DNA conformational equilibrium in the presence of Zn2+ ions in neutral and alkaline solutions

International Journal of Biological Macromolecules 50 (2012) 854–860

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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac

DNA conformational equilibrium in the presence of Zn2+ ions in neutral and alkaline solutions V.A. Sorokin a , V.A. Valeev a , E.L. Usenko a , V.V. Andrushchenko b,∗ a b

B.I. Verkin Institute for Low Temperature Physics and Engineering, National Academy of Sciences of Ukraine, 47 Lenin Avenue, Kharkov 61103, Ukraine Institute of Organic Chemistry and Biochemistry, Academy of Sciences, Flemingovo nám. 2, 16610, Prague, Czech Republic

a r t i c l e

i n f o

Article history: Received 6 October 2011 Received in revised form 10 November 2011 Accepted 12 November 2011 Available online 22 November 2011 Keywords: DNA metallization Alkaline solution DNA–metal complex DNA helix–coil transition Differential UV spectroscopy Thermal denaturation

a b s t r a c t Effect of Zn2+ ions on DNA transition from B-form to a metallized form (m-DNA) in Tris and tetraborate buffers at pH 8.5 has been studied by visible and differential UV-spectroscopy and by thermal denaturation. The results have been compared to those obtained at pH 6.5 in cacodylate buffer. It was found that in alkaline solutions Zn2+ ions induced a hypochromicity of the DNA absorption in the whole spectral range monitored, which was attributed to DNA transition from B- to the m-form. Complete metallization occurred only upon heating the DNA solutions containing more than ∼2 × 10−4 M of Zn2+ ions. Phase diagrams of the DNA–zinc complexes at pH 6.5 and 8.5 have been obtained for the first time. The m-DNA form showed higher thermal stability compared to B-DNA. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Lee and co-authors [1] have found in 1993 that natural and synthetic DNA with various nucleotide content can transit to a new metallized form (m-DNA) in alkaline solutions in the presence of ZnCl2 , NiCl2 and CoCl2 . Such a metalized state of DNA exhibits a number of unusual and peculiar properties. For example, a fast electron transfer along the macromolecule, characteristic for a metallic conductivity, was observed [2–4]. Later, the metallic conductivity was discovered in DNA fibers with length of 15 ␮m [5]. The results of these findings, combined with the ability of natural and synthetic polynucleotides to self-assemble due to complementarity, suggest a possibility of utilizing metal complexes of multi-stranded polynucleotides in nanoelectronic devices and biosensors [1,3,6–9]. Such a prospect initiated extensive experimental and theoretical studies of various properties of metallized polynucleotides, including thermodynamics of B-, A- ↔ m transitions [1–10]. While the technological outlook of this research is fairly clear, the question about a biological functionality of DNA in m-form is still open. It was suggested that m-DNA could be formed in GC blocks in DNA of higher organisms at near-physiological pH

∗ Corresponding author. Tel.: +420 220183445; fax: +420 220183578. E-mail addresses: [email protected] (V.A. Sorokin), [email protected] (V.V. Andrushchenko). 0141-8130/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2011.11.011

(pH 7.8); considering that sperm of such species has even higher pH of 8.2 and usually contains relatively high concentrations of zinc, such a possibility is even more feasible [4]. One of the essential parameters affecting the B → m transition is a solution temperature. According to the existing models, mconformation of DNA is formed when imino protons of the AT and GC basepairs are replaced by divalent metal ions, which may happed upon local opening of the double helix at elevated temperatures [1,2,9]. It was shown by the ethidium bromide fluorescence quenching experiments that increasing the temperature allows to decrease the Zn2+ concentration required for m-DNA formation [1,4,10]. According to the fluctuation model of m-DNA formation [1,2], this is an anticipated result. However, transition of DNA to m-form has been studied up to date only in a narrow temperature interval, ranging from 0 ◦ C to 37 ◦ C [1,10]. The aim of the present work was to extend this temperature interval and to obtain a complete phase diagram of DNA + Zn2+ complex. To achieve this, a dependence of the conformational transition temperature on the zinc concentration must be obtained for the whole temperature range, in which the DNA double helix is stable. Another task is to experimentally decompose the zinc-induced changes of the DNA helix–coil transition temperature (Tm ) into components according to the types of DNA–metal ion interactions [11], namely coordination with N7 sites of purines or formation of metallic intramolecular bridges in AT and GC pairs [1]. The latter can be done by obtaining the concentration relationships of Tm at different experimental conditions.

