Rapid and simple method for double staining of bacteria with 4',6-diamidino-2-phenylindole and fluorescein isothiocyanate-labeled antibodies.

Bacteria were either heat fixed on microscope slides or filtered with 0.2 micron-pore-size Nuclepore filters. The samples were stained with 4',6-diamidino-2-phenylindole (DAPI) for total staining and with polyvalent rabbit antibodies and fluorescein isothiocyanate-conjugated swine anti-rabbit antibodies for specific staining. By switching between two different optical filter packages in the microscope, only one sample was needed for determining both total and specific counts of bacteria. False-positive counts and other artifacts that occur with antibody staining were easily distinguished when individual fluorescent particles were checked for DAPI fluorescence. The method for applying the general stain to membrane filters was performed quickly and simply by using a DAPI-soaked polypropylene filter that lay beneath the Nuclepore filter which collected the sample.     

Rapid and simple method for double staining of bacteria with 4',6-diamidino-2-phenylindole and fluorescein isothiocyanate-labeled antibodies

Bacteria were either heat fixed on microscope slides or filtered with 0.2 micron-pore-size Nuclepore filters. The samples were stained with 4',6-diamidino-2-phenylindole (DAPI) for total staining and with polyvalent rabbit antibodies and fluorescein isothiocyanate-conjugated swine anti-rabbit antibodies for specific staining. By switching between two different optical filter packages in the microscope, only one sample was needed for determining both total and specific counts of bacteria. False-positive counts and other artifacts that occur with antibody staining were easily distinguished when individual fluorescent particles were checked for DAPI fluorescence. The method for applying the general stain to membrane filters was performed quickly and simply by using a DAPI-soaked polypropylene filter that lay beneath the Nuclepore filter which collected the sample.     

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)     

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.     

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.     

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.     

Dual staining of natural bacterioplankton with 4',6-diamidino-2-phenylindole and fluorescent oligonucleotide probes targeting kingdom-level 16S rRNA sequences.

A method for quantifying eubacterial cell densities in dilute communities of small bacterioplankton is presented. Cells in water samples were stained with 4',6-diamidino-2-phenylindole (DAPI), transferred to gelatin-coated slides, and hybridized with rhodamine-labeled oligonucleotide probes specific for kingdom-level 16S rRNA sequences. Between 48 and 69% of the cells captured on membrane filters were transferred to gelatin-coated slides. The number of DAPI-stained cells that were visualized with eubacterial probes varied from 35 to 67%. Only 2 to 4% of these cells also fluoresced following hybridization with a probe designed to target a eukaryotic 16S rRNA sequence. Between 0.1 and 6% of the bacterioplankton in these samples were autofluorescent and may have been mistaken as cells that hybridized with fluorescent oligonucleotide probes. Dual staining allows precise estimates of the efficiency of transfers of cells to gelatin films and can be used to measure the percentage of the total bacterioplankton that also hybridize with fluorescent oligonucleotide probes, indicating specific phylogenetic groups.     

Dual staining of natural bacterioplankton with 4',6-diamidino-2-phenylindole and fluorescent oligonucleotide probes targeting kingdom-level 16S rRNA sequences.

A method for quantifying eubacterial cell densities in dilute communities of small bacterioplankton is presented. Cells in water samples were stained with 4',6-diamidino-2-phenylindole (DAPI), transferred to gelatin-coated slides, and hybridized with rhodamine-labeled oligonucleotide probes specific for kingdom-level 16S rRNA sequences. Between 48 and 69% of the cells captured on membrane filters were transferred to gelatin-coated slides. The number of DAPI-stained cells that were visualized with eubacterial probes varied from 35 to 67%. Only 2 to 4% of these cells also fluoresced following hybridization with a probe designed to target a eukaryotic 16S rRNA sequence. Between 0.1 and 6% of the bacterioplankton in these samples were autofluorescent and may have been mistaken as cells that hybridized with fluorescent oligonucleotide probes. Dual staining allows precise estimates of the efficiency of transfers of cells to gelatin films and can be used to measure the percentage of the total bacterioplankton that also hybridize with fluorescent oligonucleotide probes, indicating specific phylogenetic groups.     

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.     

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.     

Light microscopic structure of DNA in solution studied by the 4′,6-diamidino-2-phenylindole staining method

Individual DNA molecules in solution can be visualized under a fluorescence microscope by using the DNA-binding dye 4′,6-diamidino-2-phenylindole and can be recorded on video as mobile structures (Morikawa & Yanagida, 1981). DNA in the presence of 10mm-MgCl₂ was found to adhere to the glass surface, so that 4′,6-diamidino-2-phenylindole-stained DNA can be filmed as still images. Fluorescence micrographs of DNA (bacteriophages T4, T3 and λ, yeast and chicken erythrocyte) taken by the present procedure are better in resolution than those obtained by video, showing structural details of DNA molecules hitherto not observed in solution. In the specimens prepared at the reduced shear stress, the folded particles and the short thick filaments were abundant. The shear stream extended them into the wavy and the straight thin filaments. The lengths of the thin filaments seen in viral DNA correlated well with those determined by electron microscopy. Our results suggest that a DNA molecule in solution forms a certain kind of supercoil of its own accord.     

