Fluorescence lifetime distributions of DNA-4',6-diamidino-2-phenylindole complex

Time-resolved fluorescence of 4',6-diamidino-2-phenylindole (DAPI) complexes show that for a homogeneous polymer (polyd(AT) or polyd(A).polyd(T)) at high P/D (phosphate/dye) ratio, a single exponential component adequately describes the fluorescence decay. For the AT polymers at low P/D ratio or for native DNA, the decay cannot be described by a single-exponential term. A continuous distribution of lifetime values of Gaussian shape gives a good fit to the decay data. We propose that the lifetime distribution method for the analysis of the fluorescence decay of DNA-DAPI complexes provides a useful method of characterizing the microheterogeneity of site binding.     

DNA-4'-6-diamidine-2-phenylindole interactions: a comparative study employing fluorescence and ultraviolet spectroscopy

DAPI is a drug that interacts with double-stranded nucleic acids, binding preferentially to A + T base pairs. The interaction is not intercalative, therefore providing a useful model for mimicking the effect of functional molecules in modifying specific sites, namely, A + T segments, of significance in gene expression. Knowledge of the nature of such interaction has been enriched by additional information obtained from comparative analysis of the data acquired by uv spectroscopy and fluorescence. Two classes of binding sites, defined by different apparent affinity constants and numbers of binding sites, are evident. All types of interaction are dependent on the nucleic acid/dye ratio and on the ionic strength of the medium.     

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.     

Associations between a fluorescent DNA ligand, 4',6-diamidine-2-phenylindole.2HCl (DAPI), and RNA

The experiments performed in vitro have shown that DAPI and RNA form insoluble and indigestible complexes. This seems to explain the earlier observed retardation of drug accumulation in the nucleus of a living cell in the presence of RNA.     

Selective staining by 4'', 6-diamidine-2-phenylindole of nanogram quantities of DNA in the presence of RNA on gels.

4', 6-Diamidine-2-phenylindole.2HCl (DAPI) forms fluorescent complexes with double-stranded (ds) DNA but not with ds RNA as shown by fluorescence titration. The widely used dye ethidium bromide (EB) forms fluorescent complexes with both types of nucleic acids. Also, in contrast to EB, DAPI forms much weaker fluorescent complexes with single-stranded DNA than with ds DNA. These observations were utilized to develop staining procedures for the selective visualization of ds DNA on gels. The use of DAPI in addition to EB for staining makes possible the localization of ds DNA and other species of nucleic acids on a single gel.     

Assignment of DNA binding sites for 4',6-diamidine-2-phenylindole and bisbenzimide (Hoechst 33258). A comparative footprinting study

DNA binding sites for the minor groove-binding ligands DAPI (4',6-diamidine-2-phenylindole) and Hoechst 33258 (bisbenzimide) have been analysed using DNAase I and micrococcal nuclease footprinting techniques. Both drugs appear to bind to AT-rich regions containing at least four such basepairs. Hoechst 33258 seems to bind relatively poorly to nucleotide sequences containing the alternating step TpA. However, in contrast to DAPI, it can more readily accommodate the presence of guanosine residues at the end of the binding site. We compare the DNA binding sites for DAPI and Hoechst 33258 with those determined for the related minor groove-binding ligands, berenil, netropsin and distamycin A, under comparable conditions, and discuss the importance of using different footprinting probes when analysing drug-DNA interactions.     

Flow cytometry of blastogenesis and the concomitant viability assay of lymphocytes stained with 4', 6 diamidino-2-phenylindole

The blastogenic response of lymphocytes induced by concanavalin A (Con A), phytohemagglutinin (PHA), pokeweed mitogen (PWM) and by paraperiodic acid (H5IO6) was assayed by flow cytometry of 4',6 diamidino-2-phenylindole (DAPI) stained nuclei as well as by thymidine (3H-TdR) incorporation rates. The DNA content in DAPI-stained nuclei of viable and dead human, rat and mouse lymphocytes, was readily distinguishable from each other by flow cytometric methods (FCM).     

