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.     

Sequence-selective DNA binding to the regulatory subunit of cAMP-dependent protein kinase

The fluorescence of Trp-226 in the regulatory subunit of bovine type II cAMP-dependent protein kinase is unaffected by the binding of cAMP, but is quenched by the binding of 2'-dansyl-cAMP (DNS-cAMP). Up to 67% of the fluorescence of Trp-226 can be quenched by resonant energy transfer to the DNS-cAMP bound to the first site, and 96% of the fluorescence can be quenched by saturating both sites with DNS-cAMP. The observed efficiencies of energy transfer gave a distance of 16 A between Trp-226 and the DNS-cAMP bound at the first site and a distance of 12.7 A between Trp-226 and the DNS-cAMP bound at second site. The fluorescence of Trp-226 was suppressed by incubation of RII with the self-complementary octanucleotide TGACGTCA (CRE) due to binding of the oligonucleotide to RII. A detailed study of the binding equilibrium showed that each RII(cAMP)2 molecule binds 1 molecule of CRE with Kd = 80 nM. The corresponding Kd value for cAMP-depleted RII was found to be 25-fold higher. RII was also found to bind randomly selected DNA fragments with an average Kd value much higher than that of CRE. These observations show for the first time that the binding of oligonucleotide to RII is cAMP-enhanced and sequence-selective.     

Interactions of 4'', 6-diamidine-2-phenylindole with synthetic polynucleotides.

4', 6-Diamidine-2-phenylindole forms fluorescent complexes with synthetic DNA duplexes containing AT, AU and IC base pairs; no fluorescent complexes were observed with duplexes containing GC base pairs or with duplexes containing a single AT base pair sandwiched between GC pairs. The binding site size is one molecule of dye per 3 base pairs. The intrinsic binding constants are higher for alternating sequence duplexes than for the corresponding homopolymer pairs. With the exception of the four-stranded helical poly rI which exhibits considerable fluorescence enhancement upon binding of the ligand, none of the single- or multi- stranded polyribonucleotides and ribo-deoxyribonucleotide hybrid structures form fluorescent complexes with the dye. Poly rI is the only RNA which forms a DNA B-like structure (Arnott et al. (1974) Biochem. J. 141, 537). The B conformation of the helix and the absence of guanine appear to be the major determinants of the specificity of the fluorescent binding mode of the dye. Nonfluorescent interactions of the dye with polynucleotides are nonspecific; UV absorption and circular dichroic spectra demonstrate binding to synthetic single- and double-stranded DNA and RNA analogs, including those containing GC base pairs.     

Differences in the red fluorescence of acridine orange bound to single-stranded RNA and DNA.

Fluorescence of acridine orange bound to RNA or DNA in the single-stranded form including single-stranded synthetic polyribo- or polydeoxyribonucleotides was measured in the expectation that some distinct structural characteristic between single-stranded RNA and DNA might be reflected by a specific fluorescent behaviour of bound dyes. It was found that the complex of the dye with single-stranded RNA emits a weaker red fluorescence around 650 nm than the complex with single-stranded DNA at low phosphate-to-dye ratios. The fact could be explained neither by a direct interaction of bound dyes with the 2′-hydroxyl group of ribose in RNA nor by the difference in the G-C content of the nucleic acids. On the basis of the character of dye molecules emitting the red fluorescence, it was suggested that the bases in single-stranded RNA might be buried in some hydrophobic environment that would make the dyes less likely to interact with them, compared with the bases in single-stranded DNA. It was further inferred that some conformational rigidity of single-stranded RNA may partially be responsible for the weaker red fluorescence.     

Interaction of Oxazole Yellow Dyes with DNA Studied with Hybrid Optical Tweezers and Fluorescence Microscopy

We have integrated single molecule fluorescence microscopy imaging into an optical tweezers set-up and studied the force extension behavior of individual DNA molecules in the presence of various YOYO-1 and YO-PRO-1 concentrations. The fluorescence modality was used to record fluorescent images during the stretching and relaxation cycle. Force extension curves recorded in the presence of either dye did not show the overstretching transition that is characteristic for bare DNA. Using the modified wormlike chain model to curve-fit the force extension data revealed a contour length increase of 6% and 30%, respectively, in the presence of YO-PRO-1 and YOYO-1 at 100 nM. The fluorescence images recorded simultaneously showed that the number of bound dye molecules increased as the DNA molecule was stretched and decreased again as the force on the complex was lowered. The binding constants and binding site sizes for YO-PRO-1 and YOYO-1 were determined as a function of the force. The rate of YO-PRO-1 binding and unbinding was found to be 2 orders of magnitude larger than that for YOYO-1. A kinetic model is proposed to explain this observation.     

