Volume 6 Number 11 1979
Volume 6 Number 111979
Nucleic Acids Research
Nucleic Acids Research
Interactions of 4', 6-diamidine-2-phenylindole with synthetic polynucleotides
Jan Kapuscin'ski* and Wlodzimierz Szer
Department of Biochemistry, New York University School of Medicine, 550 First Avenue, New York, NY 10016, USA Received 9 May 1979
ABSTRACT 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.
INTRODUCTION 4', 6-Diamidine-2-phenylindole*2HCL (DAPI), a fluorescent dye originally synthesized by Dann et al. (1), binds to DNA with a marked increase in fluorescent quantum yield. This property of DAPI has been utilized in a number of cytochemical investigations, including flow fluorometry (2, 3 and references in 4), and in the development of a quantitative micro method for the assay of DNA in solution (5). In enzymatic studies it was found that, at low concentrations, DAPI stimulates the template activity of DNA (B. Skoczylas, personal communication), whereas high concentrations of the dye are inhibitory (6). The activity of endonuclease Rl from E. coli is affected by DAPI in an analogous concentration-dependent way (7-9). DAPI, as well as some other cationic fluorescent ligands, e.g., ethidium bromide, exhibit essentially two modes of binding to DNA: i) relatively weak nonfluorescent electrostatic interactions and, ii) strong, presumably C) Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England
3519
Nucleic Acids Research intercalative binding which gives rise to an increase in fluorescence. Weak interactions of DAPI with nucleic acids appear to show little specificity and have been observed with both DNA and RNA (6). Strong binding of DAPI accompanied by a large fluorescence enhancement has only been observed with native DNA (4). There are indications that DAPI binds preferentially to AT-rich DNA but it is not clear whether this specificity is related to the fluorescent or nonfluorescent modes of binding (4, 6). The selectivity of the fluorescent binding with respect to AT base pairs is of particular interest since it may provide a specific probe for the analysis of the micro structure of DNA. In this investigation we have determined the specificity and the stoichiometry of the fluorescent binding mode of DAPI using synthetic polydeoxyand polyribonucleotides, both single- and multi-stranded. The nonfluorescent binding of DAPI was also examined and was found to be nonspecific.
MATERIALS AND METHODS Polynucleotides: Poly dA, poly rA, poly dT, poly rU, poly rC, poly rI and poly rG were from Mile s; poly dC, poly d (G-C) * poly d (G-C) and poly d (A-U). poly d (A-U) were from Grand Island Biochemical s; poly d (A-T) . poly d(A-T), poly dG.poly dC, poly d(I-C).poly d(I-C), poly dU and oligo dAm (m= 2 to 12) were from P-L Biochemicals; poly d(A-C).poly d(T-G) was from Boehringer and poly d(A-G)-poly d(T-C) was a generous gift from Dr. A.R. Morgan, Department of Biochemistry, University of Alberta. DAPI was synthesized according to the modified procedure of Dann et al. (1, 10); physical properties of DAPI were described previously (4). Polynucleotide concentrations were estimated spectrophotometrically either from optical densities of mononucleotides produced by enzymatic hydrolysis of the polymers or according to published molar extinction coefficients (11-13). Fluorescence measurements were recorded with a thermostated AmincoBowman SPF-125S spectrofluorimeter equipped with an IP21 photomultiplier tube and a 150 W Xenon lamp; solutions of DAPI of known concentration were used as standards. Excitation spectra (uncorrected) were recorded from 200 to 420 nm (bandpass 4.5 nm) at 200; the wavelength of the emission was 454 nm (bandpass 9 nm). For fluorimetric titrations, the excitation wavelength was 364 nm; emission was measured at 454 nm with a 9 nm bandpass in both the excitation and emission monochromators. The fluorescence intensity of the sample, corrected for dilution and for inner filter factor as well as for the fluorescence intensity of the buffer and the nucleic acid (Is), was either 3520
