Interaction study of ss-DNA and Yb3+ ions in aqueous solutions by electrochemical and spectroscopic techniques

Journal of Molecular Liquids 165 (2012) 119–124

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Interaction study of ss-DNA and Yb 3+ ions in aqueous solutions by electrochemical and spectroscopic techniques Hoda Ilkhani a, b, Mohammad Reza Ganjali b, c,⁎, Majid Arvand a, Parviz Norouzi b, c a b c

Department of Chemistry, Faculty of Science, University of Guilan, P.O. Box 1914, Rasht, Iran Center of Excellence in Electrochemistry, Faculty of Chemistry, University of Tehran, Tehran, Iran Endocrinology & Metabolism Research Center, Tehran University of Medical Sciences, Tehran, Iran

a r t i c l e

i n f o

Article history: Received 12 September 2011 Received in revised form 20 October 2011 Accepted 28 October 2011 Available online 19 November 2011 Keywords: Ytterbium DNA Voltammetry Spectroscopy Computational study

a b s t r a c t In this work, for the first time the electrochemical behavior of the Yb3+ and Yb3+ ion interactions with short single strand DNA (ssDNA) sequence at two pHs was studied. Then the UV–vis spectroscopic method was used for supporting these pieces of evidence. The interaction between Yb3+ and ssDNA has different binding modes at different pHs. The ratio between [Yb3+] and [ssDNA] is dependent to pH and pKa of DNA bases. In pH 3.7, Yb3+ binds to ssDNA mainly by electrostatic attraction. Binding number, n, of 2 of Yb3+ per ssDNA and binding constant were obtained with Cyclic Voltammetry (CV) and differential pulse voltammetry (DPV) methods, respectively. In this pH, the bases of ssDNA are totally protonated and Yb3+ interacts electrostatically with phosphate groups. The UV–vis study showed similar results. The results at pH 5.5 show that Yb3+ can bind to ssDNA with electrostatic and covalent bonds. In this pH, besides phosphate groups, the bases can be interacted to Yb3+, too. The binding number 4 of Yb3+ per ssDNA was obtained. Computational studies were done and confirmed the result of experimental data. The agreement mutually verifies the accuracy of the methods. © 2011 Elsevier B.V. All rights reserved.

1. Introduction The diverse nature of medical applications of lanthanides may come as a surprise to many researchers. Recently lanthanides have attracted researcher interests, primarily as a gadolinium-based MRI contrast agents, as hypophosphatemic agents for kidney dialysis patients, as luminescent probes in cell studies, and for palliation of bone pain in osteosarcoma [1]. The biological properties of the lanthanides are similar to calcium and they have stimulated research into their therapeutic application. The lanthanides have similar ionic radii to calcium, but by virtue of possessing higher charge densities, they have a high affinity for Ca2+ sites on biological molecules [2]. The knowledge of the DNA structure and its interactions with other compounds can lead to advances in pharmacology and diagnosis basis of many diseases [3–7]. Nucleic acids are responsible for the storage and transcription of genetic information in living cells and are involved in protein synthesis. Nucleic acids are polymers of nucleoside phosphates and hence negatively charged in neutral solutions. Then they interact strongly with metal ions. These interactions are important in nature, because

⁎ Corresponding author at: Center of Excellence in Electrochemistry, Faculty of Chemistry, University of Tehran, Tehran, Iran. Tel.: +98 21 61112788; fax: + 98 21 66405141. E-mail address: [email protected] (M.R. Ganjali). 0167-7322/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.molliq.2011.10.017

it will change the genetic material's structure and function [8]. The interaction of DNA with heavy metals, since they are involved in processes leading to DNA damage, has been extensively investigated [9]. The study of the metal–DNA interactions becomes uniquely important to understand the consequent conductive behavior and its electron transfer property. The evidence for the binding of metal ions to DNA is rather indirect and still remains a matter of discussion [10]. DNA is a negatively charged polyelectrolyte, and the binding of metal cations to it, is of importance since this plays a crucial role in the biological activity of nucleotides and nucleic acids, changing their properties in ways that depend on the nature of the metal ion. Knowledge of the mechanisms responsible for the activity of these systems requires the study of metal ion–nucleic acid interactions [11]. Among the lanthanide series, just five members are electroactive. Ytterbium is one of them. The other ones are cerium, europium, samarium, and terbium. The interaction of these metal ions with DNA was studied by several methods such as luminescent [10,12], UV–vis spectroscopy [13], NMR [14,15] and electrochemistry [16–18] but the interaction of lanthanides with a short single strand DNA sequence by electrochemical method wasn't studied. Also in recent years, computational methods are applied in different branches of chemistry. In the presented paper, we have also used B3LYP/SBKJC method to study the interaction [19]. In this paper, the electrochemical behavior of Yb3+ has been studied by Cyclic Voltammetry (CV) and differential pulse voltammetry (DPV)