Heterogeneity of binding sites of proflavin on DNA

The desorption and melting with temperature of proflavine–DNA complexes has been studied by spectrophotometry and spectrofluorometry. Two methods are described to determine at each temperature the concentration of free and bound dye. The first one is based on the quenching of fluorescence of the free dye by the iodine ion, the second on fluorescence polarization measurements. It is shown that the sites where the bound dye fluoresces are thermally less stable than those where it is quenched, in such a way that a redistribution of the dye between the two types of sites occurs at intermediate temperatures, leading to a drop in the total fluorescence. This confirms the nature of the “emitting” sites which correspond to AT-rich region, while “quenched” sites correspond to GC-rich region. The first have a larger binding constant at room temperature, but only the latter are stabilized by dye intercalation. The desorption and melting have also been followed through the relative changes of absorption. The curves obtained at different wavelengths are not superimposed which is at variance with what is observed with complexes of proflavine with poly dAT and poly dG.dC. The beginning of the desorption process corresponds to minor variations at 445 nm, the maximum of absorption of the free dye, but large changes occur at 460 nm, the maximum of the difference spectrum of the complexes proflavine–poly dAT and proflavine-poly dG.dC. The spreading of the melting curves for different wave lengths must therefore reflect the dependence of the absorption spectra of the dye on the nature of the neighboring bases. However, the action spectrum of the fluorescence, which gives the absorption spectrum of the “emitting” sites only, is identical with the total absorption spectrum of the bound dye.     

The distribution of quinacrine on chromosomes as determined by X-ray microanalysis

The distribution of quinacrine and protein sulphur has been compared with that of DNA in euchromatic and heterochromatic regions of mouse chromosomes stained with the fluorescent dye quinacrine, using X-ray microanalysis. Heterochromatin tends to bind relatively more quinacrine than euchromatin, and contains a greater concentration of sulphur. Measurements of quinacrine fluorescence, when compared with quinacrine binding, show that the excitation of fluorescence is more efficient when the dye is bound to euchromatin than when it is bound to heterochromatin. Although this observation is consistent with the hypothesis that the dull quinacrine fluorescence of mouse centromeres is due to quenching by guanine residues, two other factors should also be considered: the lower absolute amount of dye bound to the centromeres, and a concentration-dependent quenching of fluorescence.     

USE OF FLUORESCENCE POLARIZATION DETECTION FOR THE MEASUREMENT OF FLUOPEPTIDE™ BINDING TO G PROTEIN-COUPLED RECEPTORS

ABSTRACT

G protein-coupled receptors (GPCRs) represent the single largest molecular target of therapeutic drugs currently on the market, and are also the most common target in high throughput screening assays designed to identify potential new drug candidates. A large percentage of these assays are now formatted as radioligand binding assays. Fluorescence polarization ligand binding assays can offer a non-rad alternative to radioligand binding assays. In addition, fluorescence polarization assays are a homogenous format that is easy to automate for high throughput screening. We have developed a series of peptide ligands labeled with the fluorescent dye BODIPY® TMR whose binding to GPCRs can be detected using fluorescence polarization methodology. BODIPY® TMR has advantages over the more commonly used fluorescein dye in high throughput screening (HTS) assays due to the fact that its excitation and emission spectra are red-shifted approximately 50 nm relative to fluorescein. Assays based on BODIPY® TMR ligands are therefore less susceptible to interference from tissue auto-fluorescence in the assay matrix, or the effects of colored or fluorescent compounds in the screening libraries. A series of BODIPY® TMR labeled peptides have been prepared that bind to a range of GPCRs including melanin concentrating hormone, bradykinin, and melanocortin receptors. Conditions have been optimized in order to utilize a comparable amount of receptor membrane preparation as is used in a radioligand binding assay. The assays are formatted in 384-well microplates with a standard volume of 40 µL. We have compared the assays across the different fluorescence polarization (FP) readers available to determine the parameters for each instrument necessary to achieve the required precision.     

