New fluorochromes, compatible with high wavelength excitation, for flow cytometric analysis of cellular nucleic acids

The spectroscopic properties of three new dyes, EK4, VL772, and LL585, free and bound to nucleic acids, are presented, with particular emphasis on their potential use in flow cytometry. Two of these dyes, EK4 and LL585, exhibit red fluorescence, while dye VL772 exhibits yellow fluorescence. Dye LL585 exhibits specificity for DNA, relative to RNA, and a marked enhancement of fluorescence efficiency upon binding to DNA, needed for a red fluorescent DNA-specific stain for flow cytometry. The dye penetrates live cells, although uniformity of nuclear fluorescence, as evidenced by DNA flow histograms, is better if the cells are first permeabilized with Triton X-100. Dye VL772 exhibits yellow fluorescence and little DNA-RNA discrimination, but may prove useful in conjunction with dye LL585 when simultaneous assay of cellular RNA and DNA is desired. Dye EK4 shares properties of the other two dyes but fluoresces with much less efficiency. Dyes LL585 and VL772, used singly, as a pair, or in combination with blue-fluorescing DNA specific dyes, such as bisbenzimidazole derivatives, should permit new, convenient analyses of the content and organization of cellular nucleic acids.     

Photoconversion of DAPI and Hoechst dyes to green and red-emitting forms after exposure to UV excitation

The fluorescent dye 4′-6-Diamidino-2-phenylindole (DAPI) is frequently used in fluorescence microscopy as a chromosome and nuclear stain because of its high specificity for DNA. Normally, DAPI bound to DNA is maximally excited by ultraviolet (UV) light at 358 nm, and emits maximally in the blue range, at 461 nm. Hoechst dyes 33258 and 33342 have similar excitation and emission spectra and are also used to stain nuclei and chromosomes. It has been reported that exposure to UV can convert DAPI and Hoechst dyes to forms that are excited by blue light and emit green fluorescence, potentially confusing the interpretation of experiments that use more than one fluorochrome. The work reported here shows that these dyes can also be converted to forms that are excited by green light and emit red fluorescence. This was observed both in whole tissues and in mitotic chromosome spreads, and could be seen with less than 10-s exposure to UV. In most cases, the red form of fluorescence was more intense than the green form. Therefore, appropriate care should be exercised when examining tissues, capturing images, or interpreting images in experiments that use these dyes in combination with other fluorochromes.     

Interactions of pyronin Y(G) with nucleic acids

Spectral properties of pyronin Y(PY) alone or in complexes with natural and synthetic nucleic acids of various base compositions have been studied in aqueous solution containing 10 or 150 mM NaCl and 5 mM Hepes at pH 7.0. The dimerization constant (KD = 6.27 X 10(3), M-1) and the absorption spectra of the dye in monomeric and dimeric form were established. The complexes of PY with single-stranded (ss) nucleic acids show a hypsochromic shift in absorption, and their fluorescence is quenched by over 90% compared to free dye. In contrast, complexes with double-stranded (ds) RNA or DNA (binding by intercalation) exhibit a bathochromic shift in their absorption (excitation) spectrum, and their fluorescence is correlated with the base composition of the binding site. Namely, guanine quenches fluorescence of PY by up to 90%, whereas A, C, I, T, and U bases exert a rather minor effect on the fluorescence quantum yield of the dye. The intrinsic association constant of the dye to ds RNA (Ki = 6.96 X 10(4), M-1) and to ds DNA (Ki = 1.74 X 10(4), M-1) was measured in 150 mM NaCl; the binding site size was 2-3 base pair for both polymers. Implications of these findings for qualitative and quantitative cytochemistry of nucleic acids are discussed.     