Nucleic Acids Research compared with the fluorescence intensity of the free dye in solution (Id) or expressed in arbitrary units. Concentrated solutions of DAPI were added directly to the cuvette (1. 0 ml, 10 mm pathlength) in 1-20 ,l portions. The molar concentrations of the bound dye, cb, and of the free dye, cf, were calculated from the corrected fluorescence of the solutions (for details see 4, 14); r = cb/cDNA (moles of bound dye per mole of nucleotide). Scatchard plots of these data were compared with theoretical plots computed according to the excluded neighboring site model and the McGhee and von Hippel treatment (15, 16); this method has been successfully applied to analyze the interaction of intercalating ligands with nucleic acids (17, 18). Calculations were carried out on an IMSAI 8080 microcomputer. Fluorescence titrations of polynucleotides (5-10 ,uM nucleotide) with DAPI were carried out at 200 in a standard buffer containing 5 mM HEPES, pH 7. 0, 100 mM Na2SO4 unless otherwise indicated. Sodium sulfate was added because it increases the fluorescence intensity of native calf thymus DNA-DAPI complexes to a greater extent than either sodium citrate, phosphate or chloride (5). Maximum fluorescence enhancement was observed with calf thymus DNA-DAPI complexes at about 100 mM Na2SO4 (Fig. 1); at higher ionic strength fluorescence decreases as a result of increased cation concentration (4, 18). A Scatchard plot of the fluorescence titration of calf thymus DNA with DAPI in the standard buffer shows a large (about 45%) increase in the maximum amount of bound dye compared to the amount of bound dye in 100 mM NaCl (4). The reason for this effect is not clear; it may be due to increased stacking resulting from the effect of SO42 on the structure of water (19, 20). Fluorescence melting curves of DNA-DAPI complexes were carried out with a temperature increase of 1°C/min. Temperature measurements were made with a YSI thermilinear probe inserted into the cuvette. The results were corrected for the temperature dependence of DAPI fluorescence in solution (-1.6%/0C). TN-melting curves were carried out using a Zeiss PM6 spectrophotometer equipped with a thermoelectric control unit. The temperature increase was 1°C/min.
Circular dichroic (CD) spectra were recorded with a Carry 61 spectropolarimeter using 5 mm jacketed thermostated cells. Data are expressed as the degree of ellipticity, 80, uncorrected for the solvent refractive index. RESULTS Double-stranded synthetic polydeoxynucleotides. A typical Scatchard plot of the fluorescence titration of poly d(A-T) poly d(A-T) with DAPI is
3521
Nucleic Acids Research
2.0-
0 N.
'°1.50-I
-3
-2 -1 LOG M Na2 S04
Fig. 1. Effect of the concentration of sodium sulfate on the fluorescence of the DAPI-calf thymus DNA complex. Fluorescence enhancement is expressed as the ratio of observed fluorescence, Is, relative to the initial fluorescence, Io, in a buffer containing 5 mM HEPES, pH 7. 0; 10 mM NaCl. Concentrations were: calf thymus DNA nucleotide, 15.5 ,uM, and DAPI, 2.6 FM. shown in Fig. 2. Analysis of the data of 3 experiments using the McGhee and von Hippel treatment (16) yields a best fit for a binding site size, n (number of nucleotides per molecule of dye) of 6, with an intrinsic binding con+6 -1 x 10 M1 stant, Ki = 3.2 t 0.3 Ten synthetic DNA duplexes were examined by fluorescence titration in the standard buffer and the results plotted as Scatchard plots (cf. Fig. 2). It was found that all six duplexes containing AT, AU and IC base pairs either in a homopolymer pair or in an alternating copolymer form fluorescent complexes with DAPI (Table 1). In contrast, the four duplexes containing GC base pairs in either arrangement, i.e., poly dG.poly dC and poly d(G-C).poly d(G-C), and duplexes containing an AT base pair "sandwiched" between GC pairs, i. e , poly d (A-C). poly d (T-G) and poly d (A-G) * poly d (T-C) exhibit, after mixing with DAPI, less than 2% of fluorescence enhancement observed with poly 3522
Nucleic Acids Research
3
I
2L 0
-
n=8
n=4
n6
0-.2
0.1
r
Fig. 2. Scatchard plot of the fluorescence titration of poly d(A-T) -poly d(A-T) with DAPI fitted according to the McGhee-von Hippel treatment (16). Solid lines represent the function
Ki-
