Electrochemistry Communications 11 (2009) 2101–2104

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Electrochemistry Communications

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Electrochemical impedance probing of transcriptional TATA binding proteinbased on TATA box site-specific binding

Haixin Chang, Jinghong Li *

Department of Chemistry, Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology, Tsinghua University, Beijing 100084, China

a r t i c l e i n f o a b s t r a c t

Article history:Received 20 August 2009Received in revised form 31 August 2009Accepted 5 September 2009Available online 12 September 2009

Keywords:TATA binding proteinElectrochemical impedance spectroscopyDNATriplex formation oligonucleotides

1388-2481/$ - see front matter � 2009 Elsevier B.V. Adoi:10.1016/j.elecom.2009.09.004

* Corresponding author. Tel./fax: +86 10 62795290E-mail address: jhli@mail.tsinghua.edu.cn (J. Li).

Electrochemical impedance probing of TATA binding protein (TBP) based on TATA box site-specific bind-ing was described in this work. A sensitive detection of TBP was developed from TATA box DNA selfassembly on the electrode and the impedance changes induced by TBP binding. Electrochemical imped-ance spectroscopy (EIS) probing of TBP had a sensitivity of 0.8 nM with excellent selectivity. Moreover,the interferences of triplex forming oligonucleotides (TFOs) and anticancer drug daunomycin on TBPbinding to TATA box DNA were investigated by EIS. TFOs reduced the stability of TBP binding to TATAbox, but daunomycin completely inhibited the TBP binding.

� 2009 Elsevier B.V. All rights reserved.

1. Introduction

TATA binding protein (TBP) is one kind of transcriptional pro-tein which binds TATA box section in genomic sequences. TBPplays a central role in gene transcription initiation and is neededin all the three eukaryotic RNA polymerases [1,2]. Therefore prob-ing the binding of TBP to TATA box is helpful for the understandingtranscription initiation and gene regulation. Generally, the bindingof TBP to TATA box DNA is probed using fluorescent method, suchas engineered TBP protein with special residue [2], using expressedprotein with epitope tag to conjugate fluorescent antibody [3], orcombination of chromatin immunoprecipitation and DNA micro-array [4]. To avoid DNA amplification or protein labeling in fluores-cent methods, electrical detection of TBP binding to TATA box wasrecently developed based on the electron transport disturbing ofDNA upon protein binding [5].

Electrochemical impedance spectroscopy (EIS) is broadly usedto monitor interfacial changes induced by a variety of biomolecularrecognition events, such as immunoassay [6], aptamer based bio-sensing [7], DNA hybridization and replication interactions [8],specific drug bindings [9], and cell culture monitoring [10,11].However, to the best of our knowledge, no work about TBP probingis reported based on EIS. Here a sensitive and specific impedanceprobing of TBP was developed from TATA box DNA self assemblyon electrode and impedance changes induced by TBP binding.Moreover, the influences of triplex forming oligonucleotides (TFOs)

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on TBP binding to TATA box DNA duplexes was investigated be-cause of versatile potentials of TFOs in biological applications suchas gene transcription modulation, genomic mutagenesis directionand therapeutic possibilities [12–16]. The influence of anticancerdrug daunomycin on TBP binding to TATA box DNA duplexes wasalso studied by EIS.

2. Experimental sections

2.1. Materials and chemicals

Potassium ferricyanide and ferrocyanide were from Tianjin Re-agent (Tianjin, China) with analytical grade purity. Daunomycinwas from Sigma (USA). Human TBP recombinant protein (molecu-lar weight 38.54 kDa) was bought from Abnova corporation (Tai-wan), and diluted with PBS (pH 7.0) when used. Bovine serumalbumin protein (BSA) was from Dingguo Biotech (Beijing).