Fluorescence and binding properties of phenazine derivatives in complexes with polynucleotides of various base compositions and secondary structures

The interactions of two phenazine derivatives, one with a neutral chromophore (glycoside) and the other with a cationic one (quaternary salt), with various synthetic single- and double-stranded polynucleotides and natural DNA were studied by fluorescence techniques, conducting measurements of steady-state fluorescence intensity and polarization degree as well as fluorescence lifetime. These dyes show fluorescence quenching upon intercalation into the GC sequences of the double-stranded nucleic acids and an increase in fluorescence emission and lifetime upon incorporation into the AT and AU sequences. GC base pairs in continuous deoxynucleotide sequences were found to be preferred as binding sites for both phenazines, in contrast to AT base pairs. On the contrary, the continuous ribonucleotide GC sequence binds the phenazines more weakly than does the AU sequence. With regard to the interaction of the phenazines with single-stranded polynucleotides, a stacking interaction of the dye chromophores with the nucleic bases was observed. In that case the guanine residue quenches the cationic phenazine fluorescence, while the stacking interaction with the other bases results in an increase in the fluorescence quantum yield. Unlike the cationic dye, the fluorescence of the neutral phenazine was quenched by both purine bases.     

Interaction of dimeric intercalating dyes with single-stranded DNA.

The unsymmetrical cyanine dye thiazole orange homodimer (TOTO) binds to single-stranded DNA (ssDNA, M13mp18 ssDNA) to form a fluorescent complex that is stable under the standard conditions of electrophoresis. The stability of this complex is indistinguishable from that of the corresponding complex of TOTO with double-stranded DNA (dsDNA). To examine if TOTO exhibits any binding preference for dsDNA or ssDNA, transfer of TOTO from pre-labeled complexes to excess unlabeled DNA was assayed by gel electrophoresis. Transfer of TOTO from M13 ssDNA to unlabeled dsDNA proceeds to the same extent as that from M13 dsDNA to unlabeled dsDNA. A substantial amount of the dye is retained by both the M13 ssDNA and M13 dsDNA even when the competing dsDNA is present at a 600-fold weight excess; for both dsDNA and ssDNA, the pre-labeled complex retains approximately one TOTO per 30 bp (dsDNA) or bases (ssDNA). Rapid transfer of dye from both dsDNA and ssDNA complexes is seen at Na+ concentrations > 50 mM. Interestingly, at higher Na+ or Mg2+ concentrations, the M13 ssDNA-TOTO complex appears to be more stable to intrinsic dissociation (dissociation in the absence of competing DNA) than the complex between TOTO and M13 dsDNA. Similar results were obtained with the structurally unrelated dye ethidium homodimer. The dsDNA- and ssDNA-TOTO complexes were further examined by absorption, fluorescence and circular dichroism spectroscopy. The surprising conclusion is that polycationic dyes, such as TOTO and EthD, capable of bis-intercalation, interact with dsDNA and ssDNA with very similar high affinity.     

Fluorescence of proflavine--DNA complexes: heterogeneity of binding sites

Measurements of the relative quantum yield of fluorescence of proflavine bound to DNA as a function of the number of bound dyes per nucleotide and the ionic strength allow the determination of the binding constants and respective number of the two types of sites previously postulated. It is demonstrated that 2–3% of the base pairs form sites where the dye is strongly bound and fluoresces normally while in the other set of sites the binding constant is 3–4 times weaker and the fluorescence completely quenched. Comparison with complexes of Pro with double stranded polynucleotides poly (A + U), poly (I + C), poly(G + C), confirm that the strong binding sites correspond to A-T-rich regions of the DNA while the quenched sites seem to require the presence of a neighboring guanine. The role of charge transfer in quenching of fluorescence and mutagnic action is considered. An original method for the determination of free dye and bound dye, based upon the use of an external quencher is described in the Appendix.     

