摘要

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中图分类号:O657.3 文献标志码:A
Comparison of Enhancement Effect of DNA-Mediated Energy Transfer by Divalent Cations: Mg2+, Ca2+, Mn2+, Co2+, and Ni2+
Jong-Moon Kim1, Myung Duk Jang2, JIN Biao3,*, Yoon Jung Jang4,*
1. Department of Health Care and Biotechnology, Kyungwoon University, Gumi City, Gyeong-buk 730-739, Republic of Korea
2. Materials & Energy Engineering College of IT & Energy, Kyungwoon University, Gumi City, Gyeong-buk 730-739, Republic of Korea;
3. Instrumental Analysis Center, Yanbian University, Yanji 133002, China
4. College of Basic Education, Yeungnam University, Dae-dong, Gyeongsan City, Gyeong-buk 712-749, Republic of Korea
e-mail: jmkim@kw.ac.kr *Corresponding authors e-mail: jinbiao@ybu.edu.cn; jyj5014@ynu.ac.kr
Abstract

The effect of various metal ions on the DNA mediated energy transfer between simultaneously bound drugs was investigated using spectroscopic methods. It was found that addition of divalent metal ions (Mg2+, Ca2+, Mn2+, Co2+ and Ni2+) resulted in further decrease of the ethidium fluorescence intensity, while a small increase was observed in the TMPyP emission band, implying that the energy of excited ethidium was transferred to TMPyP. This DNA-mediated quenching efficiency between ethidium and TMPyP was significantly enhanced by the presence of all metal ions. Among the divalent metal ions, alkali earth metal ions and Mn2+ displayed higher quenching efficiencies than other transition metal ions. The distances required to permit the energy transfer between the two drugs in DNA were calculated as 68, 66, 62, 48 and 38 Å in the presence of 100 μmol·L-1 of Mg2+, Ca2+, Mn2+, Co2+ and Ni2+ ion, respectively. The disturbed binding conformation of TMPyP in DNA by metal ions presumably accounts for the difference.

Keyword: Energy transfer; Divalent cations; DNA; Ethidium; TMPyP
Introduction

The energy and the charge transfer between DNA-bound drugs or drug to DNA bases have been the subject of intensive studies. This interest is a result of the fact that stacked π -orbitals of DNA base pairs can serve as an effective medium for electron/charge transfer[1, 2, 3, 4, 5, 6, 7, 8]. The reported biological importance of the charge transfer in DNA has been highlighted inside the cell nucleus by the discovery of the oxidative damage done to DNA from a distance[9, 10]. As well, an understanding of the charge transport in DNA has also been shown essential for developing nanodevices, i.e. designing a nanometer-sized self assembling molecular wire[11, 12, 13]. Fluorescence resonance energy transfer (FRET) has become widely used in all applications of fluorescence, including medical diagnostics, DNA analysis, and optical imaging. The widespread use of FRET is due to the favorable distances for energy transfer, which is typically the size of a protein or the thickness of a membrane[14]. If the spectral properties of the fluorophores allow FRET, it will occur and willnot be significantly affected by the biomolecules in the sample. FRET occurs between a donor molecule in the excited state and an acceptor molecule in the ground state. The donor molecules typically emit at shorter wavelengths that overlap with the absorption spectrum of the acceptor. Energy transfer occurs without the appearance of a photon and is the result of long range dipole-dipole interactions between the donor and acceptor. The term resonance energy transfer (RET)[15] is preferred because the process does not involve the appearance of a photon. The rate of energy transfer depends upon the extent of spectral overlap of the emission spectrum of the donor with the absorption spectrum of the acceptor, the quantum yield of the donor, the relative orientation of the donor and acceptor transition dipoles, and the distance between the donor and acceptor molecules. The distance dependence of RET allows measurement of the distances between donors and acceptors. It has been reported that the energy of excited state of ethidium (Figure 1) was transferred to meso-tetrakis (N-methylpyridinium-4-yl)porphyrin (referred to as TMPyP, Figure 1) when both molecules were simultaneously intercalated between DNA base-pairs[16, 17]. The energy transfer from ethidium to TMPyP mediated by DNA reached 25~30 Å . When ethidium or a Ru(Ⅱ ) complex was covalently bound to DNA, its fluorescence intensity decreased by increasing concentration of DNA-bound Ru(Ⅱ ) complexes[18, 19]. The electron transfer was accompanied in these cases. DNA mediated energy transfer of another kind was also reported. When the minor groove of DNA was saturated by 4’ , 6-diamidino-2-phenylindole, excite state energy transferred to TMPyP or [Ru(phenanthroline)2DPPZ]2+. The latter molecule probably bound at the major groove of DNA therefore, the energy transfer occurred “ across” the DNA stem[19, 20, 21]. This note reports that the efficiency of the energy transfer from ethidium to TMPyP was enhanced significantly upon the presence of various dication (Mg2+, Ca2+, Mn2+, Co2+ and Ni2+) ions when both molecules are intercalated between DNA base-pairs.

