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.     

Polarization of fluorescently labeled myosin subfragment-1 fully or partially decorating muscle fibers and myofibrils.

Fluorescently labeled myosin heads (S1) were added to muscle fibers and myofibrils at various concentrations. The orientation of the absorption dipole of the dye with respect to the axis of F-actin was calculated from polarization of fluorescence which was measured by a novel method from video images of muscle. In this method light emitted from muscle was split by a birefringent crystal into two nonoverlapping images: the first image was created with light polarized in the direction parallel to muscle axis, and the second image was created with light polarized in the direction perpendicular to muscle axis. Images were recorded by high-sensitivity video camera and polarization was calculated from the relative intensity of both images. The method allows measurement of the fluorescence polarization from single myofibril irrigated with low concentrations of S1 labeled with dye. Orientation was also measured by fluorescence-detected linear dichroism. The orientation was different when muscle was irrigated with high concentration of S1 (molar ratio S1:actin in the I bands equal to 1) then when it was irrigated with low concentration of S1 (molar ratio S1:actin in the I bands equal to 0.32). The results support our earlier proposal that S1 could form two different rigor complexes with F-actin depending on the molar ratio of S1:actin.     

Heterodimeric DNA-binding dyes designed for energy transfer: synthesis and spectroscopic properties.

Heterodimeric dyes are described which bind tightly to double-stranded (dsDNA) with large fluorescence enhancements. These dyes are designed to exploit energy transfer between donor and acceptor chromophores to tune the separation between excitation and emission wavelengths. The dyes described here absorb strongly at the 488 nm argon ion line, but emit at different wavelengths, and can be applied to multiplex detection of various targets. The chromophores in these dyes, a thiazole orange-thiazole blue heterodimer (TOTAB), two different thiazole orange-ethidium heterodimers (TOED1 and TOED2), and a fluorescein-ethidium heterodimer (FED), are in each case linked through polymethyleneamine linkers. The emission maxima of the DNA-bound dyes lie at 662 (TOTAB), 614 (TOED 2), and 610 nm (FED). The dyes showed a > 100 fold enhancement of the acceptor chromophore fluorescence on binding to dsDNA and no sequence selectivity. In comparison with direct 488 nm excitation of the constituent monomeric dyes, in the heterodimers the fluorescence of the acceptor chromophores was greatly enhanced and the emission of the donor chromophores quenched by over 90%. The acceptor emission per DNA-bound dye molecule was constant from 100 DNA bp:dye to 20 bp:dye and decreased sharply at higher dye:DNA ratios.     

Denaturation and condensation of intracellular nucleic acids monitored by fluorescence depolarization of intercalating dyes in individual cells

The intercalating binding of planar aromatic dye molecules to nucleic acids can be analyzed using fluorescence depolarization measurements of the dye molecules excited by linearly polarized light. In this study, we investigated the conformational changes of the intracellular DNA-dye complex in single cells. Flow cytometry, combined with a newly developed double-beam autocompensation technique, permitted rapid high-precision fluorescence depolarization measurements on a large number of individual cells. The dyes ethidium bromide (EB), propidium iodide (PI), and acridine orange (AO) were used in this study. Depending on the dye-to-phosphate ratio of the nuclear acid-dye complex, as well as on the spatial dye structure itself, internal and external binding sites can be monitored by fluorescence depolarization analysis. Both energy transfer and rotation and vibration of the dye molecules cause depolarization of the fluorescence emission. Differences in the concentration-dependent dye fluorescence depolarization values between PI and EB on one side and AO on the other side can be interpreted as a denaturation and condensation of double-stranded DNA regions by AO. We further show that the fluorescence polarization measurement technique can be used in an alternative way to monitor thermal denaturation of cellular DNA.     

The metachromasia of fluorescence of acridine dyes

Summary 1. Fluorescence- and absorption spectra of a number of acridine dyes were measured at several concentrations, in different solvents and at various pH and temperatures.2. In aqueous solutions metachromatic shifts are visible with all dyes — except acridine-yellow — with increasing concentrations, even when the dye is present as di- or trivalent ions in strongly acid solutions.3. Under conditions where reabsorption of the fluorescent light is excluded: completely separated fluorescence and absorption spectra, or measurement of fluorescence in capillaries with 0.8 mm internal bore, metachromatic effects are absent.4. Reasons are given to consider the hypothesis of specific aggregation (formation of dimeric dye-particles) as doubtful. In the case of the acridine dyes the optical properties of the monomeric dye ions are sufficient to explain the metachromatic shifts.With 10 Figures in the Text     

Symmetric cyanine dyes for detecting nucleic acids

A novel approach to the design of sensitive fluorescent probes for nucleic acids detection is proposed. Suitable modifications of tri- and pentamethine cyanine dyes in the polymethine chain and/or in the heterocyclic residues can result in a significant decrease in unbound dye fluorescence intensity and an increase in dye emission intensity in the presence of DNA compared to the unsubstituted dye. The sharp enhancement in the fluorescence intensity upon dye interaction with double-stranded DNA permits the application of the modified tri- and pentamethine dyes as fluorescent probes in double-stranded DNA detection in homogeneous assays.     