V.A. Sorokin et al. / International Journal of Biological Macromolecules 50 (2012) 854–860

2. Materials and methods Sodium salt of salmon sperm DNA with molecular weight (4–6) × 106 Da and GC content of 41% as well as ethidium bromide, sodium cacodylate Na[CH3 ]2 AsO2 ·3H2 O (pH 6.5) and Tris C4 H11 O3 N (pH 8.5) buffers were purchased from Serva, Germany. Salts of ZnCl2 ·6H2 O and NaCl (chemical grade) were obtained from Reachim, Russia; sodium tetraborate buffer Na2 B4 O7 ·10H2 O (pH 8.5) was from Reanal, Hungary (Table 1). To avoid formation of hydroxides at pH 8.5 (including insoluble ones) [12], the ZnCl2 stock solution (0.03 M) was prepared in a triple-distilled water (pH 6.7). The concentration of ZnCl2 ([Zn2+ ]) was determined by weighting and controlled by the trilonometric titration. The difference in the zinc ion concentrations determined by both methods did not exceed 2%. The required concentration of ZnCl2 in the cuvette was reached by adding an appropriate amount of the zinc chloride stock solution to the buffered DNA. The pH of the DNA solutions was 8.5 and did not change by more than 0.03 pH units upon zinc chloride addition up to 6 × 10−4 M Zn2+ . The DNA phosphorous concentration (P) was (2.0 ± 0.1) × 10−5 M as determined by the molar extinction coefficient (εm = 6600 M−1 cm−1 ) at m = 38,500 cm−1 (m = 260 nm) [11]. The pH of the solutions was determined by a pH-meter pH-340 (Russia) with an error of ± 0.03 pH units.

855

Table 1 DNA helix–coil transition temperature (Tm ) and heat-induced maximum hyperchromicity of DNA absorption (hm )0 (m = 38,500 cm−1 (m = 260 nm)) in the absence of Mt2+ ions. Buffera

pHTo

Tm , ◦ C b

(hm )0

Cacodylate Tris Tetraborate

6.5 8.5 8.5 9.0

67.8–68.8 d 69.7 70.7 71.2

0.43 d 0.39 0.39 0.41

c

a Concentration of cacodylate and Tris buffers was 10−2 M, concentration of tetraborate buffer was 5 × 10−3 M. b Average value for all buffers (Tm )aver = 69.6 ◦ C. c Average value for all buffers (hm )0 aver = 0.41. d Variations of Tm and (hm )0 for different pH values are within the determination error for these parameters (1.5 ◦ C and 0.02, respectively).

by a computer connected to the spectrophotometer. The h(T) relationship describing the DNA melting was computed as follows:



h(T ) =

[εTo + ε(T )] εm



,

(2)

m

where ε(T) is the change in DNA absorption upon heating. 3. Results and discussion

2.1. Differential UV spectroscopy

3.1. Differential UV and visible spectroscopy

Differential UV (DUV) spectra A() were measured at room temperature To = 25 ± 2 ◦ C with a UV–vis spectrophotometer (Carl Zeiss, Jena, Germany) using the four-cuvette scheme [11,13]. The spectra were normalized to DNA concentration as follows:

In DNA B-form, N7 atoms of purine bases are the most sterically accessible from all the nitrogen base atoms; they also have the maximum molecular electrostatic potential [[11] and references therein, [14,15]]. Interaction of different cations, including d-ions of Mt2+ (these are the ions with d-electrons in outer orbitals) with N7 atoms of purines has been reliably documented by various techniques [14,16]. The cation-N7 interaction induces changes in energy of the nitrogen base electronic transitions. Such changes result in a shift of bases’ absorption bands and give rise to the differential spectra. It should be mentioned that the shape of such spectra is mainly determined not by electronic structure of the cations but rather by the nature of nitrogen base atoms binding these cations [11,13,17,18]. At the concentrations not exceeding ∼10−4 M in Tris buffer (Fig. 1b) and 3 × 10−5 M in tetraborate (not shown), Zn2+ ions induce differential spectra similar to those observed in the presence of Ni2+ ions (Fig. 1b). In both cases, the spectra exhibit extrema at the positions corresponding to those observed in DUV spectra of Zn2+ complexes with N7 atoms of AMP and GMP (Fig. 1a). This suggests a presence of an inner-sphere interaction of the ions with N7 of purine bases also in a double-stranded DNA. Although such an interaction is usually not considered in theoretical models describing the formation of m-DNA [9], it may play a role of an additional “trigger” for initiating the m-DNA formation. Indeed, it is believed that the only necessary condition for DNA metallization is an elevated pH, which should be around pKa of deprotonation of guanine N1 and thymine N3 iminoprotons in GC and AT pairs, respectively [1]. However, pKa values for these nucleotide atoms (9.9–10 for dTMP and 9.5–9.7 for dGMP [19]) are substantially higher than pHm (8.4–8.5) (pHm is a pH value at which 50% of DNA base pairs adopt m-form) [4]. A possible reason for such a difference could be that Mt2+ ions coordinating to N7 of purines lower the ionization constant of N1. It was shown that Zn2+ and Cu2+ ion binding to N7 of Ado, Ino and Guo nucleosides lowers pKa of N1 atom by 0.7–1.1 units [20]. As a result, the observed pHm are close to pKa values of purine deprotonation. As can be seen from Fig. 1c, higher zinc concentrations induce hypochromicity of the whole absorption spectrum of DNA. In this case the most intensive minimum in the differential spectra 5 and

εTo () =

ATo () P

(1)

At the high zinc concentrations ([Zn2+ ] ≥ 2 × 10−4 M) the DUV spectra reached the equilibrium within 30 min after the addition of the zinc solution to the DNA. Further incubation for 150 min resulted in the intensity changes (εTo ) not exceeding 100 M−1 cm−1 . The DUV spectra and εTo values reported here for different concentrations of ZnCl2 correspond to the incubation time of 180 min at T = To . It should be noted that the optical density of the DNA solutions in the long-wavelength region of the spectrum ( < 30,000 cm−1 or  > 333 nm) did not change even at T = 85 ◦ C in the whole zinc concentration range studied ([Zn2+ ] ≤ 3 × 10−4 M). This signifies that no insoluble Zn(OH)2 hydroxides capable of light scattering were formed, meaning that Zn2+ ion concentration was close to the concentration of ZnCl2 . 2.2. Visible spectroscopy Absorption spectra of ethidium bromide (EB) in solutions containing DNA and ZnCl2 were measured with M40 spectrophotometer (Carl Zeiss, Jena, Germany). 2.3. Thermal denaturation DNA melting curves were measured at  = m = 38,500 cm−1 (m = 260 nm) with the same spectrophotometer as was used for recording DUV spectra but employing the double-cuvette scheme [11]. After 180 min of the initial incubation at T = To the reference cuvette was thermostated at T = To ± 0.5 ◦ C, while the sample cuvette was slowly heated at a rate of 0.25 ◦ C/min to ensure the thermodynamic equilibrium of the heated solution. The temperature in both cuvettes was determined with an error not exceeding 0.5 ◦ C. The DUV spectra and the DNA melting curves were recorded

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6 (Fig. 1c) is located at  = 39,400 cm−1 ( = 254 nm), i.e. near the position of the absorption maximum. Although coordination of Zn2+ ions to N7 of purines is expected to induce hypochromicity of DNA absorption at  = m , its value is relatively small in the present case and does not exceed ∼200 M−1 cm−1 (Table 2). The difference between εTo values obtained at pH 6.5 (curve 1 in Fig. 2) and pH 8.5 (curves 2 and 3 in Fig. 2) increases with the rise of Zn2+ ion concentration. The most probable reason for the additional hypochromicity of DNA at pH 8.5 is a deprotonation of the thymine N3 atoms induced by zinc ions upon the formation of N3(T)-Zn2+ -N1(A) bridge, which leads to the appearance of m-DNA [1–5,7,10]. Indeed, the hypochromicity value εa × [T] equals to −543 M−1 cm−1 upon deprotonation of N3 of dTMP (Table 2). At the same time the hypochromicity observed due to deprotonation of N1 of dGMP upon the formation of N3(C)-Zn2+ -N1(G) chelate, also typical for m-form, is 7 times as low (Table 2). Since the shape of curve 1 in Fig. 2 is determined only by Zn2+ ion interaction with N7 of purines, the intersection points of curves 2 and 3 with curve 1 indicate the critical concentrations of Zn2+

Table 2 Changes in nucleotide extinction at m = 38,500 cm−1 (m = 260 nm) induced by Zn2+ ions and by deprotonation of N1 of dGMP and N3 of dTMP.