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2. Experimental

h 1.80E-04

a 8.00E-05

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for the first time. Then the interaction between ssDNA with Yb3+ has been studied at two pHs and with two methods: UV–vis spectroscopy and electrochemistry. The experimental results have proved that Yb3+ could interact with ssDNA mainly by electrostatic binding and by binding number, n, 1, but in higher pH, the interaction between Yb3+ and ssDNA is not only electrostatic but also covalent binding and the binding number is higher.

I/A

120

-1.20E-04

2.1. Apparatuses -2.20E-04

A 10-mer oligonucleotide was supplied (as lyophilized powder) from MWG-Biotech AG, with the following sequence: 3′-GGAGCTCCTG5′. The stock solutions of short ssDNA sequence (1.0×10− 2 M) were prepared by dissolving powder primer in doubly distilled water and kept frozen in −20 °C temperature. Yb2O3 was obtained from Merck Co. The stock solution of Yb 3+ (1 × 10 − 2 M) was prepared by dissolving 0.366 g of its oxide in minimum amount of nitric acid and diluted with phosphate buffer (pH 3.7 and 5.5) in 100 mL volumetric flaks. Dilute solution was prepared just before use. 2.3. Electrochemical studies Cyclic Voltammetry (CV) experiments were carried out at room temperature (23 ± 2 °C) in a potential ranging from −0.7 V to 0.0 V at various scan rates (from 5 mV s − 1 to 300 mV s − 1). Only reproducible responses were recorded. For differential pulse voltammetry (DPV), the conditions were as follows: the step potential 0.01 V, the modulation time 0.04 s, and the interval time 0.53 s, in a potential range from − 0.65 V to −0.2 V. All voltammetric experiments were performed in a single compartment glass cell of three electrode assemblies of 500 μL capacity in which platinum wire was used as counter electrode, Ag/AgCl was used as the reference electrode and platinum electrode was used as working electrode. Prior to each electrochemical experiment, the working electrode was polished successively with 0.3 μm (grain size) alumina powder (Metrohm) and then cleaned ultrasonically in water. Interaction between ssDNA and Yb 3+ was carried out in constant concentration of Yb 3+ and varying concentrations of ssDNA by CV and DPV methods. Vice versa, for UV–vis titration of ssDNA with Yb 3+, the concentration of ssDNA was kept constant and various concentrations of Yb 3+, were added, up to 3 mL final volume.

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E/V Fig. 1. CVs of Yb3+ in 0.1 M NaCl at pH 3.7 and potential range −0.5 V to −0.25 V. Yb3+ concentrations were: (a) 2.5×10− 4 M, (b) 5.0×10− 4 M, (c) 1.0×10−3 M, (d) 2.0× 10− 3 M, (e) 3.33× 10− 3 M, (f) 5.0 × 10− 3 M, (g) 6.6 × 10− 3 M, and (h) 1.0× 10− 2 M. The scan rate was 0.1 V s− 1. Inset: Plot of Ip vs. concentration of Yb3+ at the same conditions.

corrections together with entropies to convert the internal energies to Gibbs energies at 298.15 K [21].

3. Results and discussion 3.1. Electrochemical study 3.1.1. Electrochemical behavior of Yb 3+ The CVs for the ytterbium (III) nitrate phosphate buffer (pH 3.7) are shown in Fig. 1. Forward scans reveal that the anodic peak associated with the oxidation of Yb 3+ to Yb 4+ occurs at approximately −212.8 mV. On the reverse scan, the cathodic peak associated with reduction of Yb4+ to Yb 3+ occurs at approximately −530.2 mV versus Ag/AgCl. Fig. 1 shows the CVs of Yb3+ in different concentrations too. The concentration of Yb3+ ranged from 2.5 × 10− 4 M to 1.0 × 10− 2 M as can be seen in this figure, there are pair of redox peaks for Yb 3+. Anodic and cathodic peak currents changed slightly with increasing concentration. Also the peak potential is constant with increasing the concentration of Yb 3+ ion and Ipc/Ipa is very near to one. Thus, this result shows that the redox peak is reversible. Inset of this figure shows that the relationship between Ip and concentration of Yb3+, is linear.

