冀中華 朱 斌 李經(jīng)建,* 蔡生民 袁 谷 徐東升

(1北京大學化學與分子工程學院物理化學研究所,北京分子科學國家實驗室,北京100871; 2北京大學化學與分子工程學院化學生物學系,生物有機與分子工程教育部重點實驗室,北京100871)

1 Introduction

Crescent-shaped polyamides containing N-methyl pyrroles and N-methyl imidazoles are a type of small molecules that can be recognized and bound in the minor groove of DNA base-pairs with high levels of affinity and specificity.1,2A detailed interaction mechanism of combination with DNA has been reported,and a molecular recognition rule between DNA and polyamide was established.3-5The research of strategy to distinguish the four Watson-Click base-pairs by small molecules as well as polyamides may allow regulating gene expression in living cells and may have a potential application in gene therapeutics.6-8

Recently,we developed a novel polyamide-modified electrode to investigate DNA-mediated charge transport(CT)in the polyamide-bound minor groove which was sensed by a redox probe[Ru(NH3)6]3+/[Ru(NH3)6]2+.9,10The experiment results support thata broadened π-stack is formed by partial overlap of the π orbitals of the DNA base-pair and the polyamide heterocycle.This broadened π-stack provides a pathway for charge transport during the reduction of[Ru(NH3)6]3+.However,as one of the most important macromolecules,the DNA is fully charged negatively and strongly exhibits polyelectrolyte properties in solution.The electrostatic attraction is well-known as one of the binding modes for small molecule-DNA interactions. Electrostatic potentials near the surface of DNA were calculated using the classical,nonlinear,as well as modified Poisson-Boltzmann equation.11-17[Ru(NH3)6]3+was also established as a probe to quantify the surface density of DNA immobilized.18,19Although some studies investigated the ionic effects on electrostatic potentials of DNA surface,12,13,15,17but little is known about the relationship between the electrochemical potential of ligand binding DNA and the electrostatic interaction function on DNA-mediated CT.

In this paper,we focused on the effect of the ionic strength for DNA-mediated CT on an electrode modified with polyamide.The influence of the ionic strengths of the solution on the interaction between[Ru(NH3)6]3+and DNA was investigated by differential pulse voltammetry(DPV)and cyclic voltammogram(CV).

The polyamide PyPyPyγImImImβCOOH(where,Py=N-methylpyrrole,Im=N-methylimidazole,γ=γ-aminobutyric acid, β=β-alanine)was synthesized as previously described,20and the structure is shown in Scheme 1.

2 Experimental

2.1 Chemicals

The gold substrate was fabricated by sputter-coating sequentially with a 10 nm Ti adhesion layer and a 100 nm Au layer on a silicon wafer.11-Amino-1-undecanethiol and Ru(NH3)6Cl3were purchased from Sigma-Aldrich and used as received.

Scheme 1 Structure of PyPyPyγImImImβCOOH

DNAoligonucleotides with sequence 5?-TTAGGGTTAGGG-3?and its complementary sequence 3?-AATCCCAATCCC-5? were synthesized and purified by AuGCT Biotechnology Co., Ltd.(Beijing,China).Duplexes were obtained in deoxygenated 10 mmol·L-1Tris-HCl(pH 7)solution by heating to 94°C for 3 min followed by slow cooling to room temperature. According to Dervan?s recognition rule for polyamide and DNA,3-5this DNA sequence can be recognized by the polyamide(PyPyPyγImImImβCOOH).

2.2 Preparation of polyamide modified electrode

The polyamide modified electrodes were prepared as described previously by self-assembling 11-amino-1-undecanethiol on the gold substrate and covalently linked to PyPyPyγIm-ImImβCOOH.9

In order to eliminate the effect for the measurement of DNA-mediated CT from the pinhole on the gold electrode,the electropolymerization of 2-naphthol on the polyamide-modified electrode surface was employed for passivating the electrode.21Then 50 μL of 50 μmol·L-1DNA duplex solution was dropped on the passivated polyamide-modified electrode for interaction and later rinsed copiously amounts of water.