Nucleotide −1

−1

εTo × [N], M cm [N]a Coordinating atom

0.01 M Zn2+ (Fig. 1a)

Deprotonation [19]

GMP

AMP

dGMP

−110 0.2 N7

−70 0.3 N7

−78 (−60 ) 0.2 N1

dTMP b

−543 0.3 N3

a [N] represents a relative content of the corresponding nucleotide in salmon sperm DNA. b The value in parentheses is for GMP.

ions ([Zn2+ ]cr ) below which m-DNA is not formed under the ionic conditions and the incubation time studied. It is seen from Fig. 2 that the critical concentration of Zn2+ ions in Tris buffer is higher than that in tetraborate. This may be due to the complex formation between Zn2+ ions and Tris molecules, decreasing the effective Zn2+ concentration necessary for m-DNA formation. Similar complexes with Tris molecules have been also shown for Mg2+ ions [11]. On the other hand, such complexes are absent in tetraborate buffer, which results in lower zinc concentrations required for the DNA transition to m-form. However, in ‘c–d’ region of the plot (Fig. 2) the absolute value of hypochromicity |εTo | reaches its maximum of |800| ± 100 M−1 cm−1 and coincides for both buffers, signifying that the equilibrium level of the degree of DNA metallization was reached. The data presented in Fig. 3 confirm the formation of m-DNA in the presence of ZnCl2 . Upon DNA addition into EB solution a red shift of the dye spectrum is observed, which results in a decrease of the EB absorption at  = m = 20,800 cm−1 ( = 480 nm) (curve 2 in Fig. 3). A subsequent addition of 2 × 10−4 M ZnCl2 to the solution containing DNA and EB reduces the amount of EB bound to DNA (curve 3 in Fig. 3). This is caused by Zn2+ ions displacing EB molecules upon formation of m-DNA [3]. If one considers that, upon DNA addition, the decrease in the optical density of EB solution in its absorption maximum is proportional to the amount of EB bound to DNA, the degree of DNA metallization ([m]) by Zn2+ ions can be evaluated according to the following formula. [m] =

(Ai − A0 ) , Amax

(3)

where A0 and Ai are the absorptions of EB or its complexes with DNA at  = m in the absence and presence of 2 × 10−4 M Zn2+ , respectively; Amax is the maximum difference between the absorptions of EB and EB + DNA complex without Zn2+ ions (Table 3).

Fig. 1. Differential UV spectra of nucleotides (a) and DNA (b, c), induced by Zn2+ ions at T = To . (a) GMP (1) and AMP (2) in the presence of 0.01 M Zn2+ in cacodylate buffer (pH 6.5). Nucleotide concentration is 6 × 10−5 M. (b) DNA in the presence of 9.9 × 10−5 M Zn2+ (3) and 10−3 M Ni2+ (4) [11] in Tris buffer (pH 8.5). DNA concentration is 2 × 10−5 M (P). (c) DNA in the presence of 2 × 10−4 M Zn2+ in tetraborate buffer (pH 8.5) (5) and in Tris (pH 8.5) (6).

Fig. 2. Dependence of DNA absorption change at T = T0 (εTo ) and  = 38,500 cm−1 ( = 260 nm) on Zn2+ ion concentration in different buffers: cacodylate (1), Tris (2) and tetraborate (3). Critical concentrations of Zn2+ ions below which mDNA is not formed under the conditions used: ([Zn2+ ]cr )2 = (9.4 ± 0.2) × 10−5 M; ([Zn2+ ]cr )3 = 3 × 10−5 M. [m] is a fraction of B-DNA, which adopted m-form (metallization degree).