4',6-Diamidino-2-phenylindole, a fluorescent probe for tubulin and microtubules

A new fluorophor for tubulin which has permitted the monitoring of microtubule assembly in vitro is reported. DAPI (4',6-diamidino-2-phenylindole), a fluorophor already known as a DNA intercalator, was shown to bind specifically to a unique tubulin site as a dimer (KD(app) = 43 +/- 5 microM at 37 degrees C) or to tubulin associated in microtubules (KD(app) = 6 +/- 2 microM at 37 degrees C) with the same maximum enhancement in fluorescence. When tubulin polymerization was induced with GTP, the change in DAPI affinity for tubulin resulted in an enhancement of DAPI binding and, consequently, of fluorescence intensity. DAPI, whose binding site is different from that of colchicine, vinblastine, or taxol, did not interfere greatly with microtubule polymerization. It induced a slight diminution of the critical concentration for tubulin assembly due to a decrease in the depolymerizing rate constant. Moreover, DAPI did not interfere with GTP hydrolysis correlated with tubulin polymerization, but it decreased the GTPase activity at the steady state of tubulin assembly. Even at substoichiometric levels DAPI can be used to follow the kinetics of microtubule assembly.     

The binding of 4',6-diamidino-2-phenylindole to bovine serum albumin.

The binding of 4',6-diamidino-2-phenylindole (DAPI) to bovine serum albumin (BSA) has been investigated between pH 6 and 8, in 0.05 M phosphate buffer at 20 degrees C, by fluorescence titrations and the results analyzed according to a procedure previously reported (R. Favilla and A. Mazzini, Biochim. Biophys. Acta 788 (1984) 48). The dye binds to the protein with a blue shift of about 4 nm in its fluorescence emission maximum, but with an enhancement factor of 10 of its fluorescence quantum yield. The dissociation constant decreases from 100 microM to 54 microM as the pH is increased from 6 to 8, with a constant number of nearly three equivalent binding sites. The complete displacement of DAPI bound to BSA by Ca2+ suggests a possible specificity of this substantially electrostatic interaction. The fluorescence decay of DAPI bound to the protein shows a double exponential kinetics, with a tau 1 = 0.97 ns and tau 2 = 2.78 ns. These results, compared with those obtained for DAPI alone, tau 1 = 0.16 ns and tau 2 = 2.8 ns, are rationalized in terms of two different rotamers of DAPI. Both rotamers are able to bind to the protein, but only one of them undergoes an intramolecular proton transfer, from the 6-amidinium group to the indole aromatic ring, in the excited singlet state of DAPI alone. When DAPI interacts with BSA this transfer does not occur and consequently a large increase of fluorescence is observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

A molecular approach to 4′,6-diamidine-2-phenylindole (DAPI) photophysical behaviour at different pH values

The photophysical mechanisms which determine the spectral properties and decay rates of 4′,6-diamidine-2-phenylindole (DAPI) in solution and in association with nucleic acids have not yet been fully elucidated. We have performed steady-state and time-resolved fluorescence experiments on DAPI in a wide pH range to investigate the hypothesis that different ground-state conformations are responsible for the photophysical properties of the probe. Several excited-state mechanisms are investigated and it is concluded that among the proposed models, the hypothesis of ground-state heterogeneity with rapid interconversion among conformations is the only one consistent with the experiments in the entire pH range investigated.     