All the DNA sequences were synthesized by Takara Corporation(Dalian). DNA I probe is a purine rich TATA box DNA sequencewhich combines TATA box section with a well studied polypurinesequence in the Chinese hamster hprt gene (DNA I) [17]. The italicbold part in DNA I probe is the specific binding site of TBP. Under-lined sequence in DNA I probe is the target site of TFOs. The DNAsequences used are listed below:

DNA I Probe: 50-SH-GGAGGCTATAAAAGAAGAAAAAAGAGAAATC-TGAC-30

complementary: 50-GTCAGATTTCTCTTTTTTCTTCTTTTATAGCCT-CC-30

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A

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Fig. 1. (A) Nyquist plots of DNA I duplexes modified gold electrode before and afterinteraction with TBP of different concentrations. Inset: Ret versus TBP concentration.(B) Nyquist plots of DNA I duplexes modified gold electrode before and afterinteraction with BSA and TBP. Electrolyte: 10 mM PBS (pH 7.0) with 10 mM[Fe(CN)6]3�/4� and 10 mM NaCl. Frequency: from 0.1 Hz to 100 k Hz, Amplitude:10 mV, Bias potential: +0.20 V.

2102 H. Chang, J. Li / Electrochemistry Communications 11 (2009) 2101–2104

TFOs sequences for DNA I: Complementary: 50-GAGAAAAA-AGAAGAAAA-30

G mismatch 50-GAGAAAAAGGAAGAAAA-30

C mismatch 50-GAGAAAAACGAAGAAAA-30

2.2. TATA box DNA self assembly and TBP binding

DNA I probes first hybridized with their complementary targetsin 10 mM PBS buffer (pH 7.0) with 100 mM NaCl at 40 �C for over24 h to form duplexes with the ratio of targets to probes 5:1. Thenthe gold electrode was incubated with 8 lL DNA I duplexes in hu-mid environment for over 16 h followed by 15 min blocking in1 mM 6-Mercapto-1-hexanol (MCH). TATA box DNA duplexesmodified gold electrode was incubated with different concentra-tion TBP for 30 min. The binding buffer is 5 mM PBS buffer (pH7.0) with 50 mM NaCl and 4 mM Mg2+. For kinetic researches,DNA I duplexes modified electrode was incubated in 80 nM TBPwith different time. The disassociation of TBP from TATA boxDNA was conducted by washing electrodes in the PBS buffer.

To study the influence of TFOs on TBP binding to TATA boxDNA, DNA I duplexes modified gold electrode was incubated with8 lL complementary TFO or single mismatch TFOs in 10 mM PBSbuffer (pH 7.0) with 50 mM NaCl and 10 mM Mg2+ for over 16 hto form triplexes [12,18]. For daunomycin influence evaluation,the TATA box duplexes modified electrode was incubated in5 mM PBS (pH 7.0, 50 mM NaCl) with 1.77 mM daunomycin for30 min.

2.3. Electrochemical measurements

Electrochemical impedance measurements were performedusing an electrochemical system PARSTAT 2273 (Princeton AppliedResearch) with one three-electrode system. TATA box DNAmodified gold electrode was used as the working electrode witha platinum wire as the counter electrode and a KCl saturated Ag/AgCl as the reference electrode. EIS measurements were conductedin 10 mM PBS (pH 7.0) buffer with 10 mM K4[Fe(CN)6], 10 mMK3[Fe(CN)6] and 10 mM NaCl at room temperature. The EIS datawere recorded from 0.1 Hz to 100 k Hz with the amplitude of10 mV and the applied bias potential of + 0.20 V.

3. Results and discussion

As shown in Fig. 1A, the electron transfer impedance Ret in-creases with the concentration of TBP. The increases of impedancesare due to the increasing barrier for electron transfer induced bybinding of TBP. Ret has a good linear relationship with TBP concen-tration c in the range of 0.8–68.8 nM with the linear equationRet = 33.665c + 1389.8 and linear regression coefficient of 0.96. Thislabel free EIS method can detect at least 0.8 nM TBP binding toTATA box duplexes and has comparable even relatively higherdetection limit compared with complicated, labeled method ofmonitoring electron transfer interruption in DNA (3 nM) [5]. More-over the associate constant (Ka) of TBP binding to TATA box DNAduplexes can be determined from the EIS data by following equa-tion (1) [19]:

Ret;i � Ret;0

Ret;0¼ DRet

Ret;0¼ Kac ð1Þ

Ret,0 is the impedance of TATA box duplexes modified gold elec-trode, and Ret,j is the impedance after TBP binding. Detail calcula-tions show the linear relationship between DRet/Ret,0 and TBPconcentration. The regression equation is DRet/Ret,0 = 0.0438c withthe regression coefficient of 0.91. Ka can be deduced from the slope

of linear regression equation to be 4.38 � 107 M�1. The disassociateconstant Kd (22.8 nM) can be calculated from 1/Ka and is close to thedisassociate constants obtained from electrophoretic results [20].