f 4.00E-06

a -1.00E-06 lpc 1.60E+01

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1.20E+01

2.4. Computational methods Calculations on the isolated ssDNA and Yb 3+–ssDNA were performed within Gamess package [20]. Each species was initially optimized with PM3 method and, then the optimized structures were again optimized with B3LYP/SBKJC. Full geometry optimizations and frequency calculations were performed and each species was found to be minima by having no negative values in the frequency calculation. The calculations gave internal energies at 0 K. In order to obtain gas phase free energies at 298.15 K, it is necessary to calculate the zero-point energies and thermal

0.0045

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2.2. Reagents

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Electrochemical experiments were performed using AUTO LAB PGSTAT 30 electrochemical analysis system and general propose electrochemical system (GPES) 4.9005 software package (Eco Chemie, The Netherlands). A PERKIN-ELMER UV–vis spectrophotometer with a 1 cm path cell was used for spectrophotometric determinations. Also a Heidaloh MR 3001K stirrer was used in this work.

y=22.37x+0.003 R2=0.993

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E/V Fig. 2. CVs of 5.0 × 10− 4 M Yb3+ in 0.1 M NaCl at pH 3.7 and potential range −0.7 V to −0.1 V. Scan rates are (a) 5 mV s− 1, (b) 10 mV s− 1, (c) 50 mV s− 1, (d) 100 mV s− 1, (e) 200 mV s− 1, and (f) 300 mV s− 1. Inset: Plot of Ip vs. square root of scan rates at the same conditions.

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a

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log [ssDNA]

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E/V Fig. 3. (a) CVs of 1.66 × 10− 6 M Yb3+ in 0.1 M NaCl at pH 3.7 and (b) a + 2.0 × 10− 6 M ssDNA, (c) a + 4.0 × 10− 6 M ssDNA, (d) a + 5.6 × 10− 6 M ssDNA, (e) a + 7.4 × 10− 6 M ssDNA, (f) a + 9.1 × 10− 6 M ssDNA, (g) a + 1.07 × 10− 5 M ssDNA, (h) a + 1.23 × 10− 5 M ssDNA, (i) a+ 1.44× 10− 5 M ssDNA, (j) a+ 1.54× 10− 5 M ssDNA, (k) a +1.7 × 10− 5 M ssDNA, (l) a+1.86× 10− 5 M ssDNA, (m) a +2.29 ×10− 5 M ssDNA, h  and (n)i a+ 2.45× 10− 5 M ssDNA at the same conditions. Inset: Plot of log ΔI ðΔI max −ΔIÞ vs. log½ssDNA at pH=3.7.

The scan rate dependence of the peak current in phosphate buffer was reported in Fig. 2. The scan rate range was from 5 to 300 mV s − 1. The cathodic and anodic currents increased with increasing scan rate. In reversible wave, Ip (as well as the current at any other point on the wave) is proportional to υ 1/2 with Eq. (1) and the value of D0 in Eq.(1) can be determined from the slope of Ip versus υ 1/2 plate.
 = = = 3 Ip ¼ 0:4463 F =RT n AD 0 υ C 3

1

2

1

2

ð1Þ

2

The plot of Ip vs. υ1/2 was shown in the inset of Fig. 2. The regression equation is Ip = 8 × 10 − 7 υ1/2 − 2 × 10− 6 and the correlation coefficient is R = 0.997. These results indicate that the electrochemical processes are limited by diffusion control. This figure illustrates that the anodic “line” and cathodic “line” are close together and having similar slopes, indicative of relatively comparable electron transfer rate. If n = 1, A = 0.0314 cm2 and C = 5 × 10− 4 M, then D0 = 3.59 × 10 − 8 cm2 s− 1 was obtained. 7.00E-05

a 6.00E-05

Fig. 5. Plot of intense absorbance vs. different concentrations of Yb3+. (a) 1.0 × 10− 6 M ssDNA and (b) a + 6.66× 10− 7 M Yb3+, (c) a + 1.0 × 10− 6 M Yb3+, (d) a + 1.33× 10− 6 M Yb3+, (e) a + 1.66× 10− 6 M Yb3+, (f) a + 2.0 × 10− 6 M Yb3+, (g) a + 2.33 × 10− 6 M Yb3+, and (h) a + 2.66× 10− 6 M Yb3+ at pH = 3.7.