Interaction of cyanine dyes with nucleic acids. XXI. Arguments for half-intercalation model of interaction

The spectral luminescent properties of two groups of monomethine cyanine dyes were studied in the presence of DNA. The first group included five dyes with 5,6-methylenedioxy-[d]-benzo-1,3-thiazole heterocycle and their unsubstituted analogs. Five monomethine pyrylium cyanines and their N-methyl-pyridine analogs were included in the second group. In each pair the pyrylium and pyridine dyes had similar geometry but differed in charge density distribution. The results presented some evidence in favor of the half-intercalation interaction mode between the studied dyes and DNA. When the benzothiazole residue had the lowest electron donor ability between the two heterocycles in the dye molecule, its substitution with the bulky methylenedioxy group led to a significant decrease in fluorescence enhancement of the dye-DNA complex. On the contrary, when the substituents that create steric hindrance (e.g., methylenedioxy and methyl groups) were introduced into the heterocycle with the higher electron donor ability, the fluorescence enhancement value of the dye-DNA complex was virtually unchanged. The changes in the Stock's shift values upon the formation of the dye-DNA complexes were in agreement with the proposed half-intercalation model. Interestingly, in the dye-DNA complexes the pyrylium dyes probably resided in a place similar to the pyridine ones. It is possible that the benzothiazole (or benzooxazole) ring intercalated between the DNA bases and the pyrylium (or pyridine) residue was located in the DNA groove closer to the phosphate backbone.     

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.     

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.     

Increase in acridine orange (AO) fluorescence intensity of monocytes cultured in plastic tissue culture plates as measured by flow cytometry.

Although the green-red fluorescence of AO is an accepted measure of DNA-RNA content, respectively, it is actually a measure of the fluorescence of dye bound to nucleic acids, and may vary with changes in accessibility to the dye. It has been shown for example that extraction of nuclear proteins results in a marked increase in DNA stainability. Moreover, in certain cell systems the binding of fluorochromes correlates with structural modifications in chromatin that accompany cell differentiation. We report here that changes in green & red fluorescence intensity also occur in long-term monocyte cultures. The increased red fluorescence intensity observed in cultured monocytes may reflect ribosomal RNA synthesis and the increased green fluorescence enhanced AO accessibility to DNA due to changes in chromatin organization. We compared cultured monocytes from bladder cancer patients and healthy donors. The results indicate a small but statistically significantly greater increase in mean green & red fluorescence of cultured monocytes from the cancer patients. These fluorescence variations may indicate differences in the immunologic status of cancer patients and/or be related to disease state.     

Fluorescence decay studies of the DNA-3,6-diaminoacridine complexes

The interaction of several 3,6-diaminoacridines with DNAs of various base composition has been studied by steady-state and transient fluorescence measurements. The acridine dyes employed are of the following two classes: class I - proflavine, acriflavine and 10-benzyl proflavine; class II - acridine yellow, 10-methyl acridine yellow and benzoflavine. It is found that the fluorescence decay kinetics follows a single-exponential decay law for free dye and the poly[d(A-T)]-dye complex, while that of the dye bound to DNA obeys a two-exponential decay law. The long lifetime (tau 1) for each complex is almost the same as the lifetime for the poly[d(A-T)]-dye complex, and the amplitude alpha 1 decreases with increasing GC content of DNA. The fluorescence quantum yields (phi F) of dye upon binding to DNA decrease with increasing GC content; the phi F values for class I are nearly zero when bound to poly(dG) X poly(dC), but those for class II are not zero. This is in harmony with the finding that GMP almost completely quenches the fluorescence for class I, whereas a weak fluorescence arises from the GMP-dye complex for class II. The fluorescence spectra of the DNA-dye complexes gradually shift toward longer wavelengths with increasing GC content. In this connection, the fluorescence decay parameters show a dependence on the emission wavelength; alpha 1 decreases with an increase in the emission wavelength. In view of these results, it is proposed that the decay behavior of the DNA-dye complexes has its origin in the heterogeneity of the emitting sites; the long lifetime tau 1 results from the dye bound to AT-AT sites, while the short lifetime tau 2 is attributable to the dye bound in the vicinity of GC pairs. Since GC pairs almost completely quench the fluorescence for class I, partly intercalated or externally bound dye molecules may play an important role in the component tau 2.     