where n is the binding nl] site size (number of nucleotides); Ki, the intrinsic binding constant; r, moles of dye bound per polymer nucleotide and cf, concentration of free dye. Open circles are experimental points obtained from titration. The best fit is for n= 6 and Ki = 3.2 x 106 M-1. Approximate values of Ki are obtained from extrapolation of the experimental curve lim r/cf = Ki L =
C
(inr)n r-
f
r
-+
o
d(A-T) poly d(A-T) under standard conditions. An attempt to form a fluorescent complex with poly d (G-C) poly d(G-C) in 2. 0 M NaClO4 also failed. The experiment was undertaken in view of the report that this polymer undergoes a profound conformational change under these conditions (21). In a control experiment, poly d(A-T) poly d(A-T) exhibited considerable fluorescence enhancement in 2 M NaClO4. In addition to the increase in fluorescence quantum yield, polymers which form fluorescent complexes with DAPI cause a red shift in the excitation spectrum of the dye (Table 1). Certain differences in the individual fluorescence spectra were noted, depending on the polymer used, mainly with respect to the relative intensities of the two excitation maxima (examples of spectra are shown in Fig. 3). These differences are presented quantitatively in Table 1 as the ratio of intensities of the long wavelength band (about 350 nm), IBP to the short wavelength band (about 265 nm), IA. The emission spectra exhibit no polymer-dependent differences and have a maximum at about 450 nm. 3523
Nucleic Acids Research Table 1 Properties of fluorescent complexes forrmed by DAPI with polynucleotides
Polyncleotde ]'olynucleotide
ea sa
x x 10-33 max
~
Free DAPI
Maximum excitation
X,
nm
Band Band A B
K1xO6 x 10 IB/Y-Ki B/A (M 1)
nc
262
344
3.8
-
-
dA*dT
6.0260
269
357
3.6
2.0
6
d(A-T).d(A-T)
6.6262
268
359
1.8
3.2
6
dA dU
6.0 266.
267
358
2.2
2.1
6
d(A-U).d(A-U)
6.0266
268
357
2.5
3.4
6
dI*dC
53245
264
356
3.0
>2d
d(I-C).d(I-C)
6.9251
264
354
3.2
3.8
6
10.0248
259
376
2.4
2.5
200
-
-
-
-
1.6
68
rI
rI, 1. OM NaCl
Volume 6 Number 111979
Nucleic Acids Research
Nucleic Acids Research
Interactions of 4', 6-diamidine-2-phenylindole with synthetic polynucleotides
Jan Kapuscin'ski* and Wlodzimierz Szer
Department of Biochemistry, New York University School of Medicine, 550 First Avenue, New York, NY 10016, USA Received 9 May 1979
ABSTRACT 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.
INTRODUCTION 4', 6-Diamidine-2-phenylindole*2HCL (DAPI), a fluorescent dye originally synthesized by Dann et al. (1), binds to DNA with a marked increase in fluorescent quantum yield. This property of DAPI has been utilized in a number of cytochemical investigations, including flow fluorometry (2, 3 and references in 4), and in the development of a quantitative micro method for the assay of DNA in solution (5). In enzymatic studies it was found that, at low concentrations, DAPI stimulates the template activity of DNA (B. Skoczylas, personal communication), whereas high concentrations of the dye are inhibitory (6). The activity of endonuclease Rl from E. coli is affected by DAPI in an analogous concentration-dependent way (7-9). DAPI, as well as some other cationic fluorescent ligands, e.g., ethidium bromide, exhibit essentially two modes of binding to DNA: i) relatively weak nonfluorescent electrostatic interactions and, ii) strong, presumably C) Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England
3519
Nucleic Acids Research intercalative binding which gives rise to an increase in fluorescence. Weak interactions of DAPI with nucleic acids appear to show little specificity and have been observed with both DNA and RNA (6). Strong binding of DAPI accompanied by a large fluorescence enhancement has only been observed with native DNA (4). There are indications that DAPI binds preferentially to AT-rich DNA but it is not clear whether this specificity is related to the fluorescent or nonfluorescent modes of binding (4, 6). The selectivity of the fluorescent binding with respect to AT base pairs is of particular interest since it may provide a specific probe for the analysis of the micro structure of DNA. In this investigation we have determined the specificity and the stoichiometry of the fluorescent binding mode of DAPI using synthetic polydeoxyand polyribonucleotides, both single- and multi-stranded. The nonfluorescent binding of DAPI was also examined and was found to be nonspecific.