2.3 Electrochemical measurements

Electrochemical experiments were performed on CHI 706 electrochemistry workstation(CHI Instrument Co.,USA)using one compartment homemade electrolytic cell(volumetric capacity 3 mL)with a three-electrode configuration.A gold electrode with 0.21 cm2was used as the working electrode.A Pt coil was used as the counter electrode and a saturated calomel electrode(SCE)served as the reference electrode.The differential pulse voltammetry operated at:pulse amplitude 50 mV,pulse width 70 ms,potential increasement 4 mV.Water was deionized using a Milli-Q purification system(Millipore Products,Bedford,MA).All experiments were performed at ambient temperature.

3 Results and discussion

The DPVs of[Ru(NH3)6]3+/[Ru(NH3)6]2+on polyamide-DNA modified electrode are shown in Fig.1(A).The peak current decreases and the peak potential shifts negatively with the increase of KCl concentration.Fig.1(B)depicts relationship between the peak potential and square root of ionic strength(I). With increasing of solution ionic strength,the peak potential shifts negatively at middle region of ionic strength(1(mol·m-3)1/2＜I1/2＜10(mol·m-3)1/2)following the fit function:

Fig.1 (A)Differential pulse voltammetry in 0.1 mmol·L-1[Ru(NH3)6]3+/[Ru(NH3)6]2+on polyamide-DNAmodified electrode under different KCl concentrations;(B)peak potential shift as a function of square root of solution ionic strength

However,at higher ionic strength(I1/2＞10(mol·m-3)1/2)and lower ionic strength(I1/2≈0.7(mol·m-3)1/2),without KCl),the peak potentials deviate from the Eq.(1).

The peak potential Epof DPV is related to the formal potential Ep0?with the following equation,22

where n is the number of electron in the electrode reaction,DRand DOare the diffusion coefficient of the reduced form and oxidized form respectively,ΔE is the pulse amplitude(50 mV),R and F are gas constant and Faraday constant,respectively.T is absolute temperature.As T is given,the peak potential Epwill depend on the formal potential E0?and the ratio of diffusion coefficient.

Reductive peak currents from cyclic voltammograms of 0.1 mmol·L-1[Ru(NH3)6]3+at polyamide-DNA modified electrode as a function of the concentration of KCl are shown in Fig.2.It can be seen that the peak currents linearly increase with increasing the square root of scan rate and the slope of the plot is insensitive to variations in solution salt concentration under our experimental condition.The maintenance of slope of ipvs v1/2reflects the diffusion coefficient of the[Ru(NH3)6]3+basically unchanged within the variation range of KCl concentration. In general,the diffusion coefficients of both reduced and oxidized forms can be regarded as equivalent,therefore the peak potential Epof DPV is only proportional to the formal potential E0?.The formal potential incorporates the standard potential and activity coefficient of reactive species in Nernst equation,

where fIIIand fIIare activity coefficients of[Ru(NH3)6]3+and [Ru(NH3)6]2+respectively and they depend on the ionic strength of solution.E0is the standard potential of[Ru(NH3)6]3+/ [Ru(NH3)6]2+,which is a constant at given temperature.Thus the change of formal potential with the ionic strength is given by Eq.(4):

There are several electrostatic interactions in the interface between the polyamide-DNA modified electrode and solution, polyanion DNA-counter ion and polyanion DNA-[Ru(NH3)6]3+/ [Ru(NH3)6]2+.Substituting a counter ion,the electroactive [Ru(NH3)6]3+electrostatically interacts with the negatively charged phosphate groups and is adsorbed on the DNA backbone. Because of a high-affinity of hairpin polyamide with DNA (binding constant K:～107mol·L-1),23the amount of DNA binding on electrode is unchanged during varying the concentration of KCl.Therefore the charges of DNA backbone are maintained and uniformly distributed throughout the surface of modified electrode.Under this premise,we can consider the interaction between[Ru(NH3)6]3+and added KCl only and [Ru(NH3)6]3+is regarded as a center ion accordingly.With the change of KCl concentration,the ionic strength of solution will be changed,which results in the correspondant change of activity coefficient of[Ru(NH3)6]3+/[Ru(NH3)6]2+.It is well-known that the fact:practical existed electrostatic interaction induced the deviations from theoretical ideality is expressed in the term of the activity coefficient of chemical potential.In terms of Debye-Hückel theory,for 1 mol center ion,the contribution of electro-static interaction to the chemical potential of the center ion is given by:

Fig.2 Plot of the peak current(ip)of 0.1 mmol·L-1[Ru(NH3)6]3+ as a function of square root of scan rate(v1/2)at various KCl concentrations

where a is the radium of the center ion.Because 1＞＞κa,so

Use of Eq.(6)in Eq.(4)gives

where zIIIand zIIare the valences of the[Ru(NH3)6]3+and [Ru(NH3)6]2+respectively,R=NAk,NAand k are Avogadro and Boltzmann constants,respectively,with the elemental charge e=1.602×10-19C,the vacuum dielectric constant ε0=8.85×10-12C2·m-2·N-1,and the dielectric coefficient εr=78.2 for water at 25°C,ΔE0?varies linearly within the range of validity of the Debye-Hückel theory(Eq.(7)).Comparing the slope of Eq. (1)and Eq.(7)shows that there is no notable difference at the range of error under middle region of ionic strength(1(mol·which suggests that the change of chemical potential of redox ion gives rise to a negative shift of reductive potential of[Ru(NH3)6]3+when the ionic strength changes in solution.In nature,because of ionic screening from added K+,the electrostatic attraction of DNA to[Ru(NH3)6]3+is weakened with increasing the salt concentration.This screen effect increases the barrier height for the reductive reaction of [Ru(NH3)6]3+on the electrode and then rises the negative shift of DPV peak potential.Consequently the peak potential Epof DPV,a parameter of transient dynamics,relates to the chemical potential with formal potential as a“bridge”based on the Debye-Hückel theory.

Moreover,with the increase of ionic strength in solution,the activity coefficient shall deviate from the Debye-Hückel Limiting Law.So the peak potential of DPV in Fig.1b negatively deviated from the linear relationship with square root of ionic strength

However,the peak potential positively deviated from the linear relationship in the absence of support electrolyte KCl and only with Tris-HCl available in solution.The deviation extent could be evaluated by the difference between the experiment peak potential(-0.404 V)and linear relationship predicted data (-0.414 V).We believe that the positive deviation arises from the strong electrostatic effect of DNA backbone,which facilitates the charge transport between[Ru(NH3)6]3+and polyamide-DNAcomplex without the screen effect from added K+.

In conclusion the DNA mediated CT in the polyamidebound minor groove can be carried out by two steps.Firstly, the probe[Ru(NH3)6]3+is electrostatically adsorbed on a negatively charged DNA backbone.Secondly,the broadened π-stack between the DNA base-pair and the polyamide heterocycle provides a pathway for CT during the reduction of [Ru(NH3)6]3+.

4 Conclusions

The ionic strength dependence of DNA mediated charge transport was investigated on the DNA binding polyamidemodified electrode by electrochemical method.The peak potential of DPV of[Ru(NH3)6]3+shifted negatively with the increase of support electrolyte concentration.A linear relationship was obtained between peak potential and square root of ionic strength in the solution at the range of moderate salt concentration,which could be interpreted with Debye-Hückel theory using formal potential as a“bridge”.At higher salt concentration, however,the relationship between peak potential and square root of ionic strength deviated from Debye-Hückel theory prediction.Without the added salt in solution,full charged DNA duplex strongly attracted the probe[Ru(NH3)6]3+to the broadened π-stack of the DNA-polyamide complex,which facilitated DNAmediated charge transport.

Acknowledgment: Professor LIU Zhong-Fan(Institute of Physical Chemistry,College of Chemistry and Molecular Engineering,Peking University)is thanked for significant discussion.