A theory for polarized fluorescence microscopy of chromosome structures

A theory for the determination of DNA arrangements in DNA-containing specimens, using planar aromatic dye molecules as probes for plane polarization of fluorescence, has been described. At low dye-to-DNA concentrations, the dye molecules are sandwiched between the stacked bases of DNA; hence, the fluorescence from the dye bound to a local region of DNA helix is plane-polarized with the polarization direction perpendicular to the local axis of DNA. The degree of such polarization from an aligned DNA-specimen complexed with dye is determined both by the DNA orientation and the conformational state (e.g., base tilt) of DNA into that specimen. Analysis has been made of the relationship between the degree of polarization and the orientation of the emitting dipoles of dye. The dye complexes may be aligned in a mechanical shear or electric field. However, any change in the orientation distribution of the emitting dipoles due to force fields should be taken into account. With some assumptions and approximations, the magnitude and the direction of maximum polarization can be related to different orders of DNA coiling and to their various combinations. Since the measured polarization is averaged over all DNA regions of the specimen, if the magnitude of polarization is appreciable and the polarization occurs in the specific direction of the specimen, the theory helps to eliminate several probable arrangements of DNA. The predominant molecular features of the actual DNA arrangement can be determined through this process of elimination, as explained in two subsequent papers with T-even bacteriophage and chromosome systems.     

Orientation and spectral properties of two stilbazolium merocyanine dyes in stretched and unstretched polyvinyl alcohol films

Spectral properties (anisotropy coefficients calculated for absorption, emission and fluorescence decay time) of two stilbazolium merocyanine dyes have been determined to evaluate the applicability of these dyes as sensitizers in photodynamic therapy. The dyes were embedded in an anisotropic polymer matrix. Analysis of the emission decay components measured in polarized light provides information on the interactions of the dye molecules with the polymer matrix being a model of an anisotropic biological system. Different values of the emission anisotropies obtained from various polarized components of fluorescence decays have shown that the orientations of the dye molecules influence their interactions with the polymer. This means that differently oriented dye molecules located in biological systems should exhibit different interactions with membranes. The chain length and type of side groups attached as well as the salt form of the dye molecule were shown to influence the dye-polymer interactions and should be taken into account before the application of merocyanine dyes in medicine. These dyes seem to be promising optical sensors with spectral properties, including the calculated anisotropy coefficients, sensitive to the molecular environment, useful to study orientation and interaction with neighbouring molecules in biological membranes.     

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.     

A fluorescence photobleaching study of the microsecond reorientational motions of DNA.

We have conducted a polarized fluorescence photobleaching recovery (FPR) study of the rotational dynamics of ethidium azide labeled DNA. Polarized photobleaching experiments provide data on microsecond and millisecond molecular reorientation that complement the information available from nanosecond fluorescence depolarization studies. In polarized FPR experiments an anisotropic angular concentration of fluorophore is created by bleaching dye molecules in a preferred orientation with a short, intense pulse of polarized light. The sample is then weakly illuminated, and the temporal variation in the emitted fluorescence is monitored. The fluorescence signal will systematically change as molecules undergo post-bleach reorientation and the angular distribution of dye tends toward isotropy. We have observed that the time dependence of our microsecond FPR curves is also determined in part by nonrotational phenomena. To isolate the reorientational recovery we conduct our FPR experiments in two modes (called parallel and perpendicular) that differ only in the polarization of the bleaching light. A quotient function, R(t), is constructed from the data obtained in these two modes; the variation with time of this new quantity is governed solely by processes that are sensitive to the polarization of the incident light (e.g., molecular rotation). It is found experimentally that R(t) remains constant, as expected, for rotationally restricted DNA systems despite a temporal recovery in the parallel and perpendicular FPR curves. We also follow the dynamics of solutions of phage lambda DNA as revealed in the temporal dependence of R(t). This DNA system rotationally relaxes after approximately 100 microseconds and the dye/DNA complex reorients substantially during the 10-microseconds bleach period. Our FPR data are interpreted in terms of dynamic models of DNA motion.     