Reevaluation of ANS binding to human and bovine serum albumins: key role of equilibrium microdialysis in ligand - receptor binding characterization

In this work we return to the problem of the determination of ligand-receptor binding stoichiometry and binding constants. In many cases the ligand is a fluorescent dye which has low fluorescence quantum yield in free state but forms highly fluorescent complex with target receptor. That is why many researchers use dye fluorescence for determination of its binding parameters with receptor, but they leave out of account that fluorescence intensity is proportional to the part of the light absorbed by the solution rather than to the concentration of bound dye. We showed how ligand-receptor binding parameters can be determined by spectrophotometry of the solutions prepared by equilibrium microdialysis. We determined the binding parameters of ANS - human serum albumin (HSA) and ANS - bovine serum albumin (BSA) interaction, absorption spectra, concentration and molar extinction coefficient, as well as fluorescence quantum yield of the bound dye. It was found that HSA and BSA have two binding modes with significantly different affinity to ANS. Correct determination of the binding parameters of ligand-receptor interaction is important for fundamental investigations and practical aspects of molecule medicine and pharmaceutics. The data obtained for albumins are important in connection with their role as drugs transporters.     

Use of fluorescence polarization detection for the measurement of fluopeptidetm binding to G protein-coupled receptors

G protein-coupled receptors (GPCRs) represent the single largest molecular target of therapeutic drugs currently on the market, and are also the most common target in high throughput screening assays designed to identify potential new drug candidates. A large percentage of these assays are now formatted as radioligand binding assays. Fluorescence polarization ligand binding assays can offer a non-rad alternative to radioligand binding assays. In addition, fluorescence polarization assays are a homogenous format that is easy to automate for high throughput screening. We have developed a series of peptide ligands labeled with the fluorescent dye BODIPY TMR whose binding to GPCRs can be detected using fluorescence polarization methodology. BODIPY TMR has advantages over the more commonly used fluorescein dye in high throughput screening (HTS) assays due to the fact that its excitation and emission spectra are red-shifted approximately 50 nm relative to fluorescein. Assays based on BODIPY TMR ligands are therefore less susceptible to interference from tissue auto-fluorescence in the assay matrix, or the effects of colored or fluorescent compounds in the screening libraries. A series of BODIPY TMR labeled peptides have been prepared that bind to a range of GPCRs including melanin concentrating hormone, bradykinin, and melanocortin receptors. Conditions have been optimized in order to utilize a comparable amount of receptor membrane preparation as is used in a radioligand binding assay. The assays are formatted in 384-well microplates with a standard volume of 40 microL. We have compared the assays across the different fluorescence polarization (FP) readers available to determine the parameters for each instrument necessary to achieve the required precision.     

Analysis of allosteric signal transduction mechanisms in an engineered fluorescent maltose biosensor

We previously reported the construction of a family of reagentless fluorescent biosensor proteins by the structure-based design of conjugation sites for a single, environmentally sensitive small molecule dye, thus providing a mechanism for the transduction of ligand-induced conformational changes into a macroscopic fluorescence observable. Here we investigate the microscopic mechanisms that may be responsible for the macroscopic fluorescent changes in such Fluorescent Allosteric Signal Transduction (FAST) proteins. As case studies, we selected three individual cysteine mutations (F92C, D95C, and S233C) of Escherichia coli maltose binding protein (MBP) covalently labeled with a single small molecule fluorescent probe, N-((2-iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole (NBD), each giving rise to a robust FAST protein with a distinct maltose-dependent fluorescence response. The fluorescence emission intensity, anisotropy, lifetime, and iodide-dependent fluorescence quenching were determined for each conjugate in the presence and absence of maltose. Structure-derived solvent accessible surface areas of the three FAST proteins are consistent with experimentally observed quenching data. The D95C protein exhibits the largest fluorescence change upon maltose binding. This mutant was selected for further characterization, and residues surrounding the fluorophore coupling site were mutagenized. Analysis of the resulting mutant FAST proteins suggests that specific hydrogen-bonding interactions between the fluorophore molecule and two tyrosine side-chains, Tyr171 and Tyr176, in the open state but not the closed, are responsible for the dramatic fluorescence response of this construct. Taken together these results provide insights that can be used in future design cycles to construct fluorescent biosensors that optimize signaling by engineering specific hydrogen bonds between a fluorophore and protein.     

