DAPI: a DNA-Specific Fluorescent Probe

Jan Kapuscinski Department of Molecular Biology, Gdansk University, 80-822 Gdansk, Poland, and The Cancer Research Institute, New York Medical College, Elmsford, New York 7 0523

ABSTRACT. DAPI (4’.6-diamidino-2-phenylin- RNase. These observations established DAPI as dole) is a DNA-specific probe which forms a a DNA-specific fluorescent stain. At the same fluorescent complex by attaching in the minor time, these authors discredited DAPI as a quan- grove of A-T rich sequences of DNA It also forms titative measure of DNA because of a mistaken nonflnorescentintercalative complexes with dou- observation that the free dye solution is nearly bk-stranded nucleic acids. The physicochedcal as fluorescent as the DNA-dye complex. Most of the dye and complexes with nu- properties its likely this phenomenon was a result of the pres- cleic acids and history of the development of this dye as a biological stain are described. The appU- ence of sarcosyl (N-laurosylsarcosine), an an- cation of DAPI as a DNA-specMc probe for flow ionic detergent in the solution used for DNA cytometry, chromosome stainfng. DNA visnaliza- separation. Later it was shown that anionic de- tion and qnantitation in histochemistry and bio- tergents enhanced DAPI almost as chemistry is reviewed. The mechanisms of DAPI- much as DNA, i.e., about 20 times (Kapuscinski nncleic acid complex formation including minor and Skoczylas 1978). Russell et al. (1975) pub- gmove bding. intercalation and condensation lished a simple cytochemical technique for de- cuediscoseed. tecting myoplasma infection of cultured HeLa cells treated with DAPI using fluorescence mi- Key words: 4’,6-diamidino-2-phenylindolenu- croscopy. These authors published a stunning cleic acids, light absorption spectr~~~opy.fino- photograph of vaccinia within DAPI rescence spectroscopy,fIowcytometry, DNAViau- alization. DNA assay. chromosome , treated cells. These papers attracted the atten- intercalation tion of other scientists in the field and during the next two years most of DAPI’s application as a DNA-specific fluorochrome was explored. Development of DAFl The practicability of DAPI for fluorescent microscopic observation of DNA in DAPI (4‘,6-diamidino-:!-phenylindole dichlo- For personal use only. (Grossgebaueret al. 1976,Jagielski et al. 1976), ride) (Fig. 1)was first synthesized in Otto Dann’s (Schweizer 1976a,b, Schweizer and Nagl laboratory at Erlangen as one of many diami- 1976).protozoa (Hajduk 1976)and mammalian dine compounds in the search for new trypano- cells (Grossgebauer et al. 1976, Lin and Alfi cides related to the drug berenil (Dann 1976, Lin et al. 1976, Zworska et al. 1976) was et al. 1971). DAPI, to my knowledge, never went demonstrated. Detection of con- to clinical trials as a drug, but in 1975William- tamination was explored further by Jagielski et son and Fenneu used it for isolation of mito- al. (1976) and Grossgebauer et al. (1977).The chondrial DNA in cesium gradient (Williamson structure of chromatin and chromosomes and Fennelll974). They observed enhancement stained with DAPI alone (Lin and Alfi 1976, of the dye’s fluorescence when it was attached Schweizer 1976a,b, Schweizer and Nagl 1976, to DNA. This enhancement was particularly Lin et al. 1977)and in combination with actino- for mitochondxial DNA which is rich in A-T good mycin D or chromomycin (Schweizer 1976a,b), sequences. They also used DAPI for staining mithramycin (Schnedl et al. 1977) or dista- DNA of yeast cells and demonstrated that the

Biotech Histochem Downloaded from informahealthcare.com by Karlsruher Institut fuer Technologie - KIT on 10/02/12 mycin D (Schweizer et al. 1978, 1979, Buys et fluorescence is sensitive to DNase. but not to ai. 1979a,b) were studied and the high affinity of DAPI for A-T rich DNA sequences was con- bed.In addition, biochemical study revealed 10520295/95R24233/$3.00 Volume 70 BlOTECHNfC & HlSTOCHEMlSTRY Number that DAPI inhibited EcoRl restriction nuclease Copyright Q 1995 by Williams & Wilkins activity at A-T rich regions (Kania and Fanning 220 DAPI: DNA-Specific Fluorescent Probe 22 1