Polarized photoacoustic, absorption and fluorescence spectra of chloroplasts and thylakoids oriented in polyvinyl alcohol films

The polarized photoacoustic, absorption and fluorescence spectra of chloroplasts and thylakoids in unstretched and stretched polyvinyl alcohol films were measured. The intensity ratios of fluorescence bands at 674 nm, 700 nm, 730 nm and 750 nm, and the polarized fluorescence excitation spectra are strongly dependent on light polarization and film stretching. In stretched films, thylakoids exhibit predominantly 674 nm emission. The ratio of photoacoustic signal to absorption is different for light polarized parallel and perpendicular to film stretching. This difference is large in the region of chlorophyll a and carotenoids absorption in which the fluorescence excitation spectra are also strongly dependent on light polarization and film stretching. The observed spectral changes are explained by reorientation of pigment molecules influencing the yield of excitation transfer between different pigments.     

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.     

Exchange of counterions in DNA condensation

We measured the fluorescence intensity of DNA-bound fluorescent dyes YO-PRO-1 (oxazole yellow) and YOYO-1 (dimer of oxazole yellow) at various spermidine concentrations to determine how counterions on DNA are exchanged in the process of DNA condensation. A decrease of fluorescence intensity was observed with an increase of spermidine. Considering the chemical equilibrium under the competition between the dye and spermidine for counterion condensation on DNA, the theoretical curve well describes the decrease of the fluorescence intensity. These results indicate that dyes are exchanged for spermidine at the binding site on DNA; that is, the exchange of counterions occurs. The parameters associated with the decrease of the fluorescence intensity show that the relative affinity of the dye and spermidine for DNA depends on the state of DNA. Moreover, YOYO-1 prevents the DNA condensation, but the effect of YO-PRO-1 on the condensation is very slight, though both dyes intercalate for DNA; the high affinity of YOYO-1 compared to YO-PRO-1 enables prevention of the condensation.     

Cyanine dye-protein interactions: looking for fluorescent probes for amyloid structures

We ascertained the ability to detect fibrillar beta-lactoglobulin (BLG) of a series of mono-, tri-, penta-, and heptamethinecyanines based on benzothiazole and benzimidazole heterocycles, and of benzothiazole squaraine. Fluorescence properties of these cyanine dyes were measured in the unbound state and in the presence of monomeric and fibrillar BLG and compared with those for the commercially available benzothiazole dye Thioflavin T. The correlation between the chemical nature of the dye molecules and the ability of dyes to bind aggregated proteins was established. We found that meso-substituted cyanines with amino substituents in heterocycle in contrast to the corresponding unsubstituted dyes have a binding preference to fibrillar BLG and a noticeable fluorescence response in the presence of the aggregated protein. For the squaraines and benzimidazole penthamethinecyanines studied, fluorescence emission increased both in the presence of native and fibrillar protein. The trimethinecyanines T-49 and SH-516 exhibit specifically increased fluorescence in the presence of fibrillar BLG. These dyes demonstrated the same or higher emission intensity and selectivity to aggregated BLG as Thioflavin T, and are proposed for application in selective fluorescent detection of aggregated proteins.     

Trimethine cyanine dyes as fluorescent probes for amyloid fibrils: The effect of N,N′-substituents

The effect of various N,N′-substituents in the molecule of benzothiazole trimethine cyanine dye on its ability to sense the amyloid aggregates of protein was studied. The dyes are low fluorescent when free and in the presence of monomeric proteins, but their emission intensity sharply increases in complexes with aggregated insulin and lysozyme, with the fluorescence quantum yield reaching up to 0.42.     

Polarized fluorescence studies of electrically oriented DNA-dye solutions

Transient changes have been recorded in each of the four polarized components of fluorescence, when dilute solutions of dye-tagged DNA are subjected to short electric pulses. The directions of the absorption and emission transition moments, and hence of the plane of the dye molecules, relative to the DNA geometry have been estimated for eleven dyes. Data obtained for ethidium bromide and five acridine derivatives are consistent with the intercalation model generally accepted for these dyes. In addition, it is shown that neutral red, acridine red and probably auramine O also bind with their molecular planes essentially perpendicular to the long helical axis. The remaining two, hydroxystilbamidine and the bibenzimidazole derivative Hoechst 33258, give rise to effects which indicate that these molecules bind in such a manner that the absorption and emission transitions are closely associated with the grooves of the DNA helix.     

Chromatic shifts in the fluorescence emitted by murine thymocytes stained with Hoechst 33342.

