A detailed analysis of the fluorescence lifetime revealed that the dialkylamino group at the 2‐position of quinoline suppressed nonradiative decay in favor of increased fluorescence quantum yield. Moreover, these newly developed dyes were able to stain the nucleus specifically in fixed HeLa cells examined by using a confocal laser‐scanning microscope.     

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.     

A New Trace Analytic Method for the Determination of Sequence-Specific DNA with Fluorescent Probes

A new method of fluorescence spectrometry detection of single-strand DNA (ssDNA) was established by hybridizing the ssDNA with its complementary ssDNA to form double-stranded DNA (dsDNA). Our results show that the fluorescence intensity increased significantly when the nucleic acid molecular “light switch"(Ru(phen)₂dppx²⁺) or Hoechst 33258 dye interacted with dsDNA, and the fluorescence intensity also increased as the DNA concentration increased. The changing law was also studied about how the fluorescence intensity changed when the two kinds of fluorescent probes interacted with oligonucleotide of different lengths and different sequences, as well as DNA-DNA′ hybridization products. Then, the effect of the bases mismatch, varying length of DNA chain, and different DNA sequences on the fluorescence intensity were explored at the same time, by detecting the specific DNA sequence of avian influenza H1N1 virus, cauliflower mosaic virus, and hepatitis C virus. Additionally, the selectivity, linear range, and sensitivity of the two probes were compared.     

Capillary electrophoresis of double-stranded DNA fragments using a new fluorescence intercalating dye EvaGreen

EvaGreen is a new DNA intercalating dye successfully used in quantitative real-time PCR. In the present work, we firstly apply EvaGreen to the analysis of dsDNA by CE with LIF detection. Comparisons of EvaGreen dye with the commonly used dyes SYBR Green I and SYBR Gold were preformed in dsDNA analysis by CE. The linear range of dsDNA using EvaGreen was slightly wider than that using SYBR Gold and SYBR Green I, and the detection limits of dsDNA were not significantly different for the three dyes. Good separations of dsDNA fragments were obtained using the three dyes. Reproducibility of migration time and the peak area of dsDNA fragments with EvaGreen were better than those for SYBR Green I and SYBR Gold. The RSD values were 0.24-0.27% for migration time and 3.45-7.59% for peak area within the same day, 1.35-1.63% for migration time and 6.72-12.05% for peak area for three days. Our data demonstrated that EvaGreen is well suited for the dsDNA analysis by CE with LIF detection.     

Comparison of benzothiazole-based dyes for sensitive DNA detection

For efficient and quantitative DNA detection, fluorescence staining is the most often explored approach, which relies on non-covalent binding of dyes with double stranded DNA (dsDNA). Ethidium bromide (EB) is the most classic DNA stain, but suffers from its high carcinogenicity. A series of less toxic alternatives were developed, many of which contain the core structure of the benzothiazole ring. However, the relationship between the structure and the DNA detection performance was not illustrated. Herein, five benzothiazole dyes, namely thiazole orange, SYBR Green I, PicoGreen, SYBR Safe, and thioflavine-T, were compared for DNA detection through direct fluorescence and gel electrophoresis, with particular focus on the structure-performance relationship. It turned out that SYBR Green I is currently the best choice for DNA detection. The results in this work may be useful for future DNA-staining dye developments.     

Comparison of benzothiazole-based dyes for sensitive DNA detection

For efficient and quantitative DNA detection, fluorescence staining is the most often explored approach, which relies on non-covalent binding of dyes with double stranded DNA (dsDNA). Ethidium bromide (EB) is the most classic DNA stain, but suffers from its high carcinogenicity. A series of less toxic alternatives were developed, many of which contain the core structure of the benzothiazole ring. However, the relationship between the structure and the DNA detection performance was not illustrated. Herein, five benzothiazole dyes, namely thiazole orange, SYBR Green I, PicoGreen, SYBR Safe, and thioflavine-T, were compared for DNA detection through direct fluorescence and gel electrophoresis, with particular focus on the structure-performance relationship. It turned out that SYBR Green I is currently the best choice for DNA detection. The results in this work may be useful for future DNA-staining dye developments.     

