DNA Sequence-Specific Ligands: XII. Synthesis and Cytological Studies of Dimeric Hoechst 33258 Molecules

We synthesized dimeric Hoechst dye molecules composed of two moieties of Hoechst 33258 fluorescent dye with the phenolic hydroxy groups tethered via pentamethylene, heptamethylene, or triethylene oxide linkers. A characteristic pattern of differential staining of chromosome preparations from human HL60 premonocytic leukemia cells was observed for all the three fluorescent dyes. The most contrasting pattern was obtained for the bisHoechst analogue with the heptamethylene linker; its quality was comparable with the picture obtained in the case of chromosome staining with 4′,6-diamidino-2-phenylindole. The ability to penetrate into live human fibroblasts was studied for the three bisHoechst compounds. The fluorescence intensity of nuclei of live and fixed cells stained with the penta- and heptamethylene-linked bisHoechst analogues was found to differ only slightly, whereas the fluorescence of the nuclei of live cells stained with triethylene oxide-linked bisHoechst was considerably weaker than that of the fixed cells. The bisHoechst molecules are new promising fluorescent dyes that can both differentially stain chromosome preparations and penetrate through cell and nuclear membranes and effectively stain cell nuclei.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 4, 2005, pp. 385–393.Original Russian Text Copyright © 2005 by Gromyko, Popov, Mosoleva, Streltsov, Grokhovsky, Oleinikov, Zhuze.     

Pairs of fluorescent dyes as probes of DNA and chromosomes.

If two fluorescent dyes with different binding or fluorescence specificities are used simultaneously to stain DNA or chromosomes, the ratio of their fluorescent signals can provide information about base composition or base analogue substitution. Energy transfer between such dye pairs, possible if the fluorescence spectrum of one overlaps the absorption spectrum of the other, can modify observed fluorescence. Microfluorometric measurements were used to document the occurrence of energy transfer between quinacrine or 33258 Hoechst as energy donor and ethidium or 7-aminoactinomycin D as acceptor when used jointly to stain cytologic preparations of human metaphase chromosomes. Use of 7-aminoactinomycin D, a dye with G-C binding specificity, as energy acceptor permitted the identification of human chromosome regions presumptively enriched for clusters of A-T base pairs, based on the resistance of A-T specific fluorescence, from quinacrine or 33258 Hoechst, to energy transfer dependent quenching. The results provide information about basic structural features of metaphase chromosomes, and the associated methodology may prove useful in accentuating specific fluorescent polymorphic chromosome regions.     

Nuclear Staining of Colletotrichum Gloeosporioides F. SP. Malvae Conidia with Fluorescent and Nonfluorescent Stains

Six different staining techniques were evaluated for their suitability to stain nuclei of Colletotrichum gloeosporioides f. sp. malvae (C.g.m.) spores. Of the three fluorescent stains, DAPI (4',6-diamidino-2-phenylindole) and bisbenzimide (Hoechst 33258) stained spore nuclei well; mithramycin did not. To achieve consistent results with the bisbenzimide staining protocol, the spores had to be fixed prior to staining and the stain had to be supplemented with Triton X-100. Both safranin O and Giemsa were suitable nonfluorescent staining techniques; lomofungin was not. Safranin O staining was simple and rapid. However, reproducibility was better if the spore suspension and KOH droplets were rapidly mixed prior to adding the stain. There was no significant difference in the percentages of uninucleate and binucleate spores observed in spore preparations stained with DAPI, bisbenzimide, safranin O or Giemsa. Bisbenzimide and safranin O were found to be simple, rapid and reliable fluorescent and nonfluorescent techniques, respectively, for staining nuclei of C.g.m. spores.     

Different effects of 33258 Hoechst and DAPI in fluorescent staining of sister chromatids differentially substituted with bromodeoxyuridine

Summary After substitution with 5-bromodeoxyuridine (BrdUrd) for two rounds of replication, chromosomes in cytological preparations stained with 33258 Hoechst show upon epiluminescence an immediate differential sister chromatid fluorescence. When stained with DAPI, however, which has a structural resemblance to part of the 33258 Hoechst molecule, such a differential pattern of fluorescence was only induced after some delay. Upon restaining with the same dye the differential fluorescence appeared instantly. In preparations double stained with ethidium bromide and 33258 Hoechst the induction of a differential staining of sister chromatids with 33258 Hoechst was not accompanied by a differential staining with ethidium bromide. Once a differential staining was obtained with DAPI in preparations double stained with ethidium bromide and DAPI, the ethidium bromide pattern also appeared to be differential upon subsequent observation. No differentiation could be obtained with ethidium bromide alone. The observations described in the case of 33258 Hoechst staining are in agreement with a molecular quenching by BrdUrd without gross structural consequences for the DNA. In the case of DAPI staining, however, there occurs a differential photolysis of BrdUrd-substituted DNA. Besides the nature, most likely the size, of the fluorochrome molecules themselves, the state of the fixed chromatin appeared also to play a role in determining the mechanism of the sister chromatid differentiation: after prolonged incubation in buffer, BrdUrd-containing chromosomes stained with 33258 Hoechst showed a differential staining evidently caused by photolysis, indicating that they had become more susceptible to light.     