When using DAPI for such measurement, one must remember that DAPI is a base specific stain and that the resulting fluorescence de- II pends not only on the amount of DNA in the cell, NH; but also on A-T the base content. The amount of the DAPI bound in the cell depends on the de- Fig. 1. Chemical structure of 4'-6-diamidine-2-phenylindole (DAPI) dichloride. gree of chromatin condensation, which depends on the method of cell preparation. For instance, removing histone proteins by washing cells with 1976) and also inhibited the template activity 0.1 N hydrochloric acid nearly doubles the of P. aurelia (Skoczylas and Kapuscinski 1977). amount of DAPI bound to DNA (Darzynkiewicz Several authors pointed out high resistance of et al. 1984, Rundquist 1993).Another DNA- DAPI to UV illumination (e.g., Schnedl et al. spe~ificstain, Hmchst 33258. has similar SF- 1977, St6hr et al. 1977). These findings estab- tral properties and binding mechanism as lished DAPI as a valuable biological stain. DAPI. There is, however, one important differ- A simple and sensitive quantitative fluores- ence between the two fluorochromes. While the cence method for DNA assay in the presence of bromodeoxyuridine incorporated into DNA at RNA and histones was developed by Kapuscin- neutral pH has no effect on DAPI emission, ski and Skoczylas (1977).The major advantage Hoechst 33258 fluorescence is quenched by ha- of this method is that no preliminary prepara- logenated DNA (Takahama and Kagaya 1988. tions, such as separation or enzymatic RNA Latt and Langlois 1990, Hard et al. 1990).Flow degradation, are required. The lowest limit of cytometry DNA assays were used for many pur- this assay is 5 X lO-'O g/ml of DNA using a com- poses including studies of spermatozoa (Otto et mercially available spectrofluorometer (Kapus- al. 1979, Evenson et al. 19861, DNA analysis of cinski and Skoczylas 1977). Later, a similar isolated nuclei from a variety of tissues mom- method was applied to measure nanogram thwaite et al. 1980. Lee et al. 1984. Otto 1990, quantities of DNA in cellular homogenates Castro et al. 1993). analysis of phytoplankton (Bmnk et al. 1979).DAPI was also used forvisu- (Trasket al. 1982). pmtozoa(Bonalyet al, 1987. alizing DNA in the presence of proteins (Doug- Muhlpfordt and Berger 1989).bacteria (Ftobert- lass et al. 1978) and in the presence of large son and Button 1989) and (Galbraith quantities of FWA in electrophoretic gels (Ka- 1990. Ulrich 1992), analysis of cellular DNA puscinski and Yanagi 1979).

For personal use only. content of paraffin embedded pathological ma- Application of DAPI in Histology, terial (Hedley 1990. Heiden et al. 19911, and cancer detection and prognosis (Owainati et al. Analytical Cytology and Biochemistry 1987. Meyer and Coplin 1988. Chi et al. 1990. Flow cytornetry. The widest application of De Vita et al. 1991, Hatchoh et al. 1992). DAPI is in . The use of the dye as Chromosome staining. During the last 25 a quantitative DNA assay in cells was reported years, use of DAPI was reported as a biologfcal first by St6hr et al. (1977), then by G6hde et al. stain for chromosomes more than 200 times, It (1978).Since then, hundreds of papers describ- was used alone or in combination with other ing the applications of this technique have DNA ligands. most fi-equently with distamycin been published. D (Dhawale and Kessler 1993). Because of the Some applications of DAPI for quantitative large number of publications in this field, only DNA measurement in individual cells have re- selected papers are cited here. The dye has been cently been reviewed in the monograph Flow used to study chromosomes of plant (Schweizer Cytometry and Sorting (Crissman and 1976a,b. LeemannandRuch 1978,1982,1983, Biotech Histochem Downloaded from informahealthcare.com by Karlsruher Institut fuer Technologie - KIT on 10/02/12 Steinkamp 1990, Darzynkiewicz 1990, Latt and Rawlins and Shaw 1988, Beardsell et al. 1990, Langlois 1990. Pallavicini et al. 1990,Waggoner Czaban and Forer 1992. Rayburn et al. 1992, 1990).The dye can be used alone, e.g., for cell Hagemann et al. 1993)and cells. and to cycle studies of drug effects, or in multi-param- investigate chromatin structure (Schwarzacher eter cell analysis in combination with another et al. 1984, Schmid et al. 1987, Mills and fluorochrome. One of the most popular stains Massey 1992). Human chromosome aberra- combines DMI with sulforhodamine (SR 101) tions were examined (Fufita et al. 1980, Haseg- for simultaneous measurement of protein and awa et al. 1984. Mohandas et al. 1985. Sachs DNAcontent in cells (StOhr and Goerttler 1979). et al. 1987, Macera et al. 1989, Merkx et al. 222 Biotechnic & Histochemistry