MATERIALS AND METHODS Polynucleotides: Poly dA, poly rA, poly dT, poly rU, poly rC, poly rI and poly rG were from Mile s; poly dC, poly d (G-C) * poly d (G-C) and poly d (A-U). poly d (A-U) were from Grand Island Biochemical s; poly d (A-T) . poly d(A-T), poly dG.poly dC, poly d(I-C).poly d(I-C), poly dU and oligo dAm (m= 2 to 12) were from P-L Biochemicals; poly d(A-C).poly d(T-G) was from Boehringer and poly d(A-G)-poly d(T-C) was a generous gift from Dr. A.R. Morgan, Department of Biochemistry, University of Alberta. DAPI was synthesized according to the modified procedure of Dann et al. (1, 10); physical properties of DAPI were described previously (4). Polynucleotide concentrations were estimated spectrophotometrically either from optical densities of mononucleotides produced by enzymatic hydrolysis of the polymers or according to published molar extinction coefficients (11-13). Fluorescence measurements were recorded with a thermostated AmincoBowman SPF-125S spectrofluorimeter equipped with an IP21 photomultiplier tube and a 150 W Xenon lamp; solutions of DAPI of known concentration were used as standards. Excitation spectra (uncorrected) were recorded from 200 to 420 nm (bandpass 4.5 nm) at 200; the wavelength of the emission was 454 nm (bandpass 9 nm). For fluorimetric titrations, the excitation wavelength was 364 nm; emission was measured at 454 nm with a 9 nm bandpass in both the excitation and emission monochromators. The fluorescence intensity of the sample, corrected for dilution and for inner filter factor as well as for the fluorescence intensity of the buffer and the nucleic acid (Is), was either 3520
Nucleic Acids Research compared with the fluorescence intensity of the free dye in solution (Id) or expressed in arbitrary units. Concentrated solutions of DAPI were added directly to the cuvette (1. 0 ml, 10 mm pathlength) in 1-20 ,l portions. The molar concentrations of the bound dye, cb, and of the free dye, cf, were calculated from the corrected fluorescence of the solutions (for details see 4, 14); r = cb/cDNA (moles of bound dye per mole of nucleotide). Scatchard plots of these data were compared with theoretical plots computed according to the excluded neighboring site model and the McGhee and von Hippel treatment (15, 16); this method has been successfully applied to analyze the interaction of intercalating ligands with nucleic acids (17, 18). Calculations were carried out on an IMSAI 8080 microcomputer. Fluorescence titrations of polynucleotides (5-10 ,uM nucleotide) with DAPI were carried out at 200 in a standard buffer containing 5 mM HEPES, pH 7. 0, 100 mM Na2SO4 unless otherwise indicated. Sodium sulfate was added because it increases the fluorescence intensity of native calf thymus DNA-DAPI complexes to a greater extent than either sodium citrate, phosphate or chloride (5). Maximum fluorescence enhancement was observed with calf thymus DNA-DAPI complexes at about 100 mM Na2SO4 (Fig. 1); at higher ionic strength fluorescence decreases as a result of increased cation concentration (4, 18). A Scatchard plot of the fluorescence titration of calf thymus DNA with DAPI in the standard buffer shows a large (about 45%) increase in the maximum amount of bound dye compared to the amount of bound dye in 100 mM NaCl (4). The reason for this effect is not clear; it may be due to increased stacking resulting from the effect of SO42 on the structure of water (19, 20). Fluorescence melting curves of DNA-DAPI complexes were carried out with a temperature increase of 1°C/min. Temperature measurements were made with a YSI thermilinear probe inserted into the cuvette. The results were corrected for the temperature dependence of DAPI fluorescence in solution (-1.6%/0C). TN-melting curves were carried out using a Zeiss PM6 spectrophotometer equipped with a thermoelectric control unit. The temperature increase was 1°C/min.