Comparison between morphological and staining characteristics of live and dead eggs of Schistosoma mansoni

Schistosoma mansoni eggs are classified, according to morphological characteristics, as follows: viable mature and immature eggs; dead mature and immature eggs, shells and granulomas. The scope of this study was to compare the staining characteristics of different morphological types of eggs in the presence of fluorescent labels and vital dyes, aiming at differentiating live and dead eggs. The eggs were obtained from the intestines of infected mice, and put into saline 0.85%. The fluorescent labels were Hoechst 33258 and Acridine Orange + Ethidium Bromide and vital dyes (Trypan Blue 0.4% and Neutral Red 1%). When labelled with the probe Hoechst 33258, some immature eggs, morphologically considered viable, presented fluorescence (a staining characteristic detected only in dead eggs); mature eggs did not present fluorescence, and the other types of dead eggs, morphologically defined, showed fluorescence. As far as Acridine Orange + Ethidium Bromide are concerned, either the eggs considered to be live, or the dead ones, presented staining with green color, and only the hatched and motionless miracidium was stained with an orange color. Trypan Blue was not able to stain the eggs, considered to be dead but only dead miracidia which had emerged out of the shell. Neutral Red stained both live and dead eggs. Only the fluorescent Hoechst 33258 can be considered a useful tool for differentiation between dead and live eggs.     

Application of pyronin Y(G) in cytochemistry of nucleic acids

Chinese hamster ovary (CHO) cells or isolated nuclei were stained with pyronin Y(PY) and analyzed by absorption or fluorescence microscopy, as well as by flow cytometry. Specificity of the staining reaction was assayed by testing sensitivity of the stainable material to RNase or DNase. The colored complexes detected by light absorption in fixed cells stained with PY are nonfluorescent and are most likely the products of condensation of single-stranded (ss) RNA by PY; the poly(rA) and poly(rA,rG) are the most sensitive to condensation. The products of PY interaction with double-stranded (ds) nucleic acids are fluorescent and can be detected in cells by cytofluorometry. PY used alone stains both DNA and RNA, and the staining capabilities of these nucleic acids vary depending upon the PY concentration at equilibrium; at a concentration above 330 microM, the RNA stainability decreases, perhaps due to its denaturation and condensation caused by the dye. In the presence of Hoechst 33342, PY can specifically stain RNA in fixed cells or isolated cell nuclei. Because only complexes of PY with ds RNA are fluorescent, this dye can be used as a probe of RNA conformation, e.g., to monitor denaturation of RNA in situ. The RNA stainability of mitotic cells is about 25% lower than that of cells in G2 phase, which indicates that during mitosis proportionately less cellular RNA is in the ds conformation. The advantages and limitations of the two cytochemical methods for DNA/RNA detection, one based on the use of Hoechst 33342 and PY, and another employing the metachromatic properties of acridine orange, are compared.     

Aqueous solutions of some basic dyes--their use in the cytochemical detection of DNA

The paper contains results of staining DNA-aldehyde molecules with aqueous solutions of brilliant cresyl blue, thionin or neutral red, following Feulgen procedure and also reports on the use of aqueous solutions of these dyes, with primary amino group(s) in their molecules, for staining animal tissue nuclei after extraction of RNA with cold phosphoric acid. The pH of the dye solutions most suitable for optimum staining is 6.0. The time necessary for optimum staining of DNA-aldehydes and DNA-phosphate groups are 10 and 2 min respectively for tissues fixed in formalin, paraformaldehyde or Craf. Tissue fixed in Buin-fluid stain slower. The absorption curves of nuclei stained for DNA-aldehyde molecules and DNA-phosphate groups, stained with each of the three dyes are different from each other. The in vitro absorption curves of aqueous solutions of the three dyes have also been presented. Some implications of the results obtained are discussed.     