1990, Sago et al. 1991, Smeets et al. 1991, Fluorescent assay of DNA in solution. The Blennow et al. 1993) in cancer studies and for fluorescence enhancement of DAPI in the pres- cancer diagnosis (Macera et al. 1989, Huber et ence of DNA is the basis for a simple and rapid al. 1990. Callahan et al. 1992, Holden et al. method for DNA microassay in the presence of 1986). RNA and proteins (Kapuscinski and Skoczylas DNA visualization and quantitation. DAPI 1977) and cellular homogenates (Brunk et al. has been used frequently for microscopic study 1979). The sensitivity of this method reaches of DNA in chloroplasts (James and Jope 1978, 0.5 ng DNA/ml. The assay can not be performed Coleman 1979, 1984, Coleman et al. 1981, in the presence of an anionic detergent, such Hoursiangou Neubrun et al. 1982, Lawrence as sodium docasyl sulfate (SDS), which en- and Possingham 1986, Ehara et al. 1990) and hances the fluorescence of the dye (Kapuscinski in mitochondria (Williamson and Fennell 1974, and Skoczylas 1978). Also, application of DAPI Hyman et al. 1982, Hamada and Fujita 1983, is limited in the presence of RNA, which binds Miyakawa et al. 1984, Van Blerkom and Runner DAPI to form a nonfluorescent complex. If the 1984, Yamada et al. 1986, McCarthy et al. nucleic acid mixture contains more than 96% 1987, Ito Kuwa et al. 1988, Satoh and Kuroiwa RNA, DNA cannot be measured accurately (Ka- 1991), detection of protozoans including ma- touzian Safadi et al. 1989). Because the DAPI laria [Hyman and Macinnis 1979, Celada et al. forms a fluorescent complex only with A-T-se- 1983, Kawamoto et al. 1987, Matsumoto et al. quences, the calibration curves must be made 1987). spermatogenesis (Abbott and Gerbi with DNA with a GC:AT ratio similar to the DNA 1981, Sahdev et al. 1989, Bressac and Rousset measured in the sample. The other limitation is 1993) and fertilization (Goff and Coleman 1984, the presence of large amounts of tubulins or maset al. 1984, Coleman and Goff 1985, microtubules in the sample, because these are Hollenbeck and Cande 1985, Mori et al. 1988, the only proteins known to form fluorescent Rohloff et al. 1990, Nakanishi et al. 1990, 1991, complexes with DAPI (Bonne et al. 1985).A Perry 1987).The diversity and diagnostic poten- modification of this technique was also used to tial of the DAPI staining technique is illustrated assay DNA in epidermis and cultured by a recent study concerning the detection of fibroblasts (Meyer and Grundmann 1984), both spermatozoa and Pneumocystis carinii in plant tissue (Lee and Garnett 1993), prokary- the lung of an AIDS patient (Pohle and otes (whole bacteria treated with toluene with- Grossgebauer 1986). out DNA purification) (Legros and Kepes 1985)