A time-resolved fluorescence study of 4',6'-diamidine-2-phenylindole dihydrochloride binding to polynucleotides

At phosphate/dye (P/D) ratios greater than 30 the quantum yield of 4',6'-diamidine-2-phenylindole dihydrochloride (DAPI)-DNA and DAPI-poly(d(A-T)) complexes was found to be 0.62 and 0.66, respectively. Contrary to earlier reports a fluorescence enhancement of DAPI-poly(d(G-C)) complexes was observed with a quantum yield of 0.22. Time-resolved fluorescence measurements of complexes with a P/D ratio of 150:1 indicate that there were three fluorescent components in DAPI-DNA complexes with lifetimes of 3.86, 1.79 and 0.13 ns. In DAPI-poly(d(A-T)) complexes the lifetimes were 3.91, 1.20 and 0.11 ns. Also, three components with lifetimes of 3.98, 0.87 and 0.12 ns were found in DAPI-poly(d(G-C)) complexes. At low P/D ratios (< 5) another binding form of DAPI was observed which was assigned to the interaction of one or more molecules of DAPI with one previously bound to DNA. It is concluded that DAPI does not exhibit A-T binding specificity and that at high P/D ratios there are two types of binding having similar binding constants.     

Characterization of interaction between DNA and 4',6-diamidino-2-phenylindole by optical spectroscopy

We have examined the interaction between 4',6-diamidino-2-phenylindole (DAPI) and DNA using flow linear dichroism (LD), circular dichroism (CD), and fluorescence techniques. We show the presence of two spectroscopically distinct binding sites at low binding ratios with saturation values of 0.025 and 0.17, respectively. In both sites DAPI is bound with its long axis approximately parallel to the grooves of the DNA helix. Resolution of CD spectra shows that an exciton component is present at higher binding ratios, which we attribute to the interaction of two accidentally close-lying DAPI molecules. We also find evidence that DAPI, at least in the high-affinity site, binds preferentially to AT-rich regions. From the spectroscopic results, supported by structural considerations, we can completely exclude that DAPI is bound to DNA by intercalation. Binding geometries and site densities are consistent with a location of DAPI in the grooves of DNA, with the high-affinity site most probably in the minor groove.     

DNA sequence dependent binding modes of 4',6-diamidino-2-phenylindole (DAPI)

The interactions of DAPI with natural DNA and synthetic polymers have been investigated by hydrodynamic, DNase I footprinting, spectroscopic, binding, and kinetic methods. Footprinting results at low ratios (compound to base pair) are similar for DAPI and distamycin. At high ratios, however, GC regions are blocked from enzyme cleavage by DAPI but not by distamycin. Both poly[d(G-C)]2 and poly[d(A-T)]2 induce hypochromism and shifts of the DAPI absorption band to longer wavelengths, but the effects are larger with the GC polymer. NMR shifts of DAPI protons in the presence of excess AT and GC polymers are significantly different, upfield for GC and mixed small shifts for AT. The dissociation rate constants and effects of salt concentration on the rate constants are also quite different for the AT and the GC polymer complexes. The DAPI dissociation rate constant is larger with the GC polymer but is less sensitive to changes in salt concentration than with the AT complex. Binding of DAPI to the GC polymer and to poly[d(A-C)].poly[d(G-T)] exhibits slight negative cooperativity, characteristic of a neighbor-exclusion binding mode. DAPI binding to the AT polymer is unusually strong and exhibits significant positive cooperativity. DAPI has very different effects on the bleomycin-catalyzed cleavage of the AT and GC polymers, a strong inhibition with the AT polymer but enhanced cleavage with the GC polymer. All of these results are consistent with two totally different DNA binding modes for DAPI in regions containing consecutive AT base pairs versus regions containing GC or mixed GC and AT base pair sequences. The binding mode at AT sites has characteristics which are similar to those of the distamycin-AT complex, and all results are consistent with a cooperative, very strong minor groove binding mode. In GC and mixed-sequence regions the results are very similar to those observed with classical intercalators such as ethidium and indicate that DAPI intercalates in DNA sequences which do not contain at least three consecutive AT base pairs.     