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

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

Cytochemical studies of metaphase chromosomes by flow cytometry

The cytochemical properties of metaphase chromosomes from Chinese hamster and human cells were studied by flow cytometry. This technique allows precise quantitation of the fluorescence properties of individual stained chromosome types. Chromosomes were stained with the following fluorescent DNA stains: Hoechst 33258, DAPI, chromomycin A₃, ethidium bromide, and propidium iodide. The relative fluorescence of individual chromosome types varied depending on the stain used, demonstrating that individual chromosome types differ in chemical properties. Flow measurements were performed as a function of stain and chromosome concentration to characterize the number and distribution of stain binding sites. Flow analysis of double stained chromosomes show that bound stains interact by energy transfer with little or no binding competition. For most hamster chromosomes, there is a strong correlation between relative fluorescence and stain base preference suggesting that staining differences may be determined primarily by differences in average base composition. A few hamster chromosome types exhibit anomalous staining which suggests that some other property, such as repetitive DNA sequences, also may be an important determinant of chromosomal staining.     

Uptake kinetics of nucleic acid targeting dyes in S. aureus, E. faecalis and B. cereus: a flow cytometric study

For flow cytometry-based detection as well as susceptibility testing and counting, staining of the bacterial cells is essential. In an attempt to develop rapid preparatory procedures for nucleic acid staining of wild type Gram positive bacteria, the uptake of fluorescent dyes in viable S. aureus, E. faecalis, and B. cereus cells was studied by flow cytometry under conditions intended to block probe efflux and increase cell wall permeability. The aim of the study was to develop procedures which allow rapid nucleic acid staining independent of fixation, since ethanol fixation is time-consuming and may mask phenomena associated with viability and lead to uncontrolled loss and aggregation of cells. The dye uptake was measured repeatedly after treating cells with metabolic inhibitors in order to block probe efflux, or cold shock (0 degree C) to increase permeability. The probes used were mithramycin (Mi), ethidium bromide (EB), DAPI, Hoechst 33342 and Hoechst 33258. None of the procedures facilitated uptake of the dyes to a level similar to that obtained in fixed control cells in all of the species. After metabolic inhibition of B. cereus cells, DAPI and Hoechst fluorescence increased to a level similar to or above that found in fixed cells, indicating that the uptake of these dyes is limited by energy-dependent efflux. A similar increase of DAPI fluorescence was observed after cold shock suggesting the uptake of this dye to be limited also by permeability in B. cereus. The Mi and EB fluorescence increased to the level of the fixed control cells under all conditions tested, suggesting free probe influx in this species. Generally, probe uptake in S. aureus and E. faecalis was lower than in B. cereus cells, and no permeabilizing effect of cold shock was observed. In some experiments the fluorescence exceeded that of ethanol fixed control cells, indicating that the fixation may cause conformational changes in DNA.     

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.     

DNA labeling in living cells.

BACKGROUND: Live cell fluorescence microscopy experiments often require visualization of the nucleus and the chromatin to determine the nuclear morphology or the localization of nuclear compartments. METHODS: We compared five different DNA dyes, TOPRO-3, TOTO-3, propidium iodide, Hoechst 33258, and DRAQ5, to test their usefulness in live cell experiments with continuous imaging and photobleaching in widefield epifluorescence and confocal laser scanning microscopy. In addition, we compared the DNA stainings with fluorescent histones as an independent fluorescent label to mark chromatin. RESULTS: From the dyes tested, only Hoechst and DRAQ5 could be used to stain DNA in living cells. However, DRAQ5 had several advantages, namely low photobleaching, labeling of the chromatin compartments comparable to that of H2B-GFP fusion proteins, and deep red excitation/emission compatible with available genetically encoded fluorescent proteins such as C/G/YFP or mRFP. CONCLUSIONS: The DNA dye DRAQ5 is well suited for chromatin visualization in living cells and can easily be combined with other fluorophores with blue to orange emission.     

