Studies of the interaction between poly(diallyldimethyl ammonium chloride) and DNA by spectroscopic methods

Colloids and Surfaces A: Physicochem. Eng. Aspects 233 (2004) 129–135

Studies of the interaction between poly(diallyldimethyl ammonium chloride) and DNA by spectroscopic methods Yinglin Zhou, Yuanzong Li∗ The Key Laboratory of Bioorganic Chemistry and Molecular Engineering, Department of Chemical Biology, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China Received 19 May 2003; accepted 18 November 2003

Abstract The interaction of poly(diallyldimethyl ammonium chloride) (PDDA) with DNA has been studied by spectroscopic analysis of different kinds of nucleic acid probes, microscopic IR- and CD-spectroscopy. The two spectroscopic changes of ethidium bromide (EB) on its binding to DNA, namely red-shift of its maximum absorption wavelength (νmax ) and enhancement of fluorescence intensity are utilized to study the interaction of PDDA with DNA. Reversion of νmax and decrease in fluorescence of DNA–EB system on addition of PDDA show the dissociation of DNA/EB complex and the formation of the DNA/PDDA complex. At the same time, the binding constant of EB and the number of binding sites per nucleotide decrease with increase in the concentrations of PDDA, indicating non-competitive inhibition of EB binding to DNA in the presence of PDDA. From the Scatchard analysis, we obtain the association constant of PDDA to DNA is 4.7 × 104 M−1 . Contrary to EB, the binding of PDDA with DNA can greatly enhance the fluorescence intensity of groove-binding probes, Hoechst 33258 and DAPI, which can also be used to investigate the formation of the DNA/PDDA complex. The fluorescence analysis shows that the interaction of DNA with PDDA may have effect on the conformation of secondary structure of DNA. IR-spectra show that PDDA interacts with DNA through both the phosphate groups and the bases of DNA and the formation of DNA/PDDA complex causes the change of the conformation of the DNA secondary structure, which is further proved by CD-spectra. © 2003 Elsevier B.V. All rights reserved. Keywords: Poly(diallyldimethyl ammonium chloride); DNA; Interaction; Conformation; Spectroscopic methods

1. Introduction The DNA/polycation complexes are considered currently as very promising systems in genetic engineering and gene therapy for functional gene delivery into eukaryotic cells, since these complexes have the ability to facilitate the transfer of nucleic acids through biological membranes [1,2]. High density of negatively charged phosphate groups of the double helix provides the ability of DNA to form rather stable complexes with synthetic polycations [3–5]. Complexes of DNA with poly(N-ethyl-4-vinylpyridinium) cation (PEVP) [6], poly-l-lysine [7,8], or other poly- and oligocations [9] were successfully used for increasing the efficiency of transformation of cells by the plasmids and for the protection of DNA from splitting by cell nucleases [10,11]. Although their transfection efficiency remains relatively low ∗ Corresponding author. Tel.: +86-10-6275-7954; fax: +86-10-6275-1708. E-mail address: [email protected] (Y. Li).

0927-7757/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfa.2003.11.030

up to now in comparison with viral based vectors [12], they are generally considered to be safer and less immunogenic [13–15]. Therefore, the study of the interaction of such polycations with the genomic species DNA is much needed. Poly(diallyldimethyl ammonium chloride) (PDDA) is a linear cationic polyelectrolyte. The structure of PDDA is shown in Fig. 1. It is usually used as positively charged colloid to form multilayer assemblies with negatively charged colloid through layer-by-layer technique [16,17]. Dong coworkers [18] investigated the interaction of DNA with methyl green through the layer-by-layer deposited multilayer films fabricated by DNA and PDDA. Schindler and Nordmeier [19,20] has examined the stability of polyelectrolyte complexes of calf thymus DNA and some polycations including PDDA. In this study, we have examined in detail the interaction of PDDA with DNA using UV-, IR-, CD-, and fluorescence spectroscopy. The formation of the complex between DNA and PDDA has been examined by the reversion of its maximum absorption wavelength (νmax ) of the absorption spectra and loss of fluorescence intensity