Treatment of synchronized K562 cells by tetrafluoroaluminate does not modulate the fluorescence of ethidium bromide and 4'6-diamidine-2-phenylindole in case of the binding with nucleoid DNA

Earlier we showed that 4-hours treatment of cells K562 with the GTP-binding protein activator AlF4- (10 mM NaF + 20 microM AlCl3) increased the DNA fragmentation on an average to 5% of the total 3H-thymidine-labeled DNA. The viability of cells under these conditions did not change. It has been suggested that gene toxic action of AlF4- is a result of cell proliferation block induced by AlF4-. In the present work we tried to determine possible changes in the ethidium bromide and 4',6-diamidine-2-phenylindole (DAPI) fluorescence when they bind with nucleoid DNA of synchronized cells K562 treated with AlF4-. Cells K562 were incubated for synchronization with 2 mM thymidine during 15 hours. Under these conditions DNA synthesis (3H-thymidine uptake) was suppressed by 94-99%. It has been found that the treatment of "cool" thymidine-incubated cells K562 with AlF4- did not change the fluorescence of either ethidium or DAPI. The presence of phorbol-12-myristate-13-acetate (PMA) in the incubation medium did not influence the results. On the other, hand the rat thymocytes incubated with dexametazone (2 microM) during 4 hours (positive control of DNA fragmentation) demonstrated the increase in both parameters. PMA decreased the ethidium fluorescence that correspond to its (PMA) ability to suppress fragmentation of thymocyte DNA. On the base of the results we suggested that AlF4- did not induce DNA fragmentation in the cells K562 with the blocked DNA synthesis.     

Quantitative fluorescenct analysis of different conformational forms of DNA bound to the dye, 4′,6-diamidine-2-phenylindole,and sepasated by gel electrophoresis

A quantitative fluorescent method for estimating the amounts of different conformational forms of the same DNA on agarose gels is described. Supercoiled, open circular, and linear forms of PM2 DNA and fluorescent dye (4′,6-diamidine-2-phenylindole) were used. The results are compared with respective radiometric estimations and are shown to be highly reproducible.     

Property of the M-DNA probed by a minor groove binding dye 4',6-diamidino-2-phenylindole

Spectral properties including circular and linear dichroism (CD and LD) of M-DNA, a molecular electric wire, formed at a high Zn(2+) concentration have been studied using a minor groove binding drug 4',6-diamidino-2-phenylindole (DAPI) as a probe. As the Zn(2+) concentration increased, the magnitude of LD in the DNA absorption region decreased at pH 8.5, implying the aggregation of DNA, which is in contrast with the retained LD magnitude at pH 7.0. As the M-DNA formed, change in the secondary structure of DNA was observed by CD spectrum, which resembles that of the C-form DNA, although overall structure seemed to remain as a right handed double helix. The DAPI-DNA complex in the presence of high concentration of Zn(2+) ions at pH 7.0 exhibited the similar CD spectrum with that in the absence of Zn(2+) ion, consisting of type I, II and III. In contrast, at pH 8.5 at a high Zn(2+) concentration in which DNA is in its M-form, DNA bound DAPI produced only the type III CD, suggesting that DAPI binds at the surface of M-DNA: the presence of Zn(2+) ions prevents the minor groove binding of DAPI.     

4',6-Diamidino-2-phenylindole (DAPI) interacts with rare structures of GC polymers

The binding of 4',6-diamidino-2-phenylindole (DAPI) to double-stranded GC polymers either in the alternating or in homopolymer sequence was investigated using fluorescence techniques. We employed fluctuation correlation spectroscopy, which measures the diffusion coefficient of fluorescent particles, to demonstrate that the fluorescence was originating from relatively slowly diffusing entities. These entities display a very large heterogeneity of diffusing coefficients, indicating that molecular aggregation is extensive in our samples. We used frequency domain fluorometry to characterize the fluorescence lifetime of the species, while varying the GC polymer-dye coverage systematically. At very low coverage we observed a relatively bright fluorescent component with a lifetime value of approximately 4 ns. The stoichiometry of binding of this bright species was such that it can only arise from rare molecular structures, either unusual loops or large molecular aggregates. The amount and characteristics of this bright fluorescent component were different between the homo and the alternating polymer, indicating that the difference in sequence of the two polymers is responsible for the different aggregates which are then detected in the fluorescence experiment. At large GC polymer coverage we observed a relatively wide distribution of fluorescent species with short lifetime values, in the range between 0.12 and 0.2 ns. Given the stoichiometry of binding of this fluorescent component, we concluded that it could arise either from intercalative and/or non-specific binding to the DNA double-stranded molecules. We comment on the origin of the rare but brightly fluorescent binding sites and discuss the potential to detect such unusual DNA structures.     