Optical studies of complexes of quinacrine with DNA and chromatin: implications for the fluorescence of cytological chromosome preparations

The fluorescence and circular dichroism of quinacrine complexed with nucleic acids and chromatin were measured to estimate the relative magnitudes of factors influencing the fluorescence banding patterns of chromosomes stained with quinacrine or quinacrine mustard. DNA base composition can influence quinacrine fluorescence in at least two ways. The major effect, evident at low ratios of quinacrine to DNA, is a quenching of dye fluorescence, correlating with G-C composition. This may occur largely prior to relaxation of excited dye molecules. At higher dye/DNA saturations, which might exist in cytological chromosome preparations stained with high concentrations of quinacrine, energy transfer between dye molecules converts dyes bound near G-C base pairs into energy sinks. In contrast to its influence on quinacrine fluorescence, DNA base composition has very little effect on either quinacrine binding affinity or the circular dichroism of bound quinacrine molecules. The synthetic polynucleotides poly(dA-dT) and poly(dA)-poly(dT) have a similar effect on quinacrine fluorescence, but differ markedly in their affinity for quinacrine and in the circular dichroism changes associated with quinacrine binding. Quinacrine fluorescence intensity and lifetime are slightly less when bound to calf thymus chromatin than when bound to calf thymus DNA, and minor differences in circular dichroism between these complexes are observed. Chromosomal proteins probably affect the fluorescence of chromosomes stained with quinacrine, although this effect appears to be much less than that due to variations in DNA base composition. The fluorescence of cytological chromosome preparations may also be influenced by fixation effects and macroscopic variations in chromosome coiling.     

Induced circular dichroism of acridine orange bound to double-stranded RNA and transfer RNA

The induced circular dichroism (CD) in the visible region of acridine orange bound to the double-stranded RNA from cytoplasmic polyhedrosis virus and to yeast tRNA has been measured as a function of RNA phosphate-to-dye ratio (P/D), under the conditions of 0.01 M Na⁺ at pH 7.0. The shape of the CD spectrum of acridine orange bound to the double-stranded RNA was quite different from the spectrum of the dye bound to DNA. The CD spectral features of acridine orange bound to the double-stranded regions in tRNA closely resembled those of the double-stranded RNA-dye complex, suggesting that the dyes bind similarly to the two RNA's. It was further found that the CD spectrum of the tRNA-dye complex at sufficiently high P/D ratios, which is assignable to monomeric, intercalated dye to the base-paired parts in tRNA, is also distinct from the corresponding spectrum of the DNA-dye complex.     

Ionic strength perturbation kinetics of gene 32 protein dissociation from its complex with single-stranded DNA.

Equilibrium and kinetic studies of the interaction of gene 32 protein of T4 phage with single-stranded fd DNA were performed monitoring the changes in protein fluorescence. From the fluorescence titrations, it was estimated that a monomer of gene 32 protein covered six nucleotide bases on the DNA and the lower limit for the apparent association constant was 1.9 x 10(8) M-1 with a cooperative parameter of 10(3) in 0.1 M 2-amino-2-hydroxymethyl-1,3-propanediol hydrochloride (pH 7) at 25 degrees C. When an ionic strength jump was applied to the gene 32 protein-fd DNA complex using a stopped-flow apparatus, the complex underwent a dissociation into its individual components accompanied by an increase in protein fluorescence. The kinetics of the dissociation are not consistent with a single first-order process. The data, however, can be analyzed in terms of a model in which gene 32 protein molecules release cooperatively starting from either one or both ends of a cluster of proteins bound to fd DNA. This type of dissociation of gene 32 protein from single-stranded DNA is very efficient and has interesting implications: it could provide a way to facilitate a rapid "zippering" of the two complementary DNA strands during DNA replication and genetic recombination.     