Visualization by fluorescence of chloroplast DNA in higher plants by means of the DNA-specific probe 4'6-diamidino-2-phenylindole

The DNA in isolated chloroplasts was visualized by the fluorescent probe 4'6-diamidino-2-phenylindole (DAPI). When excited with light of 360 nm, the DNA-DAPI complex fluoresces brilliantly at 450 nm. Nuclei also fluoresce but their nucleoli do not. RNase and Pronase treatment of chloroplasts did not affect the fluorescence but both pre- and posttreatment of DAPI-stained chloroplasts with DNase specifically destroyed the fluorescence. DNA-DAPI complexes in the chloroplasts show up as bright dots. These are distributed uniformly within the chloroplast except for the outer margins. The fluorescent dots can be seen at different focal levels. The number of DNA dots is roughly proportional to chloroplast area which, in turn, is a function of leaf size. The number of fluorescent dots also gave the impression that large leaves with large chloroplasts contain more chloroplast DNA than nuclear DNA.     

Fluorescence resonance energy transfer and molecular modeling studies on 4',6-diamidino-2-phenylindole (DAPI) complexes with tubulin

The goal of this work was to determine the binding properties and location of 4',6-diamidino-2-phenylindole (DAPI) complexed with tubulin. Using fluorescence anisotropy, a dissociation constant of 5.2+/-0.4 microM for the DAPI-tubulin complex was determined, slightly lower than that for the tubulin S complex. The influence of the C-terminal region on the binding of DAPI to tubulin was also characterized. Using FRET experiments, and assuming a kappa2 value of 2/3, distances between Co2+ bound to its high-affinity binding site and the DAPI-binding site and 2',3'-O-(trinitrophenyl)guanosine 5'-triphosphate bound to the exchangeable nucleotide and the DAPI-binding site were found to be 20+/-2 A and 43+/-2 A, respectively. To locate potential DAPI-binding sites on tubulin, a molecular modeling study was carried out using the tubulin crystal structure and energy minimization calculations. The results from the FRET measurements were used to limit the possible location of DAPI in the tubulin structure. Several candidate binding sites were found and these are discussed in the context of the various properties of bound DAPI.     

DNA mediated resonance energy transfer from 4',6-diamidino-2-phenylindole to [Ru(1,10-phenanthroline)2L]2+

The binding site of Delta- and Lambda-[Ru(phenanthroline)2L]2+ (L being phenanthroline (phen), dipyrido[3,2-a:2'3'-c]phenazine (DPPZ), and benzodipyrido[3,2-a:2'3'-c]phenazine (benzoDPPZ)), bound to poly[d(A-T)2] in the presence and absence of 4',6-diamidino-2-phenylindole (DAPI) was investigated by circular dichroism and fluorescence techniques. DAPI binds at the minor groove of poly[d(A-T)2] and blocks the groove. The circular dichroism spectrum of all Ru(II) complexes are essentially unaffected whether the minor groove of poly[d(A-T)2] is blocked by DAPI or not, indicating that the Ru(II) complexes are intercalated from the major groove. When DAPI and Ru(II) complexes simultaneously bound to poly[d(A-T)2], the fluorescence intensity of DAPI decreases upon increasing Ru(II) complex concentrations. The energy of DAPI at excited state transfers to Ru(II) complexes across the DNA via the F?rster type resonance energy transfer. The efficiency of the energy transfer is similar for both [Ru(phen)2DPPZ]2+ and [Ru(phen)2benzoDPPZ]2+ complexes, whereas that of [Ru(phen)3]2+ is significantly lower. The distance between DAPI and [Ru(phen)3]2+ is estimated as 0.38 and 0.64 F?rster distance, respectively, for the Delta- and Lambda-isomer.     

DNA-binding geometry dependent energy transfer from 4′,6-diamidino-2-phenylindole to cationic porphyrins

The circular and linear dichroism (CD and LD) spectral properties of the meso-tetrakis(N-methylpyridinium-4-yl)porphyrin (TMPyP)–DNA complex at a [porphyrin]/[DNA] ratio below 0.015 showed that TMPyP intercalates between DNA base pairs. Contrarily, when cis–bis(N-methylpyridinium-4-yl)porphyrin (BMPyP) is associated with DNA, no CD spectrum was induced and a bisignate LD spectrum was observed. These spectral properties of both the TMPyP and BMPyP were essentially retained when the minor groove of the DNA was saturated with 4′,6-diamidino-2-phenylindole (DAPI). The fluorescence of the DNA-bound DAPI was effectively quenched by BMPyP and TMPyP. The quenching by BMPyP can be described through a pure static mechanism while TMPyP quenching produced an upward bending curve in the Stern–Volmer plot. Quenching efficiency was by far greater than predicted by the “sphere of action model”, suggesting that the DNA provides some additional processes for an effective energy transfer.     