V.A. Sorokin et al. / International Journal of Biological Macromolecules 50 (2012) 854–860

857

Table 3 Dependence of ethidium bromide absorption (m = 20,800 cm−1 ) and degree of DNA metallization on its concentration (25 ◦ C, pH 8.5, [EB] = 5 × 10−6 M, incubation time 180 min). D=

P [EB]

A0 × 103 , cm−1 a Ai × 103 , cm−1 c [m] × 102 , % a

a b c

0

2

3

4

8

12

14

16

29 (29) b 29 0

25 28 19

22 26 25

17 (17) 23 (26) 37.5 (56)

14 18 25

14 16 12.5

13 15 12.5

13 15 12.5

Indices 0 and i refer to the absence and presence of 2 × 10−4 M Zn2+ , respectively. Values given in parentheses are obtained in tetraborate buffer, the rest are in Tris. Value of [m] is calculated by formula (3).

According to Table 3, about 38–56% of DNA base pairs adopt m-form at Zn2+ concentration of 2 × 10−4 M. This result is in a reasonable agreement with the value [m] ≈ 50% obtained by fluorescence spectroscopy for DNA of various origins (with the GC content of 54% and 42%) under comparable experimental conditions [4]. 3.2. Temperature dependence of DNA absorption in the presence of Zn2+ ions DNA melting curves h(T) in cacodylate buffer at [Zn2+ ] ≤ 3 × 10−4 M (Fig. 4a), and in Tris and tetraborate buffers at [Zn2+ ] < [Zn2+ ]cr (Fig. 4b and c) exhibit the usual shape; only DNA hyperchromicity (h > 0) due to the helix–coil transition is observed upon heating. In the absence of any other conformational transitions (B → m, in particular), the maximum value of h(T) achieved upon the complete strand separation (hm )i can be used as a measure of DNA degree of helicity at T = T0 . In the absence of Zn2+ ions, independently of the buffer type (and, consequently,

of the solution pH), (hm )0 is 0.41 ± 0.02 (Table 1). This value can be considered as corresponding to the degree of helicity To = 1. Ca2+ ions, interacting only with the DNA phosphates and, therefore, incapable of inducing m-form [1], do not change the DNA helicity degree up to the highest ion concentrations used (0.002 M) (Table 4). On the other hand, Zn2+ ion interaction with N7 of DNA purines decreases the (hm )i value, which indicates an appearance of disordered single-stranded regions already at T = T0 (Table 4). The number of these regions increases with the rise of Zn2+ concentration. Interaction of Zn2+ ions with N7 of DNA purines in alkaline solutions remains the same as in neutral ones and should result in similar consequences. Furthermore, an appearance of the single-stranded conformation (along with the mentioned above decrease of pKa of iminoprotons) should facilitate the formation of m-DNA at pH 8.5. As seen from Fig. 4, for [Zn2+ ] > [Zn2+ ]cr the DNA melting curves and their temperature derivatives at pH 8.5 differ from those observed at pH 6.5. In this case in the temperature range from T0 up to Tf1 a hypochromicity (h < 0) of DNA absorption is observed instead of a hyperchromicity. Particularly, at 2 × 10−4 M Zn2+ in Tris buffer the absolute value of the hypochromic effect is twice as large as the one at T = To (Fig. 4b). Considering that hTo value of −0.12 (εTo = −800 M−1 cm−1 ) corresponds to the transition of about 50% of DNA base pairs into m-form, the heating to temperature Tf1 in the presence of [Zn2+ ] ≥ (2 ± 0.3) × 10−4 M results in almost complete DNA transition into the metallic form, both in Tris and in tetraborate buffers. The intensity of the B → m transition depends not only on the zinc concentration but also on the temperature. Thus, upon heating from T = T0 to T = Ts1 (point n on the h(T) curve in Fig. 4b) the value of d[m]/dT derivative does not exceed one percent per degree. However, at T > Ts1 this value sharply increases by almost an order of magnitude (Fig. 4b and c). The latter fact shows that at T > Ts1 the character of DNA metallization changes from fluctuational to cooperative. The cooperativity may result from the coil–helix transition rather than from an additional deprotonation of the iminoprotons during the B → m transition. Such a coil–helix transition might be possible due to renaturation of the double helix from the singlestranded regions appeared upon Zn2+ interaction with N7 at room temperature. This assumption might be supported by the fact that at high zinc concentrations the cooperativity of changes in h(T) during heating from TS1 to Tf1 is of the same order of magnitude as the cooperativity of the DNA helix–coil transition at T > Ts2 (Fig. 4b and c). 3.3. Phase diagrams

