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.     

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.     

Groove-binding unsymmetrical cyanine dyes for staining of DNA: dissociation rates in free solution and electrophoresis gels

The rates of dissociation of three non-intercalative unsymmetrical cyanine dyes, BEBO, BETO and BOXTO from mixed-sequence DNA have been studied with the DNA either free in solution or in confining porous agarose gels. The properties of the new dyes were compared to the related intercalating dyes BO, BO-PRO, TO-PRO and YO-PRO. With DNA in solution, BEBO dissociates more slowly than the monovalent BO and interestingly also more slowly than the divalent dye BO-PRO. Similarly, both BETO and BOXTO exhibit considerably slower dissociation than TO-PRO. The new dyes show biexponential dissociation kinetics in mixed-sequence DNA. The average rate of dissociation increases with increasing ionic strength, but the salt dependence of the dissociation is weaker than for the corresponding intercalating dye. The rate of dye-dissociation decreases by a factor of about 10⁵ in the gel. The rates for the dyes generally follow the pattern that we observe with the DNA in free solution, however a more accentuated stabilization was seen for intercalators than for groove-bound dyes. The results show that, in particular, BOXTO is a promising candidate as a preferentially groove-bound DNA-stain with a large enhancement of the fluorescence quantum yield upon binding to DNA, and which exhibits slow and salt-insensitive dissociation compared to corresponding intercalative dyes.     

Thermodynamics of specific and nonspecific DNA binding by two DNA-binding domains conjugated to fluorescent probes

The complexes designed in this work combine the sequence-specific binding properties of helix-turn-helix DNA-binding motifs with intercalating cyanine dyes. Thermodynamics of the Hin recombinase and Tc3 transposase DNA-binding domains with and without the conjugated dyes were studied by fluorescence techniques to determine the contributions to specific and nonspecific binding in terms of the polyelectrolyte and hydrophobic effects. The roles of the electrostatic interactions in binding to the cognate and noncognate sequences indicate that nonspecific binding is more sensitive to changes in salt concentration, whereas the change in the heat capacity shows a greater sensitivity to temperature for the sequence-specific complexes in each case. The conjugated dyes affect the Hin DNA-binding domain by acting to anchor a short stretch of amino acids at the N-terminal end into the minor groove. In contrast, the N-terminal end of the Tc3 DNA-binding domain is bound in a well-ordered fashion to the DNA even in the absence of the conjugated dye. The conjugated dye and the DNA-binding domain portions of each conjugate bind noncooperatively to the DNA. The characteristic thermodynamic parameters of specific and nonspecific DNA binding by each of the DNA-binding domains and their respective conjugates are presented.     

Binding of symmetrical cyanine dyes into the DNA minor groove

Optical methods, such as fluorescence, circular dichroism and linear flow dichroism, were used to study the binding to DNA of four symmetrical cyanine dyes, each consisting of two identical quinoline, benzthiazole, indole, or benzoxazole fragments connected by a trimethine bridge. The ligands were shown to form a monomer type complex into the DNA minor groove. The complex of quinoline-containing ligand with calf thymus DNA appeared to be the most resistant to ionic strength, and it did not dissociate completely even in 1 M NaCl. Binding of cyanine dyes to DNA could also be characterized by possibility to form ligand dimers into the DNA minor groove, by slight preference of binding to AT pairs, as well as by possible intercalation between base pairs of poly(dG)-poly(dC). The correlation found between the binding constants to DNA and the extent of cyanine dyes hydrophobicity estimated as the n-octanol/water partition coefficient is indicative of a significant role of hydrophobic interactions for the ligand binding into the DNA minor groove.     

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.     

Effects of Polyamines on the Binding of Hoechst 33258 to Calf Thymus DNA

Abstract

The ability of polyamines to displace the minor groove-binding dye Hoechst 33258 from calf thymus DNA was investigated. Polyamines displace non-specific DNA phosphate bound Hoechst in a charge-dependent fashion, but show very little ability to displace the high affinity binding of Hoechst in the minor groove of DNA. This high affinity binding is, however, sensitive to ethidium bromide and the minor groove binding drug berenil. These studies suggest that polyamines probably bind DNA in the minor groove very weakly, if at all, relative to known minor groove binding agents.     