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Y. Zhou, Y. Li / Colloids and Surfaces A: Physicochem. Eng. Aspects 233 (2004) 129–135

Fig. 1. The structures of PDDA, EB, Hoechst 33258, and DAPI.

in ethidium bromide (EB)–DNA system, and the binding constant of PDDA to DNA has been studied. Hoechst 33258 and DAPI were also chosen to investigate the influence of PDDA on the interaction between DNA with them. The structures of EB, Hoechst 33258 and DAPI are also shown in Fig. 1. At the same time, the effect of PDDA on the conformational state of DNA is examined through several spectroscopic methods, which is helpful to clarify the mechanism of the effects of PDDA on the structure and physical properties of DNA and the reasons for the fluorescence changes of different fluorescent probes caused by PDDA.

2. Experimental 2.1. Chemicals Stock solution of DNA was prepared at 0–4 ◦ C by dissolving commercially purchased herring sperm DNA (hsDNA, Beijing Jingke Reagent Company, Beijing, China) in doubly deionized water. The concentration of DNA solution was determined from UV absorption at 260 nm using a molar extinction coefficient, 6600 M−1 cm−1 . A stock solution of PDDA was prepared by dissolving the solution of PDDA (40 wt.% aqueous solution, average M.W.: 5000–20,000, Aldrich, USA) in doubly deionized water. Its working solution was 7.8 × 10−3 M. If there is no specific explanation, the concentration of PDDA means that of amine group. Fluorescent dyes, EB (Beijing Huamei Reagent Company, Beijing, China), Hoechst 33258 (Aldrich), and 4 ,6-diamidino-2-phenylindole·2HCl (DAPI, Aldrich) were used as received.

Tris–HCl buffer solution was used to control the acidity of the reaction systems. All reagents were of analytical reagent grade without further purification, and doubly deionized water was used throughout. 2.2. Methods The effect of PDDA on UV-Vis spectra of DNA/EB complex were obtained with a Hewlett Packard 8453 spectrophotometer (Agilent) by adding a certain volume of PDDA solution step by step into the solution of DNA/EB complex. Fluorescence spectra and intensity of the solutions were measured using F-4500 Fluorescence Spectrophotometer (HITACHI). A certain volume of PDDA solution (total concentration of amine groups was 0.001 M) was added into the solution of the DNA/EB complex. The fluorescence intensity was measured at 595 nm with excitation wavelength at 535 nm. Then we observed the changes of the fluorescence intensity. In the similar methods, we examined the influences of PDDA on the fluorescence intensity of Hoechst 33258–DNA and DAPI–DNA systems. The fluorescence intensity was measured at 460 nm with excitation wavelength at 350 nm for Hoechst 33258 while for DAPI, the emission wavelength is 450 nm and excitation wavelength 340 nm. Binding studies of PDDA to hsDNA was studied by fluorescence Scatchard analysis as described in the literature [21,22]. Microscopic FTIR-spectra of DNA complex with PDDA, as well as the spectra of DNA and PDDA were taken on Nicolet MAGNA-IR 750 (Nicolet) at room temperature. Solid hsDNA was used directly to obtain microscopic FTIR spectrum of DNA, while the samples of PDDA and DNA/PDDA complex were prepared as thin films by

Y. Zhou, Y. Li / Colloids and Surfaces A: Physicochem. Eng. Aspects 233 (2004) 129–135

100

Relative fluorescence intensity

volatilizing the appropriate concentrated solutions on glass slides. Then we obtained the micro solid powder of these samples from these films for the microscopic FTIR analysis. CD-spectra of native DNA, thermally denatured DNA or DNA/PDDA complexes in 10 mM Tris–HCl buffer (pH 7.3) were recorded with a 1 cm path length cuvette using JOBIN YVON-SPEX CD 6 (France Groupe Instruments S.A.) at ambient temperature and subtracted from the spectrum of buffer alone. Each measurement was the average of two repeated scans and the measured CD signals were converted to molar absorbance differences.

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Fig. 3. Effect of added PDDA on the fluorescence intensity of DNA–EB containing 10.0 ␮M EB and 40.0 ␮M DNA in 0.01 M Tris–HCl, pH 7.3.