3.1.2. Electrochemical behavior of ssDNA The CV and DPV techniques were also used in order to investigate the electrochemical behavior of 1 × 10 − 5 M ssDNA at a condition mentioned in Section 3.1.1. Any peak wasn't obtained for ssDNA in the same potential range. Williams [22] has reported that, in this pH (3.7), the transition width of ssDNA doesn't significantly change as the overstretching transition force is lowered, whereas at lower pH (near 2.5) the transition width increases by a factor of 5. This transition broadening was attributed to the growing flexibility of ssDNA associated with its charge neutralization. It has been argued that ssDNA molecule can be stretched all the way through the transition without breaking. Therefore ssDNA wasn't broken at pH 3.7, in this research. 3.1.3. Electrochemical study of the interaction of ssDNA with Yb 3+ ions The CVs of Yb 3+ were obtained in different concentrations of ssDNA. Fig. 3 shows the voltammograms of Yb 3+ and Yb 3+–ssDNA at pH 3.7. The peak currents (both the Ipc and Ipa) decrease with increasing concentration of ssDNA while both the Epc and Epa shifted to more negative potentials. The ionic strength was strictly kept constant, since the change in the rate constant could be due to the small changes in the ionic strength [23]. The phenomena of the shift of E° and the decrease of peak current implied forming a new association complex. We know there are three kinds of binding modes for small

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E/V Fig. 4. (a) DPV of 1.66 × 10− 6 M Yb3+ at pH 3.7 and (b) a + 2.0 × 10− 6 M ssDNA, (c) a + 4.0 × 10− 6 M ssDNA, (d) a + 5.6 × 10− 6 M ssDNA, (e) a + 7.4 × 10 − 6 M ssDNA, (f) a + 9.1 × 10− 6 M ssDNA, (g) a + 1.07× 10− 5 M ssDNA, (h) a + 1.23× 10− 5 M ssDNA, (i) a + 1.44× 10− 5 M ssDNA, (j) a + 1.54× 10− 5 M ssDNA, (k) a + 1.7 × 10− 5 M ssDNA, (l) a + 1.86× 10− 5 M ssDNA, and (m) a + 2.29 × 10− 5 M ssDNA, initial potential, −0.8 V, the end potential, 0.1 V, the step potential, 0.015 V, the modulation time, 0.02 s, and the interval time, 0.53 s.

a b

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wavelength/nm

0.142 0

1

2

3

4

5

Fig. 6. Plot of intense absorbance vs. (a) 1.0 × 0− 6 M ssDNA and different amounts of (b) a + 3.33 × 10− 7 M Yb3+, (c) a + 6.66 × 10− 7 M Yb3+, (d) a + 1.66 × 10− 6 M Yb3+, (e) a + 2.33 × 10− 6 M Yb3+, (f) a + 2.66 × 10− 6 M Yb3+, (g) a + 3.33 × 10− 6 M Yb3+, (h) a + 4.0 × 10− 6 M Yb3+, and (i) a + 4.33 × 10− 6 M Yb3+, at pH = 5.5.

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Table 1 Order of energy for interaction between Yb3+ and ssDNA at two pHs in different states.

State one State two State three State four

pH = 3.7 (n = 2 Yb3+) Energy/kcal/mol

pH = 5.5 (n = 4 Yb3+) Energy/kcal/mol

−610.4 −438.2 −448.2 −566.6

− 779.8 − 628.68 − 835.4 − 904.3

molecules to DNA. Among those modes, Bard [24] has reported that if E° shifted to more negative value when small molecules interacted with DNA, the interaction mode was electrostatic binding. On the contrary, if E° shifted to a more positive value, the interaction mode was intercalative binding. Fig. 3 shows that after addition of different amounts of ssDNA, the CVs of Yb 3+ shifted to a more negative value of E°, therefore the interaction between ssDNA and Yb 3+ is only electrostatic binding (pH 3.7). In this pH, the bases of ssDNA are completely protonated and cannot interact with Yb 3+ ions, but its phosphate groups have negative charge, and can interact with Yb 3+ ions by electrostatic mode. Therefore, based on this evidence, Yb 3+ ions only bind to phosphate groups. Also the binding ratio and binding constant of ssDNA–Yb 3+ complex were studied. It is assumed that the interaction of ssDNA with 3+ ions Yb 3+ only produces a single complex ssDNAm–Yb. h If Yb i and ΔI ssDNA form a single complex, then the plot of log vs. log ΔImax −ΔI [ssDNA] becomes linear with a slope of m [25]. Inset of Fig. 3 indicates a linear relationship which implies that Yb 3+ can form a single complex with ssDNA in different concentrations of ssDNA. The slope of this equation is − 0.572, the intercept is 1.953 and the correlation coefficient is R = 0.994. The slope value shows that two Yb 3+ ions