Cyanine dye labeling reagents containing isothiocyanate groups

New isothiocyanate derivatives of cyanine dyes were synthesized as fluorescent covalent labeling reagents for proteins and other biomolecules. These dyes have maximum absorbance in the red and near infrared regions of the spectrum, have high extinction coefficients and have adequate quantum yields. Incorporating two alkyl sulfonate groups in the dye structures increases their water solubility, which is beneficial for labeling biological molecules in aqueous solution. Reactivities of proteins with these new cyanines are similar to their reactivities with fluorescein isothiocyanate. These new labeling reagents are complementary to the fluorescein and rhodamine reagents, expanding the possibilities of multicolor analyses. Sheep anti-mouse-IgG antibody was labeled with a pentamethine cyanine dye (CY5.8-ITC) and used with a fluoresceinated antibody as a second reagent for detecting human T-cell subsets by flow cytometry.     

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 µM 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.     

Linear dichroism and polarized fluorescence of dye-complexed DNA fibers

Summary A simple method to obtain well orientated DNA fibers for studying the ordered binding of dyes and fluorochromes by linear dichroism and polarized fluorescence is described. The metachromatic dye toluidine blue and the intercalating fluorochromes ethidium bromide and acridine orange showed a perpendicular alignement to DNA; the minor groove binding fluorochromes 33258 Hoechst and DAPI appeared parallel. Thus, DNA fibers represent a suitable cytochemical test substrate for studying the orientation of bound dyes by polarization methods.     

Investigation of DNA binding modes for a symmetrical cyanine dye trication: effect of DNA sequence and structure.

The DNA binding behavior of a tricationic cyanine dye (DiSC3+(5)) was studied using the [Poly(dA-dT)]2, [Poly(dI-dC)]2 and Poly(dA) x Poly(dT) duplex sequences and the Poly(dA) x 2Poly(dT) triplex. Optical spectroscopy and viscometry results indicate that the dye binds to the triplex structure by intercalation, to the nonalternating Poly(dA) x Poly(dT) duplex through minor groove binding and to the alternating [Poly(dA-dT)]2 duplex by a combination of two binding modes: intercalation at low concentration and dimerization within the minor groove at higher concentration. Dimerization occurs at lower dye concentrations for the [Poly(dI-dC)]2 sequence, consistent with our previous investigations on an analogous monocationic cyanine dye. [Seifert, J.L., et al. (1999) J. Am. Chem. Soc. 121, 2987-2995] These studies illustrate the diversity of DNA binding modes that are available to a given ligand structure.     

Spectral analysis of the DNA targeting bisalkylaminoanthraquinone DRAQ5 in intact living cells.

BACKGROUND: We report on the potential DNA binding modes and spectral characteristics of the cell-permeant far red fluorescent DNA dye, DRAQ5, in solution and bound within intact cells. Our aim was to determine the constraints for its use in flow cytometry and bioimaging. METHODS: Solution characteristics and quantum yields were determined by spectroscopy. DRAQ5 binding to nuclear DNA was analyzed using fluorescence quenching of Hoechst 33342 dye, emission profiling by flow cytometry, and spectral confocal laser scanning microscopy of the complex DRAQ5 emission spectrum. Cell cycle profiling utilized an EGFP-cyclin B1 reporter as an independent marker of cell age. Molecular modeling was used to explore the modes of DNA binding. RESULTS: DRAQ5 showed a low quantum yield in solution and a spectral shift upon DNA binding, but no significant fluorescence enhancement. DRAQ5 caused a reduction in the fluorescence intensity of Hoechst 33342 in live cells prelabeled with the UV excitable dye, consistent with molecular modeling that suggests AT preference and an engagement of the minor groove. In vivo spectral analysis of DRAQ5 demonstrated shifts to longer wavelengths upon binding with DNA. Analysis of spectral windows of the dual emission peaks at 681 and 707 nm in cells showed that cell cycle compartment recognition was independent of the far red-near IR emission wavelengths monitored. CONCLUSIONS: The study provides new clues to modes of DNA binding of the modified anthraquinone molecule in vivo, and its AT base-pair selectivity. The combination of low quantum yield but high DNA affinity explains the favorable signal-to-noise profile of DRAQ5-nuclear fluorescence. The robust nature of cell cycle reporting using DRAQ5, even when restricted spectral windows are selected, facilitates the analysis of encroaching spectral emissions from other fluorescent reporters, including GFP-tagged proteins.     