3.1. The effect of PDDA on the binding of EB to DNA EB, a phenanthridine fluorescence dye forms soluble complexes with nucleic acids. The interaction of the dye with nucleic acids is associated with a red-shift of νmax and enhancement of its fluorescence intensity. It has been confirmed that EB binds to DNA by intercalation [23,24]. The spectroscopic changes of EB on its binding to DNA are often utilized to study the interaction between DNA and other substances, such as metal complexes [25,26] and cationic polymers [27,28]. Spectrum (a) of Fig. 2 shows the absorption spectrum of 30.0 ␮M EB at pH 7.3 Tris–HCl buffer. EB shows a single monomeric peak at 480 nm indicating the weak aggregating tendency of the dye [29]. On adding DNA to a polymer/dye ratio of 4.0, spectrum (a) changes to (b) with the absorption peak red-shifted from 480 to 500 nm and the decrease of the absorbance. When PDDA is step by step added into the solution, spectrum (b) changes from (c) to (e) with progressive reversion of the νmax to shorter wavelength and the increase of the absorbance. This gradual reversion of the

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Fig. 2. Absorption spectra of 30.0 ␮M EB in 0.01 M pH 7.3 Tris–HCl buffer (a); in the presence of 120.0 ␮M hsDNA (b); (c)–(g) are of the solution b in the presence of increasing PDDA 30, 45, 60, 90, and 120 ␮M, respectively.

νmax indicates that the interaction between DNA and PDDA probably releases EB from its DNA complex. However, the complete reversion of the absorption spectra at high PDDA concentration could not be confidently probed due to the appearance of the turbidity of the solution caused by the formation of the hydrophobic complex between DNA and PDDA, which is apparent from spectra (f) and (g) in Fig. 2. The curve in Fig. 3 shows the change in fluorescence of 40.0 ␮M DNA and 10.0 ␮M EB with increasing concentration of PDDA. As we can see, the curve is sigmoid with pronounced decrease of the fluorescence intensity within the concentration range of PDDA from 32 to 52 ␮M, which corresponds to the N/P ratio (molar ratio of polymer amino groups to DNA phosphates) from 0.8 to 1.3. The fluorescence intensity changes from 85 to 50% of its initial fluorescence within this range. Further increase of the concentration of PDDA up to 120 ␮M changes the fluorescence intensity sluggishly. This significant quenching of the fluorescence presumably indicates that the addition of PDDA is not in favor of the interaction between DNA and EB. We suggest that this may be due to the DNA conformational change (see Section 4) caused by the cooperative interaction of DNA with PDDA and charge screening which decreases binding strength of EB to DNA. The effect of PDDA on the fluorescence intensity of EB in the presence of DNA has also been followed by adding increasing amounts of EB to a preformed DNA/PDDA complex containing 30.0 ␮M DNA and 100.0 ␮M PDDA. The result is represented in Fig. 4c and the fluorescence of an aqueous solution of EB itself of comparable concentration and that in the presence of 30.0 ␮M DNA alone are also shown in Fig. 4a and b. As we can see, the fluorescence in (c) is higher than that in (a) but is lower than (b), indicating that EB can still bind to DNA in the presence of a large amount of PDDA and enhance its fluorescence intensity, but the fluorescence quantum yield is much lower than that in the presence of DNA alone. This may also indicate that the presence of PDDA changes the conformation of DNA to a certain extent and influences the efficient binding of EB with

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Y. Zhou, Y. Li / Colloids and Surfaces A: Physicochem. Eng. Aspects 233 (2004) 129–135 Table 1 The binding of EB to herring sperm DNA in the presence of increasing concentration of PDDA (pH 7.3 Tris–HCl buffer, ionic strength 0.01)

60 b

Fluorescence intensity

50

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Ka × 10−5

nb

0.00 0.33 0.67 2.50 5.00 6.67

10.08 9.96 9.80 1.79 1.49 1.05

0.076 0.056 0.044 0.031 0.030 0.024

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Fig. 4. The fluorescence intensity of EB alone vs. its concentration in 0.01 M Tris–HCl, pH 7.3 (a); in the presence of 30.0 ␮M DNA (b); in the presence of both 30. 0 ␮M DNA and 100 ␮M PDDA (c).