For personal use only. DAPI and staining techniques. The and for DNA deposited on filters (Wilkins and combination of DAPI and a fluorescent labeled Keamey 1984). DAPI also has been used for de- antibody is a powerful cytochemical tool for termining cell density in microtiter wells concurrent visualization of DNA and individual (McCaffrey et al. 1988). proteins in the cell. Using this technique, DNA Visualization of DNA in electrophoresis gels. and tubulin, microtubules or microfilaments Douglas et al. (1978) described a method for can be identified (Baumstark Khan et al. 1984, electrophoresis of DNA prestained with DAPI. A Roos et al. 1984. Wang et al. 1988) and malaria method for selective staining of DNA in gels in parasites (Murakami and Tanabe 1985), bacte- the presence of double stranded (ds) RNA, ria (Hoff 1988). and Herpes simplex (Ran- which is not visualized, has been proposed (Ka- dall and Dinwoodie 1986) can be detected. Pro- puscinski and Yanagi 1979). This staining duction of interferon in the cell in relation to method is several times more sensitive than cell cycle can also be studied (Tang et al. 1989). staining with ethidium bromide (Nairn et al. In situ hybridization. In recent years this 1982, Buel and Schwartz 1993). technique, combined with simultaneous DNA Biotech Histochem Downloaded from informahealthcare.com by Karlsruher Institut fuer Technologie - KIT on 10/02/12 staining with DAPI, has become very popular. Physicochemical Properties of DAPI and The detailed description of its implementation its Complexes with Nucleic Acids is beyond the scope of this article, but can be found in the following selected recent pub- DAPI is commercially available as the dichloride lications (Fan et al. 1990, Plattner et al. 1991, or diacetate salt. In the solid state and kept in Smeets et a]. 1991, Crolla et al. 1992, Ried the dark, the dye can be stored for several years et al. 1992, Boyle et al. 1992, Callahan et al. without decomposition. DAPI is readily soluble 1992, Naffeld et al. 1992, Heng and Tsui 1993, in water and concentrated frozen solutions (sev- Trask et al. 1993). eral mg/ml) are stable for months. The absorp- DAPI: DNA-Specific Fluorescent Probe 223

I I I

(nanometers) Fig. 2. Excitation and emission spectra of DNA-bound fluorescent dye Hoechst 33258 (or DAPi). Superimposed are the light source spectra of UG 1 filtered mercury arc lamp (---), the laser 351- and 364-nm emission lines (- - -1 and the transmission curve of 3-72 high pass barrier filter. Reproduced with permission from Peters (1979).

tion spectrum of DAPI has three maxima, 222, of the free dye is multi-exponential with the 259 and 340 nm, but only the 340 nm band is prevalent component at 0.26 nsec; the lifetime important for cytofluorometrics. This band is of the dye bound to DNA is approximately4 nsec well positioned for excitation by UG 1 filtered (Barcellonaand Gratton 1991).DAPI is not opti- For personal use only. mercury lamp or 351 and 364 nm laser light cally active, but in complexes with nucleic (Peters 1979) (Fig. 2). The fluorescence quan- acids, the induced extrinsic positive Cotton ef- tum yield of the free dye is very low with a maxi- fect can be seen. This effect was observed in mum of emission at 453 m; when bound to circular dichroism (CD) spectra, with a maxi- DNA there is a bathochromic shift of excitation mum above 330 m, of both fluorescent and and a hypsochromic shift of emission, and the nonfluorescent complexes of the dye with nu- fluorescence quantum yield increases more cleic acids (Kapuscinski and Szer 1979). The than 20-fold Fable 1). The fluorescence decay position of the long wavelength maximum of the

Table 1. Spectral Properties of DAPI and its Complexes with Nucleic Acids Mode x, EXlWcm'M-' ' s Q of KX106M' nm nm nm Binding

~ ~ DAPI' 340 2.70 347 453 0.04 - - Biotech Histochem Downloaded from informahealthcare.com by Karlsruher Institut fuer Technologie - KIT on 10/02/12 DAPI-ds DNA" 347 2.36 363 448 0.92 m.g. 20.0 DAPI-poly(dA)-poly(dT)b 358 2.21 350 448 0.92 m.g. 7.40 DAPI-poly(rA).poly(rU)b 3 60 1.93 350 460 0.30 int. 0.05 DAPI-poly[d(G-C)12b 360 1.93 nonfluorescent int. 0.1 2 DAPI-poly(rA) 1:2a 356 2.04 356 500 0.20 cond. 0.32' Lr,maximum absorption; E, molar absorption coefficient; A+, maximum excitation; 5,maximum emission; Q, fluorescence quantum yield; K, asmiation constant; m.g,, binding in the minor groove of DNA; int., intercalation into the double helix; cond., condensation of the complex. a Data from reference (Kapuscinski 1990) Data from reference (Tanious et al. 1992). Cooperative association constant. 224 Biotechnic & Histochemistry