Circular dichroic (CD) spectra were recorded with a Carry 61 spectropolarimeter using 5 mm jacketed thermostated cells. Data are expressed as the degree of ellipticity, 80, uncorrected for the solvent refractive index. RESULTS Double-stranded synthetic polydeoxynucleotides. A typical Scatchard plot of the fluorescence titration of poly d(A-T) poly d(A-T) with DAPI is
3521
Nucleic Acids Research
2.0-
0 N.
'°1.50-I
-3
-2 -1 LOG M Na2 S04
Fig. 1. Effect of the concentration of sodium sulfate on the fluorescence of the DAPI-calf thymus DNA complex. Fluorescence enhancement is expressed as the ratio of observed fluorescence, Is, relative to the initial fluorescence, Io, in a buffer containing 5 mM HEPES, pH 7. 0; 10 mM NaCl. Concentrations were: calf thymus DNA nucleotide, 15.5 ,uM, and DAPI, 2.6 FM. shown in Fig. 2. Analysis of the data of 3 experiments using the McGhee and von Hippel treatment (16) yields a best fit for a binding site size, n (number of nucleotides per molecule of dye) of 6, with an intrinsic binding con+6 -1 x 10 M1 stant, Ki = 3.2 t 0.3 Ten synthetic DNA duplexes were examined by fluorescence titration in the standard buffer and the results plotted as Scatchard plots (cf. Fig. 2). It was found that all six duplexes containing AT, AU and IC base pairs either in a homopolymer pair or in an alternating copolymer form fluorescent complexes with DAPI (Table 1). In contrast, the four duplexes containing GC base pairs in either arrangement, i.e., poly dG.poly dC and poly d(G-C).poly d(G-C), and duplexes containing an AT base pair "sandwiched" between GC pairs, i. e , poly d (A-C). poly d (T-G) and poly d (A-G) * poly d (T-C) exhibit, after mixing with DAPI, less than 2% of fluorescence enhancement observed with poly 3522
Nucleic Acids Research
3
I
2L 0
-
n=8
n=4
n6
0-.2
0.1
r
Fig. 2. Scatchard plot of the fluorescence titration of poly d(A-T) -poly d(A-T) with DAPI fitted according to the McGhee-von Hippel treatment (16). Solid lines represent the function
Ki-
where n is the binding nl] site size (number of nucleotides); Ki, the intrinsic binding constant; r, moles of dye bound per polymer nucleotide and cf, concentration of free dye. Open circles are experimental points obtained from titration. The best fit is for n= 6 and Ki = 3.2 x 106 M-1. Approximate values of Ki are obtained from extrapolation of the experimental curve lim r/cf = Ki L =
C
(inr)n r-
f
r
-+
o
d(A-T) poly d(A-T) under standard conditions. An attempt to form a fluorescent complex with poly d (G-C) poly d(G-C) in 2. 0 M NaClO4 also failed. The experiment was undertaken in view of the report that this polymer undergoes a profound conformational change under these conditions (21). In a control experiment, poly d(A-T) poly d(A-T) exhibited considerable fluorescence enhancement in 2 M NaClO4. In addition to the increase in fluorescence quantum yield, polymers which form fluorescent complexes with DAPI cause a red shift in the excitation spectrum of the dye (Table 1). Certain differences in the individual fluorescence spectra were noted, depending on the polymer used, mainly with respect to the relative intensities of the two excitation maxima (examples of spectra are shown in Fig. 3). These differences are presented quantitatively in Table 1 as the ratio of intensities of the long wavelength band (about 350 nm), IBP to the short wavelength band (about 265 nm), IA. The emission spectra exhibit no polymer-dependent differences and have a maximum at about 450 nm. 3523
Nucleic Acids Research Table 1 Properties of fluorescent complexes forrmed by DAPI with polynucleotides
Polyncleotde ]'olynucleotide
ea sa
x x 10-33 max
~
Free DAPI
Maximum excitation
X,
nm
Band Band A B
K1xO6 x 10 IB/Y-Ki B/A (M 1)
nc
262
344
3.8
-
-
dA*dT
6.0260
269
357
3.6
2.0
6
d(A-T).d(A-T)
6.6262
268
359
1.8
3.2
6
dA dU
6.0 266.
267
358
2.2
2.1
6
d(A-U).d(A-U)
6.0266
268
357
2.5
3.4
6
dI*dC
53245
264
356
3.0
>2d
d(I-C).d(I-C)
6.9251
264
354
3.2
3.8
6
10.0248
259
376
2.4
2.5
200
-
-
-
-
1.6
68
rI
rI, 1. OM NaCl