BACKGROUND: Many methods in flow cytometry rely on staining DNA with a fluorescent dye to gauge DNA content. From the relative intensity of the fluorescence signature, one can then infer position in cell cycle, amount of DNA (i.e., for sperm selection), or, as in the case of flow karyotyping, to distinguish individual chromosomes. This work examines the staining of murine thymocytes with a common DNA dye, Hoechst 33342, to investigate nonlinearities in the florescence intensity as well as chromatic shifts. METHODS: Murine thymocytes were stained with Hoechst 33342 and measured in a flow cytometer at two fluorescence emission bands. In other measurements, cells were stained at different dye concentrations, and then centrifuged. The supernatant was then used for a second round of staining to test the amount of dye uptake. Finally, to test for resonant energy transfer, we measured fluorescence anisotropy at two different wavelengths. RESULTS: The fluorescence of cells stained with Hoechst 33342 is a nonlinear process that shows an overall decrease in intensity with increased dye uptake, and spectral shift to the red. Along with the spectral shift of the fluorescence to the longer wavelengths, we document decreases in the fluorescence anisotropy that may indicate resonant energy transfer. CONCLUSIONS: At low concentrations, Hoechst 33342 binds to the minor groove of DNA and shows an increase in fluorescence and a blue shift upon binding. At higher concentrations, at which the dye molecules can no longer bind without overlapping, the blue fluorescence decreases and the red fluorescence increases until there is approximately one dye molecule per DNA base pair. The ratio of the blue fluorescence to the red fluorescence is an accurate indicator of the cellular dye concentration.     

Cyanine dye–protein interactions: Looking for fluorescent probes for amyloid structures

We ascertained the ability to detect fibrillar β-lactoglobulin (BLG) of a series of mono-, tri-, penta-, and heptamethinecyanines based on benzothiazole and benzimidazole heterocycles, and of benzothiazole squaraine. Fluorescence properties of these cyanine dyes were measured in the unbound state and in the presence of monomeric and fibrillar BLG and compared with those for the commercially available benzothiazole dye Thioflavin T. The correlation between the chemical nature of the dye molecules and the ability of dyes to bind aggregated proteins was established. We found that meso-substituted cyanines with amino substituents in heterocycle in contrast to the corresponding unsubstituted dyes have a binding preference to fibrillar BLG and a noticeable fluorescence response in the presence of the aggregated protein. For the squaraines and benzimidazole penthamethinecyanines studied, fluorescence emission increased both in the presence of native and fibrillar protein. The trimethinecyanines T-49 and SH-516 exhibit specifically increased fluorescence in the presence of fibrillar BLG. These dyes demonstrated the same or higher emission intensity and selectivity to aggregated BLG as Thioflavin T, and are proposed for application in selective fluorescent detection of aggregated proteins.     

Nature of the fluorescence spectra of the DNA-specific dye "Hoechst 33258" in solvents and in DNA complexes]

Fluorescence spectra of the DNA-specific dye Hoehst 33258 are obtained in a number of solvents. These spectra are interpreted as superpositions of monomeric and dimeric fluorescence bands of this compound. We show that abnormal Stokes' shift in the Hoehst fluorescence occurs only for the dimer form of this dye. It is suggested that formation of the A--T-specific complex is realized by dimers of the Hoehst 33258 located in the narrow groove of DNA.     

Development of a mechanism-based, DNA staining protocol using SYTOX orange nucleic acid stain and DNA fragment sizing flow cytometry

Accurate measurement of single DNA fragments by DNA fragment sizing flow cytometry (FSFC) depends upon precise, stoichiometric DNA staining by the intercalating dye molecules. In this study, we determined the binding characteristics of a commercially available 532 nm wavelength-excitable dye and used this information to develop a universal DNA staining protocol for DNA FSFC using a compact frequency-doubled Nd:YAG laser excitation source. Among twelve 532 nm wavelength-excitable nucleic acid staining dyes tested, SYTOX Orange stain showed the highest fluorescence intensity along with a large fluorescence enhancement upon binding to double-stranded DNA ( approximately 450-fold). Furthermore, using SYTOX Orange stain, accurate fragment-size-distribution histograms were consistently obtained without regard to the staining dye to base pair (dye/bp) ratio. A model describing two binding modes, intercalation (primary, yielding fluorescence) and external binding (secondary, involving fluorescence quenching), was proposed to interpret the performance of the dyes under different dye/bp ratios. The secondary equilibrium dissociation constant was found to be the most critical parameter in determining the sensitivity of each fluorophore to the staining dye/bp ratio. The measurements of both equilibrium dissociation constants provided us with a theoretical framework for developing a universal protocol which was successfully demonstrated over a wide range of DNA concentrations on a compact flow cytometer equipped with a frequency-doubled, diode-pumped, solid-state Nd:YAG laser for rapid and sensitive DNA fragment sizing.