Fig. 3. Absorption spectra of ethidium bromide (EB) (1) and (DNA + EB) complex in Tris buffer in the absence (2) and presence (3) of Zn2+ ions: (1) 5 × 10−5 M EB; (2) EB + 2 × 10−5 M of DNA (P); (3) EB + DNA + 2 × 10−4 M of Zn2+ .

The data of Fig. 4 allow to plot relationships of DNA conformational equilibrium on temperature and Zn2+ ion concentration (Fig. 5), similar to property–composition diagrams (phase diagrams) of solutions of simple liquids [13]. In this case, DNA plays a role of a solvent for zinc, while B-, m- and C-conformations of DNA represent the phases. In a neutral solution (Fig. 5a), in the region below “n–a” curve DNA adopts mainly B-form but also contains single-stranded

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V.A. Sorokin et al. / International Journal of Biological Macromolecules 50 (2012) 854–860

Table 4 Dependence of degree of DNA helicity () at T = T0 on concentration of Zn2+ (in cacodylate buffer, pH 6.5) and Ca2+ (in Tris, pH 8.5 [11]) in the absence of B → m transition.

a

[Zn2+ ] × 104 , M a , %

0.1 98

[Ca2+ ] × 104 , M , %

0.1 91

0.35 91 1 105

1 84 2 100

1.5 74 3 103

2 70

3 72

5 95

20 113

The degree of DNA helicity was estimated according to the formula:

=

(hm )i , (hm )0

(4)

where hm is the maximum hyperchromicity of thermally denatured DNA at  = 38,500 cm−1 ( = 260 nm). The indices 0 and i refer to the absence (see Table 1) and the presence of Mt2+ ions in a solution.

sections. The fraction of DNA base pairs in these sections ranges from several percents to as much as ∼30% at T = To and Zn2+ concentrations between 0.1 × 10−4 and 3 × 10−4 M (Table 4). Above “n–a” curve (T = Ts in Fig. 4a) the cooperative helix–coil transition starts, finishing at “v–t” curve (T = Tf in Fig. 4a). At higher temperatures all DNA exists in the state of completely disordered single strands. The data presented in Fig. 5a show that the reason for the sharp increase in cooperativity of the DNA helix–coil transition in the presence of zinc at high concentrations (Fig. 4a) is a stronger Tf decrease compared to that of Ts . This effect is caused by preferential binding of Zn2+ ions to GC pairs of DNA, thus destabilizing its double-stranded structure. Earlier, this was also demonstrated for other ions with d-electrons in outer orbitals [11,21]. The phase diagrams for alkaline solutions are more complicated (Fig. 5b and c). Similarly as at pH 6.5, the double-helix conformation is present below “n–e–L–a” curve in the whole range of studied zinc concentrations. Above “v–i–k–t” curve DNA is in Cconformation. B-form is dominated at the left of “e–f” straight line, while mainly metallic DNA is present in the region bounded by “a–L”, “L–r” and “r–s” lines. In the remaining part of the phase diagram (␥, ␥ and ␳ regions) B-, C- and m-forms of DNA coexist in a thermodynamic equilibrium with each other. In ␥ and

␥ regions elevation of a temperature leads to an increase in the fraction of m-form at a given zinc concentration. This fraction reaches its maximum value at temperatures corresponding to “e–r–s” curve (Tf1 in Fig. 4b and c). Increase of ZnCl2 concentration in Tris buffer results in a continuous decrease of both Tf1 and Ts1 . However, in tetraborate buffer the concentration derivatives of Ts1 and Tf1 change their signs at [Zn2+ ] > 1.7 × 10−4 M. This leads to widening of ␥ and ␥ regions and to narrowing of m-region (Fig. 5c). In ␥ region the process of DNA metallization is much less intense than in ␥ region. As mentioned above, this might be because in the former region the m-DNA formation proceeds by the fluctuation mechanism whereas in the latter one heating induces the cooperative C → m transition. Unlike in ␥ and ␥ regions, temperature elevation in ␳ region virtually does not change the ratio between DNA fractions in B-, m, and C-conformations, up to the temperatures inducing the double helix separation (curve “e–L” in Fig. 5b and c, corresponding to Ts2 in Fig. 4b and c). It is appropriate to mention that some other DNA forms different from B become apparent only at elevated temperature. For example, deprotonation and subsequent change in structure occurs in alkaline medium at temperatures above 40 ◦ C (pH 10.2, temperature of deprotonation Tdep = 45 ◦ C, Tm = 65 ◦ C) [22].