The binding kinetics and interaction of DNA fluorochromes used in the analysis of nuclei and chromosomes by flow cytometry

Summary The interactions and binding characteristics of DNA dyes used in the flow cytometric analysis of chromatin were studied using human chromosomes and mouse thymocyte nuclei. The kinetics of dye binding and the relationship between fluorescence intensity and dye concentration are presented. Under the conditions used, Hoechst 33258, propidium iodide and chromomycin A3 reach an equilibrium with thymocyte nuclei after approximately 5 min, 20 min and more than 1 h, respectively. The same binding kinetics are observed with Hoechst 33258 and chromomycin when nuclei are stained with a mixture of the two dyes. Sodium citrate, which improves the resolution of flow karyotypes, causes a rapid increase in Hoechst and propidium iodide fluorescence, but a decrease in the fluorescence of chromomycin. The relative peak positions of chromosomes in a flow karyotype are unaffected by sodium citrate addition. The spectral interaction between Hoechst and chromomycin is quantified. There is variation among the human chromosome types in the amount of energy transferred from Hoechst to chromomycin. By measuring the Hoechst and chromomycin fluorescence of each chromosome after Hoechst excitation, it is shown that the amount of energy transferred is correlated to the ratio of the amount of Hoechst to chromomycin bound. Although the energy transfer between the two dyes is considerable, this has little effect on the reproducibility of flow karyotype measurements. The relative peak positions of all human chromosomes in a 64×64 channel flow karyotype, except for the 13 and Y chromosomes, vary in the order of 0.5 channel over a 16-fold change in either Hoechst or chromomycin concentration. This implies that, with the present flow cytometers, variation in staining conditions will have minimal effects on the reproducibility of the relative peak positions in flow karyotypes.In honour of Prof. P. van Duijn     

Multiple-color fluorescence imaging of chromosomes and microtubules in living cells

Microscopic observation of fluorescently-stained intracellular molecules within a living cell provides a straightforward approach to understanding their temporal and spatial relationships. However, exposure to the excitation light used to visualize these fluorescently-stained molecules can be toxic to the cells. Here we describe several important considerations in microscope instrumentation and experimental conditions for avoiding the toxicity associated with observing living fluorescently-stained cells. Using a computer-controlled fluorescence microscope system designed for live observation, we recorded time-lapse, multi-color images of chromosomes and microtubules in living human and fission yeast cells. In HeLa cells, a human cell line, microtubules were stained with rhodamine-conjugated tubulin, and chromosomes were stained with a DNA-specific fluorescent dye, Hoechst33342, or with rhodamine-conjugated histone. In fission yeast cells, microtubules were stained with alpha-tubulin fused with the jellyfish green fluorescent protein (GFP), and chromosomes were stained with Hoechst33342.     

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

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

The binding kinetics and interaction of DNA fluorochromes used in the analysis of nuclei and chromosomes by flow cytometry

The interactions and binding characteristics of DNA dyes used in the flow cytometric analysis of chromatin were studied using human chromosomes and mouse thymocyte nuclei. The kinetics of dye binding and the relationship between fluorescence intensity and dye concentration are presented. Under the conditions used, Hoechst 33258, propidium iodide and chromomycin A3 reach an equilibrium with thymocyte nuclei after approximately 5 min, 20 min and more than 1 h, respectively. The same binding kinetics are observed with Hoechst 33258 and chromomycin when nuclei are stained with a mixture of the two dyes. Sodium citrate, which improves the resolution of flow karyotypes, causes a rapid increase in Hoechst and propidium iodide fluorescence, but a decrease in the fluorescence of chromomycin. The relative peak positions of chromosomes in a flow karyotype are unaffected by sodium citrate addition. The spectral interaction between Hoechst and chromomycin is quantified. There is variation among the human chromosome types in the amount of energy transferred from Hoechst to chromomycin. By measuring the Hoechst and chromomycin fluorescence of each chromosome after Hoechst excitation, it is shown that the amount of energy transferred is correlated to the ratio of the amount of Hoechst to chromomycin bound. Although the energy transfer between the two dyes is considerable, this has little effect on the reproducibility of flow karyotype measurements. The relative peak positions of all human chromosomes in a 64 X 64 channel flow karyotype, except for the 13 and Y chromosomes, vary in the order of 0.5 channel over a 16-fold change in either Hoechst or chromomycin concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Examination of living and fixed gametes and early embryos stained with supravital fluorochromes (Hoechst 33342 and 3,3′-dihexyloxacarbocyanine iodide)