Fig.1 Molecular structures of cationic structure (upper) and TMPyP (lower)

1 Experimental
1.1 Salt solutions

Chloride salts of divalent cations were obtained from Aldrich Chemical(Seoul, Korea) and used without further purification. Stock solutions containing 500 μ mol· L-1 of metal salt and 5 mmol· L-1 of sodium cacodylate were adjusted to pH 7.0.

1.2 DNA and other materials

Calf thymus DNA was purchased from Sigma-Aldrich (Seoul, Korea). DNA was dissolved in 5 mmol· L-1 cacodylate buffer and used without further purification. TMPyP was purchased from Frontier Scientific (Logan, UT) and ethidium from Sigma and used without any further purification. The concentrations were spectrophotometrically determined using their extinction coefficients: A258 nm=6 700 cm-1· L· mol-1, A480 nm=5 850 cm-1· L· mol-1, and A421 nm=245 000 cm-1· L· mol-1 for DNA, Ethidium, and TMPyP, respectively. TMPyP were always added last, immediately before measurement, since the mixing order potentially affects the binding mode of TMPyP to DNA[22].

1.3 Absorption, circular dichroism and fluorescence measurements

Absorption spectra were recorded on a Cary 100 (Varian, Australia) and circular dichroism spectra (referred to as CD) were measured on a J810 (Jasco, Tokyo, Japan) spectropolarimeter, respectively. Fluorescence spectra were recorded using a Jasco FP-777 fluorometer. The fluorescence emission spectra of ethidium in the presence and absence of TMPyP was recorded with an excitation of 527 nm, the maximum excitation for DNA-bound ethidium. Excitation and emission wavelengths of 527 and 592 nm, respectively, were used in the fluorescence quenching experiment.

The fluorescence quenching of DNA bound ethidium with TMPyP was analyzed through Stern-Volmer plots[15].

F0F=1+KSV[Q](1)

In this equation, F0 and F denote the fluorescence intensities of the fluorophore, DNA-bound ethidium, in the absence and presence of quenchers, respectively. [Q] is the concentration of quencher, TMPyP. The quenching constant, KSV represents the equilibrium constant for the formation of the nonfluorescent fluorophore-quencher complex in the static quenching process.

2 Results and discussion

It has been well known that the fluorescence intensity of ethidium increases significantly upon intercalation between DNA base-pairs. Pasternack et al. reported that energy transfer was evoked through DNA from simultaneously bound ethidium to TMPyP, which was proven by the opposite change of their emission intensities[23]. Here, we found that addition of metal ions seemed to reinforce the efficiency of DNA mediated energy transfer from ethidium to TMPyP as shown in figure 2. Addition of divalent metal ions (Mg2+, Ca2+, Mn2+, Co2+ and Ni2+) resulted in further decrease of the ethidium fluorescence intensity, while a small increase was observed in the TMPyP emission band, implying that the energy of excited ethidium was transferred to TMPyP and the efficiency of energy transfer increased by the presence of divalent metal ions (Mg2+, Ca2+, Mn2+, Co2+ and Ni2+). Trend of decrease in intensity is bigger in the case of Mg2+and Ca2+ ions than that of Mn2+, Co2+ and Ni2+ ions. When the ratio of ethidium fluorescence intensity in the absence of TMPyP (F0) to its presence (F) was plotted with respect to TMPyP concentration, an upward bending curve was observed (Figure 3, square). This DNA-mediated quenching efficiency between ethidium and TMPyP was significantly enhanced by the presence of all metal ions. As shown in Stern-Volmer plot, the quenching efficiency was lowest in the absence of divalent metal ion (Figure 3, curve a). Among the divalent metal ions, alkali earth metal ions and Mn2+ displayed higher quenching efficiencies than other transition metal ions.

Fig.2 Fluorescence emission spectra of ethidium bound to DNA-TMPyP complex in the absence (curve a) and the presence of 100 μ mol· L-1 of Mg2+ (curve b), Ca2+ (curve c), Mn2+ (curve d), Co2+ (curve e) and Ni2+ (curve f). The concentrations of DNA, TMPyP and ethidium are 100, 2.5 and 0.5 μ mol· L-1, respectively. The emission spectra were recorded at an excitation of 530 nm. Slit widths were 5 nm for both excitation and emission

Fig.3 Stern-Volmer plot for the fluorescence quenching of the ethidium bound to DNA by TMPyP in the absence and in!the presence of 100 μ mol· L-1 divalent metal ion (Mg2+, Ca2+, Mn2+, Co2+ and Ni2+). The concentrations, curve assignment and conditions are identical to those in Figure 2

Observed upward bending curves can be fit by an equation proposed by Pasternack and his coworkers[23].