Inhibition of peroxyoxalate chemiluminescence by intercalation of fluorescent acceptors between DNA bases

We have examined the ability of different fluorescent DNA dyes to become chemically excited by the peroxyoxalate chemiluminescent reaction. The intercalating dyes ethidium bromide and propidium iodide, and the bis-intercalating dyes ethidium homodimer-1, benzoxazolium-4-pyridinium dimer-1 and benzoxazolium-4-quinolinium dimer-1, exhibit an intense chemiluminescence when they are excited by the bis(2,4,6-trichlorophenyl)oxalate (TCPO)-H2O2 reaction in the absence of DNA. However, the chemiluminescence of these dyes is very low when they are bound to double-stranded DNA (dsDNA). In contrast, the minor groove-binding dye Hoechst 33258 excited by the TCPO-H2O2 reaction shows approximately the same chemiluminescence intensity when it is free in solution or complexed with dsDNA. Structural alterations or partial dissociation of dsDNA-bis-intercalating dye complexes produced by the addition of acetone, NaCl, MgCl2 or the cationic surfactant cetyltrimethylammonium bromide increases the chemiluminescence intensity. A moderate chemiluminescence intensity is observed when bis-intercalating dyes are complexed with single-stranded DNA. Our results indicate that the energy from the intermediates produced in the peroxyoxalate chemiluminescent reaction cannot be efficiently transferred to fluorescent dyes complexed with DNA; chemiexcitation is almost completely inhibited when dyes are buried in the dsDNA structure by intercalation between the base pairs.     

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 °C for more than 1 year and 40 μM solution was stable for 45 days (at least) at 4 °C.     

Orientation of dye molecules in DNA-based films with chain alignment and judgment of their DNA-binding modes

Novel composite films of chain-oriented DNA, which contain the DNA-binding dyes aligned in specific orientation, were successfully prepared by drying the solution under a horizontal magnetic field. Most of the dye-DNA composite films showed linear dichroism, as revealed by polarized ultraviolet-visible (UV-vis) spectroscopy. The intercalators, ethidium bromide and acridine orange, were fixed in chain-oriented DNA films in a similar binding manner as in solutions. Also, Hoechst 33258 and 4',6-diamidino-2-phenylindole were found to be aligned along the minor groove, even in the solid films. Thus, our new method of preparing dye-DNA composite films with chain orientation is useful for aligning small molecules, and it will provide views of the novel anisotropic materials expected in various application fields. We used this method to prepare composite DNA films with newly designed original compounds. Seven of nine dyes were judged to bind obviously to DNA as intercalators by polarized UV-vis spectroscopy. The DNA-binding manners were further analyzed by fluorescence anisotropy measurements. On the basis of the curves for the rotational angle dependence of the anisotropy, we were able to estimate the angles between the transition-dipole moments of dyes and the aligned chain axis of DNA. Interestingly, two original compounds were found to be in the tilted forms with regard to the plane of base pairs. We emphasize here that the method using aligned dye-DNA films is very convenient for identifying the binding modes of the compounds for double-stranded DNA.     

New asymmetric monomethine cyanine dyes for nucleic-acid labelling: absorption and fluorescence spectral characteristics

Absorption spectra and fluorescence properties of a series of newly synthesized asymmetric monomethine cyanine dyes are studied. The dyes carry one or two positive charges. They are devoid of their own fluorescence in solution and become fluorescent upon binding to nucleic acids only. The fluorescence maxima of the new dyes are localized between 530 and 650 nm. The wavelength and intensity of fluorescence are dependent on molecular structure of the dye, type of nucleic acid and the concentration of both nucleic acid and salts. Some of the dyes are capable of distinguishing between single-stranded and double-stranded (ds) polynucleotides giving fluorescence maxima localized at different wavelengths. Detection threshold for dsDNA for most of the dyes is comparable to that of ethidium bromide. The sensitivity of the dye-dsDNA complexes to NaCl concentrations show that the new dyes interact with dsDNA by both intercalation and electrostatically.     

Selective interactions of a few acridinium derivatives with single strand DNA: study of photophysical and DNA binding interactions

Novel acridinium derivatives 1-3, wherein steric factors have been varied systematically through substitution at the ninth position of the acridinium ring, were synthesized and their interactions with single strand and double strand DNA have been investigated through photophysical, biophysical, and microscopic techniques. The acridinium derivative 1 exhibited quantitative fluorescence yields (phi f approximately =1) and high lifetime of 35 ns, while significantly lower fluorescence yields of 0.11 and 0.02 and lifetimes of 3.5 and 1.2 ns were observed for 2 and 3, respectively. The derivatives 1 and 2 having 2-methylphenyl and 2,4-dimethylphenyl substituents at the ninth position of the acridinium ring showed selective interactions with single strand DNA (ssDNA) with association constants of KssDNA = 6.3-6.6 x 10(4) M(-1), while negligible interactions were observed with double strand DNA (dsDNA). In contrast, the derivative 3 with 2,6-dimethylphenyl substitution showed negligible interactions with both ssDNA and dsDNA. Studies with a series of 19-mer oligonucleotides indicate that these derivatives exhibit significant selectivity for the sequences rich in guanosine (ca. 3-fold) as compared to the cytosine-rich sequences. These derivatives with high water solubility and the ability to distinguish between ssDNA and dsDNA through changes in fluorescence emission can be used as fluorescent probes for understanding the role of ssDNA in various biological processes and to study various DNA-ligand interactions.     