bind to even ssDNA. Also the value of binding constant, βs, 2.05 × 10 7 M − 1 can be obtained in this equation. The phenomena mentioned above were further studied by DPV which were shown in Fig. 4. The applied potential range is from −0.8 V to 0.1 V. The DPV of 1.66 × 10 − 6 M Yb 3+ solution is shown in curve (a) and other curves are related to DPV of the same solution with different amounts of ssDNA (ssDNA concentrations are 1.98 × 10 − 6 M, 4.0 × 10 − 6 M, 5.6 × 10 − 6 M, 7.4 × 10 − 5 M, 9.1 × 10 − 5, 1.07× 10− 5 M, 1.23× 10− 5 M, 1.44 ×10− 5 M, 1.54 ×10− 5 M, 1.70 × 10− 5 M, 0.86× 10− 5 M, 2.09× 10− 5 M respectively). This figure also shows that the peak currents decreased with increasing concentration of ssDNA. 3.1.4. pH effect Electrochemical behavior of Yb 3+ at higher pH was also studied with CV, but we couldn't obtain CVs of Yb 3+ in this pH. Because with increasing [OH− ], the insoluble hydroxide complexes of ytterbium, (YbðOHÞ3 , YbðOHÞ2þ , YbðOHÞþ 2 ), can be produced. The surface of electrode was coated with these compounds and then Yb 3+ couldn't reach to the electrode surface. Then, the peaks shifted to higher potentials and the currents of peaks slightly decreased. Therefore we couldn't obtain any remarkable peak in this pH, and used UV–vis spectroscopy method. 3.2. Spectroscopic study 3.2.1. Effect of Yb 3+ ions on ssDNA spectrum The UV–vis absorbance spectrum of 1 μM ssDNA and its titration with different concentrations of Yb 3+ at pH 3.7 are displayed. The UV–vis spectrum of ssDNA shows an intense absorbance at 259 nm. Intense absorbance gradually increased and peak shifted to 264 nm

Fig. 7. The most stable model of interaction between Yb3+ ions and ssDNA at pH 3.7.

H. Ilkhani et al. / Journal of Molecular Liquids 165 (2012) 119–124

(red shift), when ssDNA solution titrated with varied concentrations of Yb 3+ solution. Because there would be an absorbance increasing at 259 nm upon increasing of Yb 3+, we added Yb 3+ to working and reference cells, indicating that the increase of absorbance was not derived from the high concentration of Yb 3+, but from the interaction of Yb 3+ with ssDNA. These results indicated that ssDNA could form a complex with Yb 3+. Fig. 5 shows that the titration curve of 1.0 × 10− 6 M ssDNA with different amounts of Yb 3+ was linear until it approximately reaches to two Yb3+ ions for each ssDNA. After this point no excess Yb 3+ was bound. 3.2.2. pH effect In pH 5.5 since the final volume of the reaction system was 3 mL, ssDNA concentration could maintain constant at 1 μM and the ratio of [Yb 3+]/[ssDNA] was 0, 0.333, 0.666, 1.66, 2.33, 2.66, 3.33, 4.00 and 4.33, respectively. Thus an absorption titration was performed under maintaining a constant ssDNA concentration and varying Yb 3+ concentrations. Inset of Fig. 6 shows the absorption spectra of ssDNA (259 nm) with increasing amounts of Yb3+. With increasing concentration of Yb3+ the intense peak was increased and red shift occurred. These results support the notion that there exists an interaction model of binding, between Yb3+ and ssDNA base pairs or phosphate groups. The relationship between Yb3+ concentration and intense absorbance is plotted in Fig. 6. This figure shows that, the reaction of Yb3+ with ssDNA was linear until Yb 3+ concentration received 4.0× 10− 6 M of Yb 3+ and after this point, intense absorbance is almost constant. Therefore approximately four Yb3+ ions were added to each ssDNA. These pieces of evidence indicate that in this pH, besides binding phosphate linkages [26–28], Yb 3+ directly coordinates electron