Fig. 4. Plots of DNA hyper- and hypochromicity vs concentration of Zn2+ ions (top row) and their temperature derivatives (bottom row). (a) cacodylate buffer: (1) [Zn2+ ] = 0; (2) 10−4 M; (3) 3 × 10−4 M. Ts and Tf are the temperatures of the beginning and the end of the helix–coil transition. Ts0 and Tf0 correspond to DNA without Zn2+ ions. (b) Tris buffer: (1) 6.5 × 10−5 M Zn2+ ; (2) 2 × 10−4 M. Ts1 is the temperature above which a sharp increase in the value of the derivative dh(T)/dT is observed. Tf1 is the temperature of the end of the B → m transition. Ts2 and Tf2 are the same as Ts and Tf in (a). [m] is the DNA metallization degree. (c) tetraborate buffer: (1) 3 × 10−5 M Zn2+ ; (2) 6 × 10−5 M; (3) 1.2 × 10−4 M; (4) 1.7 × 10−4 M. The rest of the designations are the same as in (b).

V.A. Sorokin et al. / International Journal of Biological Macromolecules 50 (2012) 854–860

Fig. 5. Phase diagrams of DNA complexes with Zn2+ ions in cacodylate (a), Tris (b) and tetraborate (c) buffers. B and m designate a double-stranded DNA adopting Band m-forms, respectively; C designates a single-stranded DNA. ␥, ␥ and ␳ are the regions where B-, C- and m-forms of DNA coexist in the thermodynamic equilibrium with each other. Plots of temperature vs Zn2+ ion concentration are represented as follows: Ts2 : “n-a” (panel a) and “n-e-L-a” (panels b and c) curves; Tf2 : “v-t” (panel a) and “v-i-k-t” (panels b and c) curves; Ts1 : “e-x” (panel a) and “e-h-x” (panels b and c) curves; Tf1 : “e-r-s” (panels b and c) curves.

3.4. Components of the thermal stability

Mt2+

ion-induced changes of DNA

Relationships of Tm on Mt2+ ion concentrations (Fig. 6a), experimentally obtained under different conditions, allow to decompose (in additive approximation) the changes of Tm on the individual components arising from different types of Zn2+ –DNA interactions (Fig. 6b). Four important points can be deduced from such decomposition. (i). The P([Mt2+ ]) curve, obtained earlier by us [11], is common for DNA complexes with Ca2+ ions in cacodylate (pH 6.5) and Tris (pH 8.5) buffers as well as for DNA complexes with Mg2+ ions in cacodylate and tetraborate (pH 9.0) buffers. These ions do not interact with DNA nitrogen bases and m-DNA is not formed in their presence [1]. Hence, the P([Mt2+ ]) curve represents Tm changes induced exclusively by Coulomb interaction of Mt2+ ions (including Zn2+ ) with negatively charged oxygen atoms of DNA phosphate groups. Such an interaction results in a monotonic increase in DNA thermal stability. (ii). The BZn curve is a plot ıTm ([Zn2+ ]), which represents the innersphere interaction of Zn2+ ions with N7 of DNA purines. As in the case of other d-ions (Ni2+ and Cd2+ [11]), this interaction leads to a decrease in the thermal stability of DNA. The effect is