Comparing mono- and divalent DNA groove binding cyanine dyes -- binding geometries, dissociation rates, and fluorescence properties

The unsymmetrical cyanine dyes BOXTO-PRO and BOXTO-MEE were derived from the DNA groove binder BOXTO, by adding a positively charged or a non-ionic hydrophilic tail to BOXTO, respectively. The main objective was to obtain more efficient DNA probes, for instance in electrophoresis and microscopy, by slowing down the dissociation of BOXTO from DNA. The interactions with mixed sequence DNA was studied with fluorescence and absorbance spectroscopy, stopped-flow dissociation and gel electrophoresis. Both the derivatives are groove bound as BOXTO, and have similar fluorescence properties when bound to mixed sequence DNA in free solution. BOXTO-PRO exhibits a slower dissociation than BOXTO from DNA, whereas the dissociation rate for BOXTO-MEE is faster and, unexpectedly independent of the ionic strength. During gel electrophoresis both BOXTO-PRO and BOXTO-MEE exhibit a faster dissociation rate than BOXTO. Still, BOXTO-PRO seems to be a good alternative as DNA probe, especially for applications in free solution where the dissociation is slower than for the corresponding intercalator TOPRO-1.     

Characterization of SYBR Gold nucleic acid gel stain: a dye optimized for use with 300-nm ultraviolet transilluminators

The highest sensitivity nucleic acid gel stains developed to date are optimally excited using short-wavelength ultraviolet or visible light. This is a disadvantage for laboratories equipped only with 306- or 312-nm UV transilluminators. We have developed a new unsymmetrical cyanine dye that overcomes this problem. This new dye, SYBR Gold nucleic acid gel stain, has two fluorescence excitation maxima when bound to DNA, one centered at approximately 300 nm and one at approximately 495 nm. We found that when used with 300-nm transillumination and Polaroid black-and-white photography, SYBR Gold stain is more sensitive than ethidium bromide, SYBR Green I stain, and SYBR Green II stain for detecting double-stranded DNA, single-stranded DNA, and RNA. SYBR Gold stain's superior sensitivity is due to the high fluorescence quantum yield of the dye-nucleic acid complexes ( approximately 0.7), the dye's large fluorescence enhancement upon binding to nucleic acids ( approximately 1000-fold), and its capacity to more fully penetrate gels than do the SYBR Green gel stains. We found that SYBR Gold stain is as sensitive as silver staining for detecting DNA-with a single-step staining procedure. Finally, we found that staining nucleic acids with SYBR Gold stain does not interfere with subsequent molecular biology protocols.     

Methylene blue binding to DNA with alternating GC base sequence: continuum treatment of salt effects

Methylene blue (MB), an efficient singlet oxygen generating photoactive dye, binds to DNA and allows photosensitized reactions to be used for sequence-specific cleavage of the DNA backbone. Intercalation and groove binding are possible binding modes of the dye, depending on base sequences and environmental conditions. In a recent modeling study of methylene blue binding to a double stranded DNA decamer with an alternating GC sequence, six structural models for intercalation structures and for minor and major groove binding have been obtained. By estimating the binding energies (including electrostatic reaction field contributions of a salt-free aqueous solvent), symmetric intercalation at the 5'-CpG-3' and 5'-GpC-3' steps was found as the predominant binding mode, followed by a slightly weaker binding of the dye in the minor groove. In this study, the stability of the modeled structures has been analysed as a function of salt concentration. The results of finite difference numerical solutions of the non-linear Poisson-Boltzmann equation show that the stabilizing effect of salt is larger for free DNA than for the modeled MB-DNA complexes. Accordingly, the estimated binding energies decrease with increasing ionic strength. A slightly higher stabilization of the groove binding complexes results in comparable binding energies for symmetric intercalation and minor groove binding at high salt concentration. Both results are in qualitative agreement with experimental data.     