CD spectrum depends on the base composition and Skoczylas 1978). depending on NaCl con- of the nucleic acid and on the dye/nucleic acid centration (0.1 and 0.01 M, respectively). The ratio (Kubista et al. 1987, Wilson et al. 1990a. enthalpy of this interaction is also favorable (AH Eriksson et al. 1993). < -5 kcal/mol) (Chandra and Mildner 1980a,b); the binding of DAPI stabilizes double stranded Mechanism of DAPI Interactions DNA against thermal denaturation (Kapuscin- with Nucleic Acids, other Polyanions ski and Skoczylas 1978). Based on these data and results of a study of solvent effects, interca- and Proteins lation was suggested as a mechanism for forma- Despite the extensive use of DAPI as a biological tion of the DAFT-double stranded DNA fluor- stain and for DNA assay, the mechanism of its escent complex (Kania and Fanning 1976, interaction with nucleic acids has only recently Kapuscinski and Skoczylas 1978, Chandra and been clarified. The ability of DAPI to form a Mildner 1980a,b, Schweizer et al. 1978, Masotti highly fluorescent complex with DNA was de- et al. 1981, Kapuscinski and Szer 1979). scribed in the 1970s (Williamson and Fennell There were also strong arguments against 1974, Russell et al. 1975).The requirements of an intercalation mechanism, however (Kubista ds DNA and base specificity (AT-rich sequences) et al. 1987). The unwinding of supercoiled DNA for formation of the fluorescent complex with induced by DAPI (Stepien et al. 1979, Manzini DAPI were also established early by both bio- et al. 1983)and the sedimentation of linear DNA chemical (Willamson and Fennell 1974, Kania bound to DAPI are not consistent with a com- and Fanning 1976. Chandra et al. 1977. Kapus- plex formed by intercalation (Waring 1970). cinski and Skoczylas 1977. Kapuscinski and Based on these data and those of linear dichro- Skoczylas 1978) and histological (Schweizer ism, circular dichroism and fluorescence spec- 1976a,b, Schnedl et al. 1977, Hajduk 1976) troscopy Kubista et al. (1987) rejected the idea methods. Studies using synthetic poly- and oli- that DAPI is bound to DNA by intercalation. gonucleotides revealed that double stranded They observed that binding geometries and site DNAcontaining dA-dT,dA-dU, dA-BrdU and dl- densities are consistent with DAPI located in dC (but not dG-dC) sequences enhanced DAPI the grooves of DNA, with the high-affinity site fluorescence (Kania and Fanning 1976, Lin et probably in the minor groove as is the case for al. 1977. Kapuscinski and Szer 1979), and that netropsin and Hoechst 33258 dye (Portugal and the binding sites must contain at least three Waring 1988).Earlier CD studies published

For personal use only. consecutive base pairs (Kapuscinski and Szer (Manzini et al. 1983)led to the same conclusion. 1979). Both single and double stranded RNA The location of the binding site in the minor form complexes with DAPI. but the fluorescence groove of the B-DNA molecule can explain the of these complexes (with the exception of strong A-T base specificity of the DAPI-DNA poly(r1)) is much weaker than the fluorescence fluorescent complex (Manzini et al. 1983, Portu- of DAPI double stranded DNA (Kapuscinski and gal and Waring 1988, Kubista et al. 1987).This Szer 1979, Kapuscinski and Yanagi 1979).The model was confirmed by x-ray diffraction of a affinity of DAPI for double stranded DNA is very single crystal of DAPI bound to the synthetic high (maand Fanning 1976); the apparent B-DNA oligonucleotide C-G-C-G-A-A-T-T-C-G- association constants (KPp)in the range of lo5- C-G (Larsen et al. 1989).According to these au- lo7M-' were reported (Kapuscinski and Skoczy- thors, the fluorescent complex is nearly isomor- las 1978. Bierzynski et al. 1978, Chandra and phous with the native DNA molecule with one Mildner 1980a.b. Morikawa et al. 1981, Masotti DAPI and 25 water molecules per DNA double et al. 1981, Barcellona et al. 1981, Dall'Asta et helix. DAPI is inserted edgewise into the narrow al. 1981. Masotti et al. 1982, Manzini et al. minor groove, displacing the ordered spine of Biotech Histochem Downloaded from informahealthcare.com by Karlsruher Institut fuer Technologie - KIT on 10/02/12 1983, Barcellona et al. 1986, Bumma et af. hydration. DAPI and a single water molecule 1988, Tanious et al. 1992). Most investigators together span the four A-T base pairs at the reported two kinds of binding sites for natural center of the duplex. The indole nitrogen forms DNA. The different S, values reported by these a bifurcated hydrogen bond with the authors can be explained by Merent experi- oxygen atoms of the two central base pairs, just mental and calculation methods. as netropsin and Hoechst 33258 do. The prefer- The Gibbs free energy change (AGO) of DAPI ence of all three of these drugs for A-T regions binding to calf thymus DNA is between -9 (Man- of B-DNA is due to three factors: 1) the minor zini et al. 1983) and - 11 kcal/mol (Kapuscinski groove associated with A-T regions is narrower DAPI: DNA-Specific Fluorescent Probe 225