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Fig. 6. Plots of the DNA helix–coil transition temperature vs Mt2+ ion concentration (a) and their components corresponding to different types of Mt2+ interaction with the polynucleotide (b and c). (a) DNA in the presence of Zn2+ in cacodylate (1), Tris (2) and tetraborate (3) buffers; in the presence of Ca2+ in cacodylate and Tris, and in the presence of Mg2+ in cacodylate and tetraborate buffers [11] (4). (b) Curve BZn represents Zn2+ ion interaction with N7 atoms of purines in a double-helical DNA. It is obtained by subtracting curve 4 from curve 1. Curve BNi is the same but for Ni2+ ions [11]. Curve P represents Mt2+ ion interaction with DNA phosphate groups [11]. Curve m represents substitution of iminoprotons in AT and GC pairs of DNA with Zn2+ ions upon m-DNA formation. It is obtained by subtracting curve 1 from curve 3. (c) Interaction of Zn2+ –Tris (TrisZn ) and Ni2+ –Tris (TrisNi , [11]) complexes with DNA. Curve TrisZn is obtained by subtracting curve 3 from curve 2. Curve TrisZn is shifted downward by 2.7 ◦ C.

much greater for Zn2+ than for Ni2+ ions (cf. BZn and BNi curves in Fig. 6b). The decrease in Tm should enhance the fluctuational opening of the double helix and, as a result, promote the formation of m-DNA at T = T0 . This may explain a higher efficiency of Zn2+ ions in facilitating the m-DNA formation compared to Ni2+ ions [4]. (iii). Formation of intramolecular metal-mediated bridges results in the thermal stabilization of the double helix ([m]([Zn2+ ]) curve, Fig. 6b), which is comparable with stabilization upon the metal ion interaction with phosphates. The thermodynamic origin of these effects is the same in both cases; it is the fact that for Zn2+ ions the binding constants are higher for a double-stranded than for a single-stranded DNA [13,21]. In the case of the interactions with the phosphates the difference in the binding is caused by a lower charge density on the surface of the single-stranded polymer compared to the double-stranded helix. In the case of m-DNA this difference is due to a lower (in absolute value) free energy of Zn2+ ions separately binding to N1 of purines and to N3 of pyrimidines in the single-stranded DNA compared to the N1(A)-Zn2+ -N3(T) and N1(G)-Zn2+ -N3(C) chelate formation energy in the double helix. It is worth mentioning that the [m]([Zn2+ ]) plot estimated by us is qualitatively consistent with the data of Lee et al. [1] obtained, however, at different ionic conditions (20 mM sodium borate, pH 9.0).

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(iv). Finally, the data in Fig. 6c show that similarly to other small ions, such as Ni2+ and Mg2+ [11], Zn2+ ions in Tris buffer have an additional DNA destabilizing effect, which is not present in tetraborate and cacodylate buffers. 4. Conclusions Interaction of Zn2+ ions with DNA was studied in alkaline solutions at pH 8.5 in Tris and tetraborate buffers as well as in neutral solutions at pH 6.5 in cacodylate buffer employing differential UV–vis spectroscopy and thermal denaturation methods. The concentration of Zn2+ ions was varied from 10−5 M to 3 × 10−4 M. The studies were performed in the temperature range from 25 ◦ C to 70 ◦ C. A transition of B-DNA to a metallized form (m-DNA) induced by Zn2+ ions and facilitated by elevated temperature was observed in alkaline solutions. It was found that high concentrations of Zn2+ ions induce DNA hypochromicity at pH 8.5. The most probable reason for the hypochromicity is a deprotonation of thymine N3 upon the formation of intramolecular chelates N1(A)-Zn2+ -N3(T). Formation of such chelates enables the transition of DNA blocks enriched with AT pairs into m-form. Deprotonation of N1(G) in GC pairs should not significantly affect the absorption spectrum of DNA. In addition to increased pH of a solution, a coordination of dions with N7 of purine bases is an important factor promoting the formation of m-DNA. Such coordination facilitates the metallization process due to intensification of fluctuational openings of the double helix and subsequent formation of the single-stranded sections. There is a threshold temperature above which the DNA metallization efficiency increases by an order of magnitude due to formation of the metal-mediated bridges, which induce the coil → double helix transition. Similarly to Ni2+ and Mg2+ [11], Zn2+ ions in Tris buffer induce a decrease in DNA thermal stability in addition to that observed in cacodylate and tetraborate buffers. Acknowledgements V.V.A. gratefully acknowledges financial support from the Grant Agency of the Czech Republic (grant number P208/10/0559).

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