DAPI (4',6-diamidino-2-phenylindole) binds differently to DNA and RNA: minor-groove binding at AT sites and intercalation at AU sites.

The interaction of DAPI and propidium with RNA (polyA.polyU) and corresponding DNA (polydA.polydT) sequences has been compared by spectroscopic, kinetic, viscometric, Tm, and molecular modeling methods. Spectral changes of propidium are similar on binding to the AT and AU sequences but are significantly different for binding of DAPI. Spectral changes for DAPI with the DNA sequence are consistent with the expected groove-binding mode. All spectral changes for complexes of propidium with RNA and DNA and for DAPI with RNA, however, are consistent with an intercalation binding mode. When complexed with RNA, for example, DAPI aromatic protons signals shift significantly upfield, and the DAPI UV-visible spectrum shows significantly larger changes than when complexed with DNA. Slopes of log kd (dissociation rate constants) versus-log [Na+] plots are similar for complexes of propidium with RNA and DNA and for the DAPI-RNA complex and are in the range expected for an intercalation complex. The slope for the DAPI-DNA complex, however, is much larger and is in the range expected for a groove-binding complex. Association kinetics results also support an intercalation binding mode for the DAPI-RNA complex. The viscosity of polyA.polyU solutions increases significantly on addition of both propidium and DAPI, again in agreement with an intercalation binding mode for both molecules with RNA. Molecular modeling studies completely support the experimental findings and indicate that DAPI forms a very favorable intercalation complex with RNA. DAPI also forms a very stable complex in the minor groove of AT sequences of DNA, but the stabilizing interactions are considerably reduced in the wide, shallow minor groove of RNA. Modeling studies,thus,indicate that DAPI interaction energetics are more favorable for minor-groove binding in AT sequences but are more favorable for interaction in RNA.     

Applications of fluorochromes to pollen biology. I. Mithramycin and 4',6-diamidino-2-phenylindole (DAPI) as vital stains and for quantitation of nuclear DNA

The two DNA-specific fluorochromes DAPI and mithramycin have been found to be extremely useful dyes in studies of pollen development and growth. Both fluorochromes stain nuclei brilliantly either in fixed or in living tricellular and bicellular angiosperm pollen, thereby permitting rapid scanning for pollen abnormalities and easy observation of nuclear details. These water soluble dyes can be incorporated into the germination medium for studies of pollen germination in vitro, facilitating observation of the movement of generative, sperm and tube nuclei during pollen growth. In fixed pollen, the fluorochromes bind quantitatively with DNA and thus may be used to quantitate ploidy changes and to study cell cycles during pollen development, germination and fertilization.     

Mithramycin- and 4'-6-diamidino-2-phenylindole (DAPI)-DNA staining for fluorescence microspectrophotometric measurement of DNA in nuclei, plastids, and virus particles

The properties of two DNA-specific fluorochromes, 4'-6-diamidino-2-phenylindole (DAPI) and mithramycin, have been analyzed as reagents to quantitate cellular DNA by fluorescence microspectrophotometry. Optimal staining conditions and concentrations, and the effects of other cellular materials to which the dyes bind, have been evaluated in measurements of the DNA of rat, chick, and yeast nuclei, Gonyostomum chloroplasts, and T4 particles. Use of either fluorochrome permits a high degree of resolution of different DNA quantities in nuclei and in cell organelles, and the DAPI-DNA complex is sufficiently fluorescent to permit quantitation of the DNA content in genomes as small as those of individual T4 bacteriophage particles. Fluorescence of mithramycin- or DAPI-stained DNA is proportional to DNA quantity when DNA of the same has composition is compared. Quantitation does not appear to be affected discernably by the state of the DNA, whether in different stages of the cell cycle, in condensed chromosomes, or in noncycling, differentiated nuclei. The use of chicken red blood cells is recommended as an internal monitor for variations in staining conditions.     

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.     

DNA binding properties of DAPI (4',6-diamidino-2-phenylindole) analogs having an imidazoline ring or a tetrahydropyrimidine ring: groove-binding and intercalation.

DAPI analogs containing an imidazoline ring or a tetrahydropyrimidine ring have been synthesized to study DNA binding properties. Spectroscopic (absorption, CD, flow dichroism and fluorescence) and viscosity measurements indicate that DAPI analogs interact with DNA both by intercalation and by groove binding. The solution structures of complexes between DAPI analog and DNA oligomers have been characterized by proton NMR spectroscopy.