than G-C regions of B-DNA, leading to a snug Most intercalators, including DAPI at high fit of the flat aromatic rings between the walls concentrations, condense and precipitate poly- of the groove, 2) the more negative electrostatic anions including both single and double potential within the minor groove in A-T re- stranded nucleic acids (Kapuscinski and Dar- gions, attributable in part to the absence of elec- zynkiewicz 1990, Kapuscinski 1990). Nucleic tropositive guanine amine groups along the acids in solution behave as random coil struc- floor of the groove, and 3) the steric advantage tures because of repulsive ionic phosphate- of the absence of those amine groups, pennit- phosphate interactions. After neutralization, ting the dye molecule to sink deeper into the the polymer collapses and forms compact (con- groove. Groove width and electrostatic factors densed) structures. Neutralization of DNA can are regional and define the relative re- be achieved by multi-cations with valences 2 3, ceptiveness of a section of DNA since they oper- such as Co[NH31sS3,spennidinet3 or sperminef4. ate over several contiguous base pairs. The simple cations (Widom and Baldwin 1980, Wi- steric factor is local, varying from one base pair dom and Baldwin 1983),or by aromatic cations (e.g., intercalators) with valences 1 with an to the next, hence it is a means of fine tuning 2 ability to form a complex with nucleic acid bases sequence specificity (Larsen et al. 1989). by stacking interactions (Kapuscinski and Dar- There presently is little doubt about the struc- zynkiewicz 1983, 1984a). The mechanism of ture of the fluorescent complex DAPI forms with nucleic acid condensation induced by these two double stranded DNA, i.e., that the fluoro- types of ligand is different. The most signiticant chrome molecule is bound in the minor grove difference is that the aromatic cation-induced of consecutive (3-4 base pairs) A-T-rich se- condensation is preceded by denaturation of quences. It does not mean that DAPI does not double helix regions of nucleic acids (Kapuscin- bind to other sequences of double stranded ski and Darzynkiewicz 1984a,b).while the sec- DNA, or double stranded RNA. These com- ondary structure of nucleic acids is preserved plexes, however, are much less fluorescent. In in the condensed product with simple cations the series of elegant papers reporting results of (Widom and Baldwin 1980).In both cases, how- several physicochemical techniques including ever, the electrostatic repulsive forces are re- NMR, light absorption spectroscopy,viscosime- duced, which leads to spontaneous condensa- try, stopped-flow kinetics, and molecular mod- tion (collapse) of the polymer (Manning 1980. eling methods, Wilson et al. (1989, 1990a,b) Kapuscinski and Darzynkiewicz 1984a.b). The