F0/F=exp{2σ[Q]/([DNA]-2[E+])}(2)

Where [Q] is the concentration of quencher, TMPyP in the current case and [E+] is the concentration of ethidium. The symbol σ denotes the minimum number of base-pairs between ethidium and TMPyP required to permit the energy transfer between them. The number of base-pairs calculated from the result shown in Figure 3 according to equation (2) was 10.6 or 36 Å in the absence of divalent metal ions, which is slightly larger than the reported value of 25~30 Å , conceivably due to difference in the experimental condition. The calculated energy transfer distances upon metal ion addition was in direct proportion to the quenching efficiencies. It reaches 20.1 base-pairs or 68 Å , 19.3 base-pairs or 66 Å , 18.3 base-pairs or 62 Å , 14.2 base-pairs or 48 Å and 11.3 base-pairs or 38 Å in the presence of 100 μ mol· L-1 of Mg2+, Ca2+, Mn2+, Co2+ and Ni2+ ion, respectively. Thus, it is obvious that the enhancement is originated from the binding of divalent metal ion to DNA.

DNA-metal cation interactions and their effects on DNA structure have been investigated by a variety of techniques, including sedimentation equilibrium measurements[24]. It has been reported that transition metal ion bound M-DNA exhibited enhanced ability for electron transfer up to 500 base-pairs long even the efficiency was less than 5%[25]. In this case of M-DNA, transition metal ions are considered to bind to the bases via coordinate covalent bonding and bridge the two opposite bases[26, 27]. Thus, the enhancement of electron transfer efficiency is assumed that transition metal ions directly participate in the electron transfer mechanism or help the π — π stacking of base-pairs. On the other hand, alkali earth metal ions have been known to bind mainly to the phosphate group via electrostatic interaction. Thus direct involvement of Mg2+ and Ca2+ ion in energy transfer mechanism can be excluded. In order to verify the influence of divalent metal ions to the binding of drugs to DNA, CD spectrum of TMPyP were obtained in figure 4. In the absence of divalent metal ions, a negative Soret absorption band appeared at around 450 nm (curve a), which is a typical signal for intercalation of TMPyP between DNA base-pairs[28].

Fig.4 CD spectrua of DNA-TMPyP-ethidium complex in the presence (curve bf) and absence (curve a) of 100 μ mol· L-1 divalent metal ions. The presence of 0.5 μ mol· L-1 ethidium did not alter the spectrum. The concentrationsand curve assignment are identical to those in Figure 2

Addition of alkali earth metal ions (Mg2+ and Ca2+) (100 μ mol· L-1) induced a bisignate signal in the Soret absorption band; a positive signal at around 425 nm and a negative signal at 450 nm, which is regarded as a moderate stacking of TMPyP at the outside of DNA[28]. Addition of transition metal ions leads to stronger positive signals so that the negative signal at 450 nm was buried. Therefore, one can assume that binding of metal ions may influence the topology of DNA or the binding conformation of TMPyP to DNA. Considering the DNA absorption region, addition of alkali earth metal ions slightly reduced the DNA CD intensity at around 280 nm (figure 4a and b), but the spectral shape was almost preserved, indicating that there is no significant change in the DNA morphology. Whereas, transition metal ions induced not only severe intensity decrease but also the change of spectral shape. This may propose that the addition of transition metal ions brought a severe conformational change of DNA, such as DNA bending or base-pair disturbance. However, this assumption was excluded by the comparison of linear dichroism (referred to as LD) spectrum of DNA in the presence of metal ions (data not shown). All metal ions induced negligible decrease of LD intensity of DNA; Ni2+ showed the largest decrease of LD intensity of 15% without considerable change of spectral shape. This observation indicates that metal ions did not induce considerable change in DNA morphology or stacking of DNA base pairs. Hence, the spectral changes of CD of TMPyP-DNA complex are presumably caused mainly by the change of binding conformation of TMPyP in DNA.

The main factor of DNA-mediated energy transfer is known to be the stacked π -orbitals of base-pairs. Hence, the preservation of base-pair stacking in DNA might be critical to the energy transfer efficiency. It is well known that both ethidium and TMPyP intercalate between DNA-base pairs, indicating that the π -orbitals of these molecules indeed lie parallel with DNA base-pairs. Generally, intercalators are apt to stabilize DNA stem, since intercalation enhances the stacking effect of DNA base-pairs. Therefore, metal ions are supposed to bind not to intercalators but to DNA, and somehow enhance the quenching efficiency. In the comparison of binding conformation of TMPyP to DNA based on the CD spectral change in figure 4, considerable part of DNA was preserved when Mg2+ and Ca2+ were added, so that the energy transfer efficiency was highly enhanced. Whereas, Mn2+, Co2+ and Ni2+ displayed much smaller efficiencies and this can be explained by the significant change in the TMPyP binding conformation in DNA.

3 Conclusion

Binding of metal ions displayed an enhancement effect on DNA-mediated energy transfer between simultaneously intercalated molecules. The magnitude of enhancement was remarkable by the presence of alkali earth metal ions comparing to that from in the presence of transition metal ions. This difference can be explained by the binding conformation of intercalated donor or acceptor molecules which were differently altered by the binding site of those metal ions; alkali earth metal ions bind to the phosphate group of DNA while transition metal ions to the DNA base.

The authors have declared that no competing interests exist.

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