Actinomycin D and 7-aminoactinomycin D binding to single-stranded DNA

The potent RNA polymerase inhibitors actinomycin D and 7-aminoactinomycin D are shown to bind to single-stranded DNAs. The binding occurs with particular DNA sequences containing guanine residues and is characterized by hypochromic UV absorption changes similar to those observed in interactions of the drugs with double-stranded duplex DNAs. The most striking feature of the binding is the dramatic (ca. 37-fold) enhancement in fluorescence that occurs when the 7-aminoactinomycin is bound to certain single-stranded DNAs. This fluorescence of the complex is also characterized by a 40-nm hypsochromic shift in the emission spectrum of the drug and an increase in the emission anisotropy relative to the free drug or the drug bound to calf thymus DNA. The fluorescence lifetimes change in the presence of the single-stranded DNA in a manner compatible with the intensity difference. Thus, there is an increase in the fraction of the emission corresponding to a 2-ns lifetime component compared to the predominant approximately 0.5-ns lifetime of the free drug. The 7-aminoactinomycin D comigrates in polyacrylamide gels with the single-stranded DNAs, and the fluorescence of the bound drug can be visualized by excitation with 540-nm light. The binding interactions are characterized by association constants of 2.0 x 10(6) to 1.1 x 10(7) M-1.     

The interactions between the fluorescent dye thiazole orange and DNA

The interaction of the fluorescent dye thiazole orange (TO) with nucleic acids is characterized. It is found that TO binds with highest affinity to double-stranded (ds) DNA [log(K) ≈ 5.5 at 100 mM salt], about 5–10 times weaker to single-stranded polypurines, and further 10–1000 times weaker to single-stranded polypyrimidines. TO binds as a monomer to dsDNAs and poly(dA), both as a monomer and as a dimer to poly(dG) and mainly as a dimer to poly(dC) and poly(dT). The fluorescence quantum yield of TO free in solution is about 2 · 10⁻⁴, and it increases to about 0.1 when bound to dsDNA or to poly(dA), and to about 0.4 when bound to poly(dG). Estimated quantum yields of TO bound to poly(dC) and poly(dT) are about 0.06 and 0.01, respectively. The quantum yield of bound TO depends on temperature and decreases about threefold between 5 and 50°C. © 1998 John Wiley & Sons, Inc. Biopoly 46: 39–51, 1998     

Flow cytometry of DNA content using oxazine 750 or related laser dyes with 633 nm excitation

The laser dyes oxazine 750 (OX750), LD700, and rhodamine 800 (R800) can be used in an instrument employing a low-power helium-neon laser source for flow cytometry of DNA content in ethanol-fixed or detergent-permeabilized cells. Cells in near-isotonic medium are stained with 10-30 microM dye, and fluorescence excited at 633 nm is measured at wavelengths above 665 nm. The dyes do not appear to stain RNA, and the intensity of DNA staining is not changed when 2 microM Hoechst 33342 is added to cells simultaneously with a red-excited dye. The effects on fluorescence of addition of DNA to LD700 or R800 in aqueous solution are strongly influenced by the base composition of the DNA; binding mechanisms remain to be determined.     

Simultaneous analysis of mitochondrial activity and DNA content in Ehrlich ascites tumor cells by dual parameter flow cytometry

Ehrlich ascites tumor cells were permeabilized using low concentrations of digitonin, 8 micrograms/10(6) cells. Permeabilization was monitored by the assay of lactate dehydrogenase released into the incubation medium and of hexokinase partially bound to mitochondria. Integrity of the cellular organelles was unaffected as determined by assay of the mitochondrial enzyme glutamate dehydrogenase. Cells were stained with rhodamine 123 as a mitochondrial specific dye and propidium iodide/mithramycin as DNA specific dyes. The green fluorescence of bound rhodamine 123 versus red fluorescence of DNA in individual cells was analysed by dual parameter flow cytometry. Incubation of cells with inhibitors of mitochondrial energy metabolism, such as, potassium cyanide and carbonyl cyanide m-chlorophenylhydrazone abolished binding of rhodamine 123. Flow cytometric data allowed a correlation between cell position in the mitotic cycle with total mitochondrial activity. In addition, comparison of the characteristics of propidium iodide and ethidium bromide staining further elucidated the molecular basis of the staining with the positively-charged fluorescent dye rhodamine 123.