Synthesis and DNA binding profile of monomeric,dimeric, and trimeric derivatives of crystal violet

Monomeric, dimeric, and trimeric derivatives of the triphenylmethane dye crystal violet (1a–1f) have been synthesized for the purpose of evaluating their affinity and sequence selectivity for duplex DNA. Competitive ethidum displacement assays indicate that 1a–1f have apparent association constants for CT DNA in the range of 1.80–16.2 × 10⁷ M⁻¹ and binding site sizes of 10–14 bp. Viscosity experiments performed on ligand 1f confirmed that these dyes associate with duplex DNA by a non-intercalative mode of binding. Circular dichroism and competition binding studies of the tightest binding ligand 1e with known major and minor groove binding molecules suggest that these dye derivatives likely occupy the major groove of DNA. Data from the binding of 1e to polynucleotides indicate close to an order of magnitude preference for associating with AT rich homopolymers over GC rich homopolymers, suggesting a shape-selective match of the sterically bulky ligand with DNA containing a wider major groove.     

Investigation of DNA Binding Modes for a Symmetrical Cyanine Dye Trication: Effect of DNA Sequence and Structure

Abstract

The DNA binding behavior of a tricationic cyanine dye (DiSC₃₊(5)) was studied using the [Poly(dA-dT)]₂, [Poly(dI-dC)]₂ and Poly(dA)?Poly(dT) duplex sequences and the Poly(dA) ?2Poly(dT) triplex. Optical spectroscopy and viscometry results indicate that the dye binds to the triplex structure by intercalation, to the nonalternating Poly(dA)?Poly(dT) duplex through minor groove binding and to the alternating [Poly(dA-dT)]₂ duplex by a combination of two binding modes: intercalation at low concentration and dimerization within the minor groove at higher concentration. Dimerization occurs at lower dye concentrations for the [Poly(dI-dC)]₂ sequence, consistent with our previous investigations on an analogous monocationic cyanine dye. [Seifert, J.L., et al. (1999) J. Am. Chem. Soc. 121, 2987–2995] These studies illustrate the diversity of DNA binding modes that are available to a given ligand structure.     

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.     

Fluorescence polarization study of DNA--proflavine complexes

The binding of proflavine to DNA has been studied by measuring the polarization and intensity of emission of DNA–dye complexes. Such measurements also permit the determination of the fluorescence of the bound dye as a function of the degree of binding. Techniques of emission spectroscopy permit the study of complexing at high phosphate to dye ratios, and we have examined complexes formed at up to 12,300:1 phosphates to dye. At high phosphate to dye ratios, we find that equilibrium plots of the binding data show only one type of binding. Reports in the literature of multiple binding constants are shown to be due to the incorrect assumption that the fluorescence of the bound dye is independent of the amount bound. The emission properties can be qualitatively accounted for by assuming that nearest-neighbor interaction between bound dyes quenches the fluorescence. We report that, within experimental error, the binding constant is insensitive to the base content of the DNA. The DNA-dye complexes show a temperature dependent depolarization, the cause of which is, as yet, unknown. Heat denaturation of the DNA–dye complex may be followed on a Perrin plot.     

Spectroscopic studies of the multiple binding modes of a trimethine-bridged cyanine dye with DNA

The interaction between DNA and a benzothiazole-quinoline cyanine dye with a trimethine bridge (TO-PRO-3) results in the formation of three noncovalent complexes. Unbound TO-PRO-3 has an absorption maximum (λ_max) of 632 nm, while the bound dyes (with calf thymus DNA) have electronic transitions with λ_max = 514nm (complex I), 584nm (complex II) and 642 nm (complex III). The blue shifts in the electronic transitions and the bisignate shape of the circular dichroism bands indicate that TO-PRO-3 aggregates with DNA. Complex I has a high dye:base pair stoichiometry, which does not depend on base sequence or base modifications. The bound dyes exhibit strong interdye coupling, based on studies with a short oligonucleotide and on enhanced resonance scattering. From thermal dissociation studies, the complex is weakly associated with DNA. Studies with poly(dGdC)₂ and poly(dIdC)₂ and competitive binding with distamycin demonstrate that complex II is bound in the minor groove. This complex stabilizes the helix against dissociation. For complex III, the slightly red-shifted electronic transition and the stoichiometry are most consistent with intercalation. Using poly(dAdT)₂, the complexes have the following dye mole fractions (X_dye): X_dye = 0.65 (complex I), 0.425 (complex II) and 0.34 (complex III).