DNA and the ability to enhance the fluorescence quantum yield. Fluorescence Scatchard plots for the binding of EB to DNA in the presence of varying concentrations of PDDA are obtained according to Eq. (1) [30,31]. Eq. (1) expresses the binding of EB to DNA in the presence of PDDA (P). 
rEB KEB = (n − rEB ) (1) cEB 1 + K P cP Here rEB is the ratio of bound EB to total nucleotide concentration; cEB , concentration of free EB; n, maximum value of rEB , KEB , and KP , intrinsic binding constant for EB and PDDA to DNA, respectively, and cP , concentration of free PDDA polymer. Using fluorescence to determine rEB , binding isotherms are determined in the presence of PDDA and the corresponding Scatchard plots constructed. As shown in Fig. 5 and Table 1, with the addition of PDDA, both the slope that is K and the intercept on the abscissa that is n (number of binding sites per nucleotide) decrease with increase in the concentrations of PDDA, indicating non-competitive inhibition of EB binding [32] in the presence of PDDA.

From the Eq. (1), we can obtain the relationship shown below KEB Kobs = (2) 1 + K P cP The reciprocal of Eq. (2) produces a straight-line relationship   1 KP 1 cP + = (3) Kobs KEB KEB The concentration of free PDDA has been approximated by the total PDDA concentration at the start of each titration when the concentration of PDDA is much larger than that of DNA. The data obtained from Table 1 is plotted according to Eq. (3) and the linear-regression equation is 1/Kobs = 0.042cP + 9.00 × 10−7 (r = 0.996, n = 3). The binding constant of PDDA to DNA is estimated to be 4.7×104 M−1 . 3.2. The effect of PDDA on the interaction between groove-binding probes and DNA Hoechst 33258 binds to DNA in minor groove of A–T base pair rich area, causing the increase of its own fluorescence [33,34]. Fig. 6 shows, at pH 7.3 in Tris–HCl buffered solution, the addition of PDDA greatly enhances 300 5

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Fig. 5. Fluorescence Scatchard plots for the binding of EB to DNA in the absence (a) and presence (b–e) of increasing concentration of PDDA 10, 20, 75, and 150 ␮M in 0.01 M Tris–HCl, pH 7.3.

Fig. 6. Excitation (a) and emission (b) spectra of 1.5 ␮M Hoechst 33258 in 0.01 M Tris–HCl, pH 7.3. (1) in the absence of DNA and PDDA; (2) in the presence of 40 ␮M DNA; (3)–(5) are of the solution 2 in the presence of increasing PDDA 16, 32, and 40 ␮M, respectively.

Y. Zhou, Y. Li / Colloids and Surfaces A: Physicochem. Eng. Aspects 233 (2004) 129–135 28 26 fluorescence intensity

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Fig. 8. The effect of PDDA on the fluorescence intensity of DAPI–DNA system containing 1.0 ␮g ml−1 DAPI and 80 ␮M DNA in 0.01 M Tris–HCl, pH 7.3.

formation of the complex between DNA and other cationic polymers. Another groove-binding probe, DAPI has the similar phenomenon to that of Hoechst 33258 when it interacts with DNA in the presence of PDDA. Fig. 8 shows the effect of PDDA on the fluorescence intensity of DAPI–DNA system. As we can see, the fluorescence intensity of the system increases with the addition of PDDA when the concentrations of DAPI and DNA remain unchanged and reaches the maximum when the N/P ratio is 1.0, meaning the formation of 1:1 DNA/PDDA complex is also in favor of the increase of the fluorescence quantum yield of DAPI. 3.3. Microscopic IR-spectra Fig. 9 demonstrates microscopic FTIR-spectra of DNA and DNA complexes with PDDA. The main absorption bands of DNA are at: 1063 cm−1 , corresponding the symmetric stretching vibration of the phosphate groups;

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Fig. 7. The effect of N/P ratio on the fluorescence intensity of Hoechst 33258–DNA system in three different concentrations of DNA in 0.01 M Tris–HCl, pH 7.3. Concentrations: Hoechst 33258, 1.5 ␮M; DNA, (␮M) (a) 20; (b) 40; (c) 80.