Photobleaching of asymmetric cyanines used for fluorescence imaging of single DNA molecules

The photobleaching of the cyanine dyes YO and YOYO has been investigated for both free and DNA-bound dyes, using absorption and fluorescence spectroscopy coupled with fluorescence microscopy. For the free dyes, the nature of the reactive species involved in the photodegradation process is different for the monomer and the dimer, as shown by scavenger studies. For DNA-bound dyes, photoinduced fading of the visible absorption band occurs by different pathways depending on the drug binding mode and can be attenuated by appropriate scavengers. However, none of these scavengers were found to have any significant effect on the photobleaching of dye fluorescence. It appears that the reduction of fluorescence intensity comes from a quenching of the dye fluorescence by modified DNA bases, possibly 8-oxo-7,8-dihydro-2'-deoxyguanosine.     

Acridine-viologen dyads: selective recognition of single-strand DNA through fluorescence enhancement

Tolylacridine-viologen dyads show distinct fluorescence emission changes in the presence of double-strand DNA (dsDNA) and single-strand DNA (ssDNA) depending on the position of the linkage. The para isomer shows fluorescence quenching in the presence of both dsDNA and ssDNA, while the ortho isomer interacts selectively with ssDNA with enhancement in fluorescence intensity.     

Identification of intermediate species in protein-folding by quantitative analysis of amplitudes in time-domain fluorescence spectroscopy

In protein-folding studies it is often required to differentiate a system with only two-states, namely the native (N) and unfolded (U) forms of the protein present at any condition of the solvent, from a situation wherein intermediate state(s) could also be present. This differentiation of a two-state from a multi-state structural transition is non-trivial when studied by the several steady-state spectroscopic methods that are popular in protein-folding studies. In contrast to the steady-state methods, time-resolved fluorescence has the capability to reveal the presence of heterogeneity of structural forms due to the ‘fingerprint’ nature of fluorescence lifetimes of various forms. In this work, we establish this method by quantitative analysis of amplitudes associated with fluorescence lifetimes in multiexponential decays. First, we show that we can estimate, accurately, the relative population of species from two-component mixtures of non-interacting molecules such as fluorescent dyes, peptides and proteins. Subsequently, we demonstrate, by analysing the amplitudes of fluorescence lifetimes which are controlled by fluorescence resonance energy transfer (FRET), that the equilibrium folding-unfolding transition of the small single-domain protein barstar is not a two-step process.     

Binding interaction of cationic phenazinium dyes with calf thymus DNA: a comparative study

Absorption, steady-state fluorescence, steady-state fluorescence anisotropy, and intrinsic and induced circular dichroism (CD) have been exploited to explore the binding of calf thymus DNA (ctDNA) with three cationic phenazinium dyes, viz., phenosafranin (PSF), safranin-T (ST), and safranin-O (SO). The absorption and fluorescence spectra of all the three dyes reflect significant modifications upon interaction with the DNA. A comparative study of the dyes with respect to modification of fluorescence and fluorescence anisotropy upon binding, effect of urea, iodide-induced fluorescence quenching, and CD measurements reveal that the dyes bind to the ctDNA principally in an intercalative fashion. The effect of ionic strength indicates that electrostatic attraction between the cationic dyes and ctDNA is also an important component of the dye-DNA interaction. Intrinsic and induced CD studies help to assess the structural effects of dyes binding to DNA and confirm the intercalative mode of binding as suggested by fluorescence and other studies. Finally it is proposed that dyes with bulkier substitutions are intercalated into the DNA to a lesser extent.     

Synthesis of chloro-substituted analogs of Thiazole orange - Fluorophores for flow cytometric analyses

Synthesis, absorption and fluorescence properties of a series of asymmetrical monomethine cyanine dyes, chloro-containing analogs of Thiazole orange, are reported. Their staining ability was studied by flow cytometry. The saturating concentrations of each dye that gives a stable staining intensity have been determined. The ability of dyes B9, B11, B13 to stain live macrophages and apoptotic splenocytes was investigated. Positive signal in nucleus of adherent macrophages detected by fluorescent microscopy showed good specificity of B9, B11 and B13 dyes for DNA. In apoptotic assay cells positive for Annexin V were stained more brightly with the dyes B9, B11 and B13 than with propidium iodide. Despite that B13 showed high DNA selectivity it induces apoptosis of splenocytes and it is not suitable for detection of dead cells. The other synthesized chloro-containing analogs of Thiazole orange B9 and B11 can be successfully used for flow cytometric analyses of DNA content in live cells and for analyses of cell apoptosis.