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donor groups on the nucleotide bases at ssDNA molecules [29]. Our results were also consistent with the previous studies on the effects of other lanthanide ions on ssDNA. 3.3. Computational methods In this section, to recognize interactive sites of ssDNA, computational methods were used. In pH 3.7 all of the negative charge sites on ssDNA bases were protonated, so Yb 3+ only could interact with phosphate group of ssDNA. Because experimental methods showed that two Yb 3+ ions can interact with ssDNA, interaction energy between them was calculated with different models. In these models Yb3+ was imagined with various chemical valences. So, 4 states were improved. 1) One of the Yb 3+ ions interacts with 5 phosphate groups in one side of ssDNA (3′) and the other Yb 3+ ion interacts with 4 phosphate groups in another side of ssDNA (5′). 2) One of the Yb 3+ ions interacts with 3 phosphate groups in one side of ssDNA (3′) and the other Yb 3+ ion interacts with 6 phosphate groups in another side of ssDNA (5′). 3) One of the Yb 3+ ions interacts with 5 phosphate groups in the middle of ssDNA and the other Yb 3+ ion interacts with 4 phosphate groups in two sides of ssDNA (3′ and 5′). 4) One of the Yb 3+ ions interacts with 2 phosphate groups in the middle of ssDNA and 2 phosphate groups in one side of ssDNA (3′) and the other Yb 3+ ion interacts with 3 phosphate groups in the middle of ssDNA and 2 phosphate groups in another side of ssDNA (5′).

Fig. 8. The most stable model of interaction between Yb3+ ions and ssDNA at pH 5.5.

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Calculated interaction energy for each of these models was shown in Table 1. Interaction energy for most stable model (Fig. 7) is −610.4 kcal/mol. These results demonstrate that one of the Yb 3+ ions interacts with 5 phosphate groups and the other interacts with 4 phosphate groups. In pH = 5.5, both phosphate groups and bases of ssDNA can interact with Yb 3+. In this pH one of the nitrogens of adenine, guanine and cytosine that is not protonated, can interact with Yb 3+ ions. But other nitrogen of these bases and all of thymine's nitrogens are protonated so it cannot interact with Yb 3+. Same as before, for calculation of interaction energy, four states can be improved. 1) Three of the Yb 3+ ions interact with phosphate groups and active site of bases with 4 chemical links (for each Yb 3+ ion) and the last ion interacts with phosphate groups and active sites of bases with 5 chemical links. 2) Two Yb 3+ ions only interact with active site of bases (4 chemical links) and the two others interact only with phosphate groups (one of them with 4 and the other with 5 chemical links). 3) One of the Yb3+ ions interacts with active site of bases in the middle of chain, one of them interacts with active site of bases in two sides of chain and two remaining ions interact with phosphate groups of ssDNA. 4) Two ions interact only with active site of bases (with 3 and 5 chemical links) and two ions interact only with phosphate groups (with 4 and 5 chemical links). Different states are presented in Table 1. In pH = 5.5 the most stable state is the fourth state (Fig. 8) and interaction energy is −904.3 kcal/mol. This investigation shows that total interaction energy at pH 5.5 is lower than pH 3.7, therefore interaction between Yb 3+ and ssDNA at this pH is more probable and more stable. Therefore, near the biological pH, the possibility of interaction between Yb 3+ ions and ssDNA, is higher. 4. Conclusion In this study, interaction between Yb 3+ and ssDNA was studied using both electrochemical method and UV–vis spectroscopy in different pHs. The electrochemical behavior of Yb 3+ was investigated at various concentrations and scan rates. There is a linear correlation between peak current (Ip) and square root of scan rate. These pieces of evidence indicate that the kinetics of process was diffusion-controlled. The interaction between Yb3+ and ssDNA has different binding modes in different pHs. The result of electrochemical study shows that, in pH 3.7, Yb 3+ ions bind to ssDNA mainly by electrostatic attraction and binding number, m, is obtained with two Yb3+ ions per ssDNA and binding constant, βs, of 2.05 × 10− 7 M− 1. In this pH the bases of

ssDNA are totally protonated and Yb3+ ions interact electrostatically with phosphate groups. The UV–vis study confirms these results. In pH 5.5, the results are completely different. In this pH, Yb3+ ions bind to ssDNA with both electrostatic and covalent bonds and besides phosphate groups, the bases also can be interacted with Yb 3+. The binding number is obtained with four Yb 3+ ions per ssDNA. Then Yb 3+ can bind to ssDNA and forms a stable single complex. The ratio between [Yb 3+] and [ssDNA] is dependent to pH and pKa of bases. Also interaction between Yb 3+ and ssDNA and active sites of ssDNA was recognized with computational methods in two pHs. These methods selected the most stable state for this interaction at different pHs. References [1] [2] [3] [4] [5]

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