Influence of water chlorination on the counting of bacteria with DAPI (4',6-diamidino-2-phenylindole).

Counting bacteria in drinking water samples by the epifluorescence technique after 4',6-diamidino-2-phenylindole (DAPI) staining is complicated by the fact that bacterial fluorescence varies with exposure of the cells to sodium hypochlorite. An Escherichia coli laboratory-grown suspension treated with sodium hypochlorite (5 to 15 mg of chlorine liter-1) for 90 min was highly fluorescent after DAPI staining probably due to cell membrane permeation and better and DAPI diffusion. At chlorine concentrations greater than 25 mg liter-1, DAPI-stained bacteria had only a low fluorescence. Stronger chlorine doses altered the DNA structure, preventing the DAPI from complexing with the DNA. When calf thymus DNA was exposed to sodium hypochlorite (from 15 to 50 mg of chlorine liter-1 for 90 min), the DNA lost the ability to complex with DAPI. Exposure to monochloramine did not have a similar effect. Treatment of drinking water with sodium hypochlorite (about 0.5 mg of chlorine liter-1) caused a significant increase in the percentage of poorly fluorescent bacteria, from 5% in unchlorinated waters (40 samples), to 35 to 39% in chlorinated waters (40 samples). The presence of the poorly fluorescent bacteria could explain the underestimation of the real number of bacteria after DAPI staining. Microscopic counting of both poorly and highly fluorescent bacteria is essential under these conditions to obtain the total number of bacteria. A similar effect of chlorination on acridine orange-stained bacteria was observed in treated drinking waters. The presence of the poorly fluorescent bacteria after DAPI staining could be interpreted as a sign of dead cells.     

4',6-Diamidino-2-phenylindole (DAPI) induces bundling of Escherichia coli FtsZ polymers inhibiting the GTPase activity

FtsZ (Filamentous temperature sensitivity Z) cell division protein from Escherichia coli binds the fluorescence probe DAPI. Bundling of FtsZ was facilitated in the presence of DAPI, and the polymers in solution remained polymerized longer time than the protofilaments formed in the absence of DAPI. DAPI decreased both the maximal velocity of the GTPase activity and the Michaelis-Menten constant for GTP, indicating that behaves like an uncompetitive inhibitor of the GTPase activity favoring the GTP form of FtsZ in the polymers. The results presented in this work support a cooperative polymerization mechanism in which the binding of DAPI favors protofilament lateral interactions and the stability of the resulting polymers.     

Binding of Hoechst 33258 and 4',6'-diamidino-2-phenylindole to self-complementary decadeoxynucleotides with modified exocyclic base substituents.

Fluorescence titrations have been carried out to determine the association constants (Ka) for binding of the dyes Hoechst 33258 and DAPI to the self-complementary decamer d(CTGAATTCAG) and nine duplex derivatives with exocyclic substituent changes in the six central base pairs. Many Ka values are in the range (2-5) x 10(8) (duplex M)-1 at 5.5 degrees C. Replacement of the leftmost adenine by 2-aminopurine in the sequence decreases Ka for Hoechst 33258 by a factor of 170. When the centermost adenine is replaced by 2-aminopurine, Ka for Hoechst 33258 and DAPI is too small to be evaluated. When the centermost adenine is replaced by purine, Ka for both dyes increases, but this very stable duplex-Hoechst 33258 complex is nonfluorescent. The measured affinities are compared to expectations derived from X-ray studies with dodecamer-dye complexes having an identical central binding sequence (Pjura et al., 1987; Teng et al., 1988; Larsen et al., 1989).