We have examined living and fixed gametes and early embryos of surf clams, sea urchins, and hamsters stained with the supravital dyes Hoechst 33342 for DNA and 3,3′-dihexyloxacarbocyanine iodide (DIOC₆) for mitochondria and endoplasmic reticulum. Hoechst staining (10 μM) was confined exclusively to egg and sperm chromatin and, in living marine specimens, did not interfere with sperm motility, fertilization, or nuclear activity during meiosis or early embryogenesis. Although Hoechst staining did not appear to affect the motility of hamster sperm, only zonae-free eggs inseminated. Because chromatin retained Hoechst 33342 stain during fertilization, the paternally and maternally derived chromosomes of living and fixed preparations fluoresced and their number, organization, and location within the zygote cytoplasm could be determined. Hence, polyspermy and other nuclear abnormalities were amenable to examination in these stained preparations. DIOC₆ staining (8.7 μM) was restricted primarily to the mitochondria of spermatozoa. Eggs stained with DIOC₆ (0.87 to 8.7 μM) were brightly fluorescent because of their size and the presence of large numbers of mitochondria and other DIOC₆-positive organelles. Sea urchin and surf clam sperm stained with DIOC₆ fertilized unstained eggs and the location of the incorporated sperm mitochondrion up to first cleavage was followed. Although hamster sperm stained with DIOC₆ were less motile than unstained sperm, they were capable of inseminating only zonae-free eggs. These observations demonstrate that staining with supravital fluorochromes provides a rapid and useful method to analyze macromolecular and organelle changes in a variety of living and fixed gametes and embryos.     

A comparison of the effect of 5-bromodeoxyuridine substitution on 33258 Hoechst- and DAPI-fluorescence of isolated chromosomes by bivariate flow karyotyping

Application of the fluorescent DNA-intercalator propidium iodide for stabilization of the mitotic chromosome structure during isolation of chromosomes from V79 Chinese hamster cells and subsequent staining with the fluorochromes 33258 Hoechst or DAPI allowed bivariate flow karyotyping of isolated chromosomes. Fluorescence of 33258 Hoechst bound to isolated chromosomes containing 5-bromodeoxyuridine (BrdUrd) was quenched in comparison with the fluorescence of control chromosomes. Despite structural relationship and similarity of both absorption and fluorescence spectra of DAPI and 33258 Hoechst, reduction of fluorescence of DAPI-stained isolated chromosomes was not observed, by contrast with findings in conventional cytological metaphase preparations. It could be obtained, however, by preirradiation of the chromosomes with near-UV in the presence of DAPI. This led to a progressive destruction of the chromosomes. Destruction also occurred without BrdUrd, though at a slower rate. Preirradiation of chromosomes in the presence of 33258 Hoechst hardly affected the integrity of the chromosomes. Preirradiation of a 33258 Hoechst solution and its subsequent use as a stain resulted in a considerably decreased fluorescence of chromosomes. For DAPI this effect was small. Thus, whereas 33258 Hoechst itself is much more sensitive to near-UV irradiation than DAPI, DAPI bound to DNA in chromosomes renders the DNA much more sensitive to irradiation than 33258 Hoechst bound to DNA. Presumably, these differences can at least partly be reduced to the different molecular sizes of the dyes.     

A comparison of the effect of 5-bromodeoxyuridine substitution on 33258 Hoechst- and DAPI-fluorescence of isolated chromosomes by bivariate flow karyotyping

Summary Application of the fluorescent DNA-intercalator propidium iodide for stabilization of the mitotic chromosome structure during isolation of chromosomes from V79 Chinese hamster cells and subsequent staining with the fluorochromes 33258 Hoechst or DAPI allowed bivariate flow karyotyping of isolated chromosomes. Fluorescence of 33258 Hoechst bound to isolated chromosomes containing 5-bromodeoxyuridine (BrdUrd) was quenched in comparison with the fluorescence of control chromosomes. Despite structural relationship and similarity of both absorption and fluorescence spectra of DAPI and 33258 Hoechst, reduction of fluorescence of DAPI-stained isolated chromosomes was not observed, by contrast with findings in conventional cytological metaphase preparations. It could be obtained, however, by preirradiation of the chromosomes with near-UV in the presence of DAPI. This led to a progressive destruction of the chromosomes. Destruction also occurred without BrdUrd, though at a slower rate. Preirradiation of chromosomes in the presence of 33258 Hoechst hardly affected the integrity of the chromosomes. Preirradiation of a 33258 Hoechst solution and its subsequent use as a stain resulted in a considerably decreased fluorescence of chromosomes. For DAPI this effect was small. Thus, whereas 33258 Hoechst itself is much more sensitive to near-U.V irradiation than DAPI, DAPI bound to DNA in chromosomes renders the DNA much more sensitive to irradiation than 33258 Hoechst bound to DNA. Presumably, these differences can at least partly be reduced to the different molecular sizes of the dyes.In honour of Prof. P. van Duijn     

DNA-specific dimeric bisbenzimidazole

A dimeric analogue of the fluorescent dye Hoechst 33258 was synthesized. It was shown to differentially stain human chromosome preparations and bind to double-stranded DNAs.