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the fluorescence intensity of Hoechst 33258–DNA system. Meanwhile, the emission maximum wavelength of Hoechst 33258 blue-shifts from 490 to 460 nm, but the excitation wavelength changes little. Clearly, the negatively charged phosphates of DNA can be neutralized by the positively charged PDDA, leading to the formation of the hydrophobic complex, which may place Hoechst 33258 in a more hydrophobic environment, causing the blue-shift of the emission maximum. At the same time, the formation of DNA/PDDA complex may change the conformation of DNA, which makes its interaction with Hoechst 33258 more efficiently. The PDDA bound to DNA may also serve as an energy protection shell for Hoechst 33258 in the groove of DNA where it is protected against quenching by aqueous solvent. We have also found that in the presence of Hoechst 33258 and PDDA, the fluorescence intensity of thermally denatured DNA is much smaller than that of native DNA, meaning the interaction between DNA and PDDA does not make DNA denatured. Fig. 7 shows the effect of the N/P ratio on the fluorescence intensity of Hoechst 33258–DNA system in three different DNA concentrations. It can be seen when the concentrations of DNA remain 2.0×10−5 , 4.0×10−5 , and 8.0×10−5 M separately, the fluorescence intensities of Hoechst 33258–DNA systems are enhanced greatly with the increase of the concentration of PDDA and reach the maximum when the N/P ratios are equal or near to 1.0, which indicates that near 1:1 complex is formed between DNA and PDDA. The fluorescence intensities decrease a little with the further increase of the concentration of PDDA. Since the excessive PDDA corresponding to DNA may cause the precipitation of DNA/PDDA complex in the solution, which is not in favor of the interaction between DNA and Hoechst 33258, thus decreasing the fluorescence intensity to a certain extent. As we can see, the change of fluorescence intensity from Hoechst 33258 is an indication of the formation between DNA and PDDA, which may be developed to examine the

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Fig. 9. Microscopic FTIR spectra of DNA and a mixture of DNA and PDDA.

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1222 cm−1 , the antisymmetric stretching vibration of the phosphate group; 1017 cm−1 , the vibration of C=N of ribose; 1693 cm−1 , the vibrations of C6 =O of guanine and C4 =O of thymine [35,36]. The wavenumbers of the absorption bands of DNA in our experiment have some deviation to that of DNA in solution using special CaF2 cuvette [37]. This is easy to explain since DNA in the latter can interact with water, CaF2 and other substrates in solution, which may shift the absorption bands. PDDA itself has no absorption in the above absorption bands. As we can see, PDDA induces the dramatic changes in the DNA spectrum. Changes are apparent in the shift of the characteristic absorption bands of the phosphate groups and bases (the absorption band at 1063, 1222, 1693 cm−1 shift to 1091, 1236, 1658 cm−1 , separately), which is evidence that PDDA interacts with both the phosphate groups and nitrogenous bases of DNA [37]. Moreover, the interaction of PDDA with DNA alters the B-conformation of DNA, which is seen from the disappearance of the B-form marker bands at 1017 cm−1 .

to the helicity of B-DNA [38]. The CD-spectra of thermally denatured DNA have the positive band at 276 nm similar to that of native DNA, but have no negative band. In the presence of 5.0 × 10−5 M PDDA both the positive peak and the negative peak of the CD spectra of DNA shifted to higher wavelengths (to 285 and 254 nm, respectively), accompanying the decrease of the peaks, which indicates a transition of the secondary DNA structure from a B- to a C-form [39,40]. Further increase of the concentration of PDDA induces further red-shift and decrease in the peaks of the CD spectra of DNA, but the CD peaks become unapparent, which may be due to the condensation of a large number of hydrophobic complex. As we can see, the spectra of DNA/PDDA complex are different from that of denatured DNA, meaning the interaction between DNA and PDDA does not cause the denaturizing of DNA, which is consistent with the results found in fluorescence experiments.