Using 2-aminopurine fluorescence to detect bacteriophage T4 DNA polymerase-DNA complexes that are important for primer extension and proofreading reactions

The fluorescence of the base analogue 2-aminopurine (2AP) was used to probe bacteriophage T4 DNA polymerase-induced conformational changes in the template strand produced during the nucleotide incorporation and proofreading reactions. 2AP fluorescence in DNA is quenched by 2AP interactions with neighboring bases, but T4 DNA polymerase binding to DNA substrates labeled with 2AP in the templating position produces large increases in fluorescence intensity. Fluorescence lifetime studies were performed to characterize the fluorescent complexes. Three fluorescence lifetime components were observed for unbound DNA substrates as reported previously, but T4 DNA polymerase binding modulated the amplitudes of these components and created a new, highly fluorescent 10.5 ns component. Experimental evidence for correlation of fluorescence lifetimes with functionally distinct complexes was obtained by forming complexes under different reaction conditions. T4 DNA polymerase complexes were formed with DNA substrates with matched and mismatched primer ends and with A+T- or G+C-rich primer-terminal regions. dTTP was added to binary complexes to form ternary DNA polymerase-DNA-nucleotide complexes. The effect of temperature on complex formation was studied, and complexes were formed with proofreading-defective T4 DNA polymerases. Complexes characterized by the 10.5 ns lifetime were demonstrated to be formed at the crossroads of the primer-extension and proofreading pathways.     

Self-association of glucagon as measured by the optical properties of rhodamine 6G

The fluorescence of rhodamine 6G is completely quenched in glucagon solutions in 0.6 M K2HOP4 at pH 10.6. The absorption of rhodamine 6G is red-shifted by the same reaction. A single rhodamine 6G molecule appears to be bound to a hydrophobic patch in the center of the trimer of glucagon. Since the glucagon monomer has almost no organized structure this site exists only in the associated trimer form of glucagon. The self-association of glucagon to the trimer has been determined from the variation in rhodamine 6G fluorescence and absorption measured over a 60-fold range of dye concentration. The self-association constant agrees with values determined by other methods in the absence of dye. The binding isotherms of rhodamine 6G to glucagon shift with glucagon concentration and exhibit negative cooperativity.     

Quantitative determination of anthracycline antibiotic binding with DNA]

A new method of quantitative intravital assessment of anthracyclines accumulation and binding with DNA in alive cells has been developed. For this purpose DNA specific fluorescent dye Hoechst 33258 was used. Extent of fluorescence quenching of bound with DNA dye correlated with binding of daunomycin with cell DNA after mixing solutions of the drug and cells. It allowed us to determine quantity of drug molecules bound with DNA in the cells during the time.     

A small-molecule-linked DNA–graphene oxide-based fluorescence-sensing system for detection of biotin

In this paper, we establish a novel fluorescence-sensing system for the detection of biotin based on the interaction between DNA and graphene oxide and on protection of the terminal of the biotinylated single-stranded DNA fluorescent probe by streptavidin. In this system, streptavidin binds to the biotinylated DNA, which protects the DNA from hydrolysis by exonuclease I. The streptavidin–DNA conjugate is then adsorbed to the graphene oxide resulting in the fluorescence being quenched. Upon the addition of free biotin, it competes with the labeled biotin for the binding sites of streptavidin and then the exonuclease I digests the unbound DNA probe from the 3′ to the 5′ terminal, releasing the fluorophore from the DNA. Because of the weak affinity between the fluorophore and graphene oxide, the fluorescence is recovered. Under optimal conditions, the fluorescence intensity is proportional to the concentration of biotin in the concentration range of 0.5–20 nmol/L. The detection limit for biotin is 0.44 nmol/L. The proposed fluorescence-sensing system was applied to the determination of biotin in some real samples with satisfactory reproducibility and accuracy. This work could provide a common platform for detecting small biomolecules based on protein–small molecule ligand binding.     

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.     

Interaction of phenosafranine with nucleic acids and model polyphosphates: II. Characterisation of phenosafranine binding to DNA