(1) Arcamone,F.;Nicolell,V.;Penco,S.;Orezzi,P.;Pirelli,A. Nature 1964,203,1064.doi:10.1038/2031064a0

(2) Zimmer,C.;Wahnert,U.Prog.Biophys.Mol.Biol.1986,47, 31.doi:10.1016/0079-6107(86)90005-2

(3)White,S.;Szewczyk,J.W.;Turner,J.M.;Baird,E.E.;Dervan, P.B.Nature 1998,391,468.doi:10.1038/35106

(4) Kielkopf,C.L.;White,S.;Szewczyk,J.W.;Turner,J.M.; Baird,E.E.;Dervan,P.B.;Rees,D.C.Science 1998,282,111. doi:10.1126/science.282.5386.111

(5) Dervan,P.B.Bioorganic&Medicinal Chemistry 2001,9,2215. doi:10.1016/S0968-0896(01)00262-0

(6) Blasko,A.;Browne,K.A.;Bruice,T.C.J.Am.Chem.Soc. 1994,116,3726.doi:10.1021/ja00088a008

(7) Gottesfeld,J.M.;Neely,L.;Trauger,J.W.;Baird,E.E.; Dervan,P.B.Nature 1997,387,202.doi:10.1038/387202a0

(8) Olenyuk,B.;Jitianu,C.;Dervan,P.B.J.Am.Chem.Soc.2003, 125,4741.doi:10.1021/ja0213221

(9) Ji,Z.H.;Li,J.J.;Zhu,B.;Yuan,G.;Cai,S.M.Electrochem. Commun.2007,9,1667.doi:10.1016/j.elecom.2007.03.020

(10) Cao,R.G.;Zhu,B.;Liu,W.;Li,J.J.;Xu,D.S.Acta Phys.-Chim.Sin.2010,26,1119. [曹瑞國,朱 斌,劉 巍,李經(jīng)建,徐東升.物理化學學報,2010,26,1119.]doi:10.3866/ PKU.WHXB20100427

(11) Boschitsch,A.H.;Fenley,M.O.J.Comput.Chem.2004,25, 935.doi:10.1002/(ISSN)1096-987X

(12) Markus,D.;Felips,J.A.;Chistian,H.;Marcelo,L.C.J.Phys. Chem.B 2001,105,10983.doi:10.1021/jp010861+

(13) Misra,V.K.;Honig,B.Proc.Natl.Acad.Sci.U.S.A.1995,92, 4691.doi:10.1073/pnas.92.10.4691

(14) Gavryushov,S.;Zielenkiewicz,P.J.Phys.Chem.B 1999,103, 5860.doi:10.1021/jp983081i

(15) Polozov,R.V.;Dzhelyadin,T.R.;Sorokin,A.A.;Ivanova,N. N.;Sivozhelezov,V.S.;Kamzolova,S.G.Journal of Biomolecular Structure&Dynamics 1999,16,1135.doi: 10.1080/07391102.1999.10508322

(16) Gavryushov,S.;Zielenkiewicz,P.Biophys.J.1998,75,2732. doi:10.1016/S0006-3495(98)77717-3

(17) Grandison,S.;Penfold,R.;Vanden-Broeck,J.M.Phys.Chem. Chem.Phys.2005,7,3486.

(18) Steel,A.B.;Herne,T.M.;Tarlov,M.J.Anal.Chem.1998,70, 4670.doi:10.1021/ac980037q

(19)Yu,H.Z.;Luo,C.Y.;Sankar,C.G.;Sen,D.Anal.Chem.2003, 75,3902.doi:10.1021/ac034318w

(20)Xiao,J.H.;Yuan,G.;Huang,W.Q.;Chan,A.S.C.;Lee,K.L. D.J.Org.Chem.2000,65,5506.doi:10.1021/jo000135n

(21)Boon,E.M.;Ackson,J.N.M.;Wightman,M.D.;Kelley,S.O.; Hill,M.G.;Barton,J.K.J.Phys.Chem.B 2003,107,11805. doi:10.1021/jp030753i

(22) Bard,A.J.;Faulkner,L.R.Electrochemical Methods Fundamentals and Applications,2nd ed.;John Wiley&Sons Inc.:New York,2001;p 290.

(23) Pilch,D.S.;Poklar,N.;Baird,E.E.;Dervan,P.B.;Breslauer, K.J.Biochemistry 1999,38,2143.doi:10.1021/bi982628g