For personal use only. and Tanious et al. (1992)provided evidence that condensation of the ffuorochrome-nucleic acid DAPI binds to other than the continuous A-T complexes has profound effects on their spec- sequences of DNA and to double stranded RNA tral properties, resulting in part from limited by intercalation. These authors stated that at contact with the solvent. For example, conden- continuous dG-dC and mixed (e.g., poly(dA- sation of the acridhe-RNA complex is responsi- dC).poly (dG-cYT)) sequences, the 2-NH2 group ble for red luminescence (Kapuscinski and Dar- of guanine sterically inhibits DAPI binding in zynkiewicz 1983, 1984b. Kapuscinski et al. the minor groove of DNA. Also, in such se- 1982),and the same process results in the lumi- nescence quenching of the pyronin Y-RNA com- quences, the depth of the minor groove is re- plexes (Darzynkiewia and Kapuschski 1988). duced and its width is increased compared to Condensed complexes of DAPI-RNA have an A-T sequences; the energetics of this favors in- emission max3mum at approximately 500 nm tercalation over minor groove binding (Tanious compared to 448 nm for the complex with dou- et al. 1992). The binding strength of DAPI to ble stranded DNA (Fig. 3); the ff uorescence the dG-dC sequences is in the range typically quantum yield of this condensed complex is Biotech Histochem Downloaded from informahealthcare.com by Karlsruher Institut fuer Technologie - KIT on 10/02/12 observed for strong intercalators such as quina- only about of the yield of the highly fluores- crine, ethidium or propidium. The same au- cent DAPI-double stranded DNA complex (Ka- thors also provided evidence that DAPI binds puscinski 1990, Skoczylas 1988). strongly to rA-rU sequences by intercalation The formation of yellow fluorescence is not rather than to the minor groove ('I'anious et al. limited to DAPI condensed complexes with nu- 1992). DAPI should be viewed, therefore, as an cleic acids. Other polyanions including muco- intercalator #at has unusual and favorable in- polysaccharides and polyphosphates are also teractions in the minor groove at dA-dT se- precipitated within cells (e.g., in vacuoles and quences (Wilson et al. 1989). on the nuclear envelope) and outside the 226 Biotechnic & Histochemistry I .- x 0.2

360 460 560 WAVELENGTH, nm Fig. 3. Fluorescence emission spectrum (excitation 340 nm) of DAPI (2 pM) normalized to 1 (-4,its complex with double stranded DNA (D/P = 0.01) (reduced five times from the original amplitude), (--4,its complex with poly(rA) at DIP = 0.1 (----) and at D/P = 0.5 (- - -). F, and Ff are the fluorescence intensity of the complex and free dye, respectively. Reproduced with permission from Kapuscinski (1 990).

plasma membrane (Grossgebauer 1979a,b, lower affinity of these proteins for DAPI com- 1980, Allan and Miller 1980, Grossgebauer and pared to DNA. The changes in the fluorescence Kupper 1981, Tijssen et al. 1982).The large ba- spectra of DAPI bound to tubulin (or microtu- thochromic emission shifts of these non-nucleic bules) are similar to those observed for changes For personal use only. acid complexes, similar to and sometimes larger observed for DAPI vs. DAPI-DNA complex, i.e., than that observed for condensed DAPI-RNA a bathochromic shift (+ 8 nm) for excitation, and complex (Kapuscinski 1990) has been reported a hypsochromic shift (-24 nm) for emission, (Allan and Miller, 1980, Tijssen et al. 1982, Kjel- compared to the spectra of the free dye. The dstad et al. 1991). fluorescence quantum yield of DAPI increases Among proteins, only tubulins are known to several times after binding to these proteins, form fluorescent complexes with DAPI (Bonne but it is much lower than that observed for DAPI et al. 1985, Heusele and Bonne 1985, Heusele double stranded DNA complex (Bonne et a€. et al. 1987. Ortiz et al. 1993).Tubulin is a major 1985). protein of microtubules, which are important cytoskeletal constituents of eukaryotic cells. ACKNOWLEDGMENTS The stoichiometry of the complex is one DAPI molecule per tubulin dimer (Bonne et al. 1985). I am grateful to Drs. 2. Darzynkiewicz and F. Bonne et al. (1985) proposed that the DAPI- Traganos of The Cancer Research Institute Biotech Histochem Downloaded from informahealthcare.com by Karlsruher Institut fuer Technologie - KIT on 10/02/12 binding site on tubulin is located partly in the NYMC for helpful discussion and suggestions. carboxyl-terminalregion of tubulin in which the Supported in part by grants DS 0010-4-0002- last 40 positions have 19 acidic side chains. 4 and KBN G P203 04406. These authors also observed energy transfer from tryptophan located close to this binding REFERENCES site to DAPI. The apparent association con- Abhtt, A. G. and Gerbi, S. A. 1981. Spermatogenesis stantsare2.3 X 104and17 X 104M-lfortubulin in Sciara coprophh 11. Precocious chromosome and microtubules, respectively (based on data orientation in meiosis 11. Chromosoma 83: from Bonne et al. (1985)),which indicates a 19-27. DAPI: DNA-Specific Fluorescent Probe 227

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