The binding of phenosafranine (PS) to DNA was studied by a combination of spectroscopic methods (absorption and fluorescence) together with hydrodynamic measurements (sedimentation and viscosity), Analysis of spectroscopic binding curves revealed that the strength of binding of PS to DNA is generally lower than that of proflavine. These measurements enabled recognition of several modes of interaction between PS and native DNA: strong monomer binding prevailing at high DNA phosphate/dye ratios (p) comprising binding outside the DNA helix as well as intercalation; two modes of dimer binding at lower values of p; and probably also weak surface-binding of monomers as p approaches unity. Longer surface-bound aggregates of PS were not detected because of the low tendency of the dye to form aggregates, though the presence of dimeric species distinct frorn pure surface-stacked PS dimer was indicated by various observations. It occurs over a broad range of p values Starting at p ≈110 for ionic strengths 10^?3–10^?1. Thermal denaturation data indicate that this species is bound more strongly than pure surface-bound stacked dimer. Its dimeric character may be explained in terms of interaction of an intercalated dye molecule with an adjacent outside-bound one as suggested for acridines by Armstrong et al. Various properties of this species are discussed. Both strong and weak modes or binding of PS to DNA are sensitive to the presence of organic solvents. The effectiveness of solvents to destabilise the complexes substantially coincides with their capacity to alter the water activity. Viscometric investigations reveal that in the region of strongest bindins (p ? 15) the elongation of the DNA helix by approximately 0.18 nm per bound PS molecule is accompanied by a strong negative change in persistence length, i.e. bending. Similar bending is also found at higher levels of binding (p ? 15) induced by less lightly bound PS molecules, in which region, however, the unusually high elongation of approximately 0.34 nm per bound PS molecule is observed.     

A fluorescent derivative of ribosomal protein S18 which permits direct observation of messenger RNA binding.

70 S Escherichia coli ribosomes were reacted with the fluorescent dye N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonic acid for 10 min under mild conditions. The resulting ribosomes were fully active. 30 S subunits isolated from these particles were also fully active. They contain approximately 0.7 eq of fluorescent dye. Nearly all of it is attached to protein S18. Competitive reaction with N-ethylmaleimide implies that the fluorescent dye is located at cysteine 10 of the protein. The labeled 30 S particles will recombine with 50 S subunits to form stable 70 S particles. Thus the procedures we have developed allow the large scale preparation of an active fluorescent conjugate of the 70 S ribosome. The fluorescence of the 70 S particles is sensitive to the binding of mRNA, showing both quenching and a shift in emission spectra. Thus it affords a simple way to quantitate mRNA binding directly. In pilot studies without tRNA, the binding constant of the initiation triplet codon adenylyl-(3' leads to 5')-uridylyl-(3' leads to 5')-guanosine to 70 S ribosome was found to be an order of magnitude larger than that of polyuridylic acid.     

Fluorescence resonance energy transfer (FRET) and competing processes in donor-acceptor substituted DNA strands: a comparative study of ensemble and single-molecule data

We studied the fluorescence resonance energy transfer (FRET) efficiency of different donor-acceptor labeled model DNA systems in aqueous solution from ensemble measurements and at the single molecule level. The donor dyes: tetramethylrhodamine (TMR); rhodamine 6G (R6G); and a carbocyanine dye (Cy3) were covalently attached to the 5'-end of a 40-mer model oligonucleotide. The acceptor dyes, a carbocyanine dye (Cy5), and a rhodamine derivative (JA133) were attached at modified thymidine bases in the complementary DNA strand with donor-acceptor distances of 5, 15, 25 and 35 DNA-bases, respectively. Anisotropy measurements demonstrate that none of the dyes can be observed as a free rotor; especially in the 5-bp constructs the dyes exhibit relatively high anisotropy values. Nevertheless, the dyes change their conformation with respect to the oligonucleotide on a slower time scale in the millisecond range. This results in a dynamic inhomogeneous distribution of donor/acceptor (D/A) distances and orientations. FRET efficiencies have been calculated from donor and acceptor fluorescence intensity as well as from time-resolved fluorescence measurements of the donor fluorescence decay. Dependent on the D/A pair and distance, additional strong fluorescence quenching of the donor is observed, which simulates lower FRET efficiencies at short distances and higher efficiencies at longer distances. On the other hand, spFRET measurements revealed subpopulations that exhibit the expected FRET efficiency, even at short D/A distances. In addition, the measured acceptor fluorescence intensities and lifetimes also partly show fluorescence quenching effects independent of the excitation wavelength, i.e. either directly excited or via FRET. These effects strongly depend on the D/A distance and the dyes used, respectively. The obtained data demonstrate that besides dimerization at short D/A distances, an electron transfer process between the acceptor Cy5 and rhodamine donors has to be taken into account. To explain deviations from FRET theory even at larger D/A distances, we suggest that the pi-stack of the DNA double helix mediates electron transfer from the donor to the acceptor, even over distances as long as 35 base pairs. Our data show that FRET experiments at the single molecule level are rather suited to resolve fluorescent subpopulations in heterogeneous mixture, information about strongly quenched subpopulations gets lost.     

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.