4. Discussion 3.4. Circular dichroism The circular dichroism of nucleic acid results from the interaction of the transition dipoles in the component of the helical structure, so circular dichroism of the perturbed DNA is one of the choicest methods to monitor the conformational changes brought about by the interacting host molecules. The effect of PDDA on the conformation of secondary structure of DNA were studied by keeping the concentration of hsDNA at 8.0 × 10−5 M while varying the concentration of PDDA from 5.0 × 10−5 to 9.4 × 10−5 M in a buffer solution of 10 mM of Tris–HCl. The spectrum of the hsDNA and those with the additives were monitored from 220 to 320 nm. As shown in Fig. 10, the UV-CD spectrum of DNA exhibits a positive absorption band at 276 nm due to the base stacking and a negative band at 248 nm due

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Wavelength (nm) Fig. 10. Effect of PDDA on the CD-spectra of DNA in 0.01 M Tris–HCl, pH 7.3; (a) 80 ␮M DNA, (b) 80 ␮M DNA + 50 ␮M PDDA, (c) 80 ␮M DNA + 94 ␮M PDDA, (d) thermally denatured DNA, 80 ␮M.

From the above fluorescence analysis we can see clearly that the formation of the complex between DNA and PDDA is not in favor of the interaction between DNA and intercalator, but is in favor of the interaction between DNA and groove-binding probes. These suggest that the binding of PDDA on DNA may have specific influence on the secondary structure of DNA. IR spectra prove that the interaction between DNA and PDDA alters the conformation of B-form DNA. CD-spectra further prove that the formation of the complex between DNA and PDDA changes the conformation of second structure of DNA from B- to C-form. C-form DNA was first observed at low relative humidity and in the absence of excess salt for LiDNA [41], and later for MgDNA [42], and NaDNA [43]. Compared with B-DNA (right-handed, one turn per 10 residues, rise per bp is 3.3–3.4 Å), C-DNA consists of a non-integral helix with about 9.33 residues per turn in rise per bp 3.31 Å [44]. Maybe it is the minor change of the conformation of DNA that is not in favor of the stacking interaction between DNA base pair and planar EB molecule, which lead to the decrease of both the binding constant of EB to DNA and the fluorescence of EB in the presence of PDDA. Since minor groove structure is also changed when DNA changes from B- to C-form, the interaction between groove-binding dyes and DNA can be changed. According to references [44], the width and depth of minor groove in B-DNA are 5.7 and 7.5 nm. But for C-DNA, they are 4.8 and 7.9 nm, respectively. Compared to B-DNA, the minor groove of C-DNA is narrower and deeper, which may be in favor of the interaction between groove-binding probes and DNA. This will increase the binding strength of the probes and also protect Hoechst 33258 and DAPI against quenching by the other substance in the solution, leading to the enhanced fluorescence intensity of the groove-binding probes.

Y. Zhou, Y. Li / Colloids and Surfaces A: Physicochem. Eng. Aspects 233 (2004) 129–135

IR experiment proves that PDDA interacts with both the positively charged phosphate groups and nitrogenous bases of DNA. Unlike simple cation, PDDA is cationic polyelectrolyte, so it has multiple binding sites in one molecule with DNA, which leads to cooperative static interaction between PDDA and DNA and the effect on the conformation of DNA structure. From the Scatchard analysis, the binding constant of PDDA to DNA is 4.7 × 104 M−1 , which is much higher than that expected for a simple cation. For example, a binding constant of Na+ is 80 M−1 [45], Mg2+ is 103 M−1 [45], and Mn2+ 2000 M−1 [46]. All of these suggest a synergetic effect among the monomers when they are in a same polymer chain.

5. Conclusion From our experiments, it can be concluded that the complex between DNA and PDDA is easily obtained when DNA is mixed with PDDA. PDDA interacts with both the positively charged phosphate groups and nitrogenous bases of DNA and this interaction leads to the conformation change of DNA from B- to C-form. We think that the change of the conformation of DNA mediates the binding strength of both intercalator EB molecule and groove-binding dyes with DNA. At the same time, from the Scatchard analysis of EB in the presence of DNA and PDDA, we can obtain the association constant of PDDA to DNA, which is helpful to understand the physicochemical properties of the DNA/PDDA complex.

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