The specificity of EIS on monitoring TBP binding to TATA boxduplexes was investigated with BSA as the referring protein. Theduplexes modified electrodes were first incubated in BSA followedby incubation in TBP. As shown in Fig. 1B, dramatic impedance in-creases occurred when incubated in 80 nM TBP. However, no sig-nificant impedance changes were observed after 30 minincubation in 5 lM BSA, indicating quite good selectivity of EISdetection of TBP.

To study the influences of TFOs on TBP binding to TATA box DNAduplexes, time dependent EIS measurements of TBP binding and dis-association were conducted. As shown in Fig. 2, impedance Ret in-creased with binding time in the TBP binding process andsaturated at an equilibrium point, and then decreased with the in-crease of washing time in disassociation process. Disassociate rateconstant kd can be calculated from the impedance data in disassoci-

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Fig. 2. Nyquist plots of TBP binding to DNA I duplexes (A), DNA I duplexes/complementary TFO (B), DNA I duplexes/C mismatch TFO (C), TBP and DNA I duplexes/G mismatchTFO (D). TBP concentration: 80 nM. Insets in A, B, C are the calculated Ret in binding and washing process. Electrolyte: 10 mM PBS (pH 7.0) with 10 mM [Fe(CN)6]3�/4� and10 mM NaCl. Frequency: from 0.1 Hz to 100 kHz, Amplitude: 10 mV, Bias potential: +0.20 V.

H. Chang, J. Li / Electrochemistry Communications 11 (2009) 2101–2104 2103

ation process by plotting lnRet/Ret,eq with disassociation time, whereRet,eq is the impedance at equilibrium point [19]. For TATA box DNAduplexes, through the plot of lnRet/Ret,eq with disassociation time,the linear relationship can be described by linear regression equa-tion lnRet/Ret,eq = �1.62 � 10�4 t with the regression coefficient of0.95, and kd is calculated to be 1.62 � 10�4 s�1 from the slope ofthe equation. Similarly, the linear regression equation for TATAbox DNA duplex in the presence of complementary TFO, C mismatchTFO, G mismatch TFO are lnRet/Ret,eq = �3.02 � 10�4 with the regres-sion coefficient of 0.95 , lnRet/Ret,eq = �1.45 � 10�4 t with the regres-sion coefficient of 0.98, lnRet/Ret,eq = �1.68 � 10�4 t with theregression coefficient of 0.98, and the corresponding kd values are3.02 � 10�4 s�1, 1.45 � 10�4 s�1 and 1.68 � 10�4 s�1, respectively.

As compared in Fig. 3A, disassociate rate constant kd of TBP fromTATA DNA duplexes in the presence of complementary TFO isabout twice as many as that of TBP from TATA DNA duplexes.For one single mismatch TFOs, no matter C or G mismatch, kd mea-sured is very close to TATA DNA duplexes. It is consistent with thefact that one single mismatch in TFO prohibits the triplex forma-tion between duplexes and TFOs [18]. These results indicate triplexformation at AAAG section in TATA box reduces the stability of TBPbinding to TATA box but does not fully inhibit the TBP binding toTATA box. It may be ascribed to the fact that TFOs just interactswith TATA DNA duplexes by weak reverse hoogsteen hydrogen

bonds with no much change of the base pairs stack in TATA DNAduplexes [12].

In contrast with TFOs, anticancer drug daunomycin completelyinhibits the TBP binding to TATA box. For untreated TATA box DNAI duplexes, the impedance increased with the incubation of TBP.After treated with daunomycin, the impedance of TATA box du-plexes modified electrode showed no increase when incubatedwith TBP as shown in Fig. 3B. These results demonstrated that dau-nomycin completely inhibited the TBP binding to TATA box du-plexes. The reasons may come from the disturbing of base pairsstacks in TATA box section induced by the intercalations of dauno-mycin in duplexes, resulting in the conformation changes and dis-tortion in TATA box recognition site. Therefore the intercalated,distorted TATA box may be not coordinated with TBP and not rec-ognized by TBP [1,2].

4. Conclusion

In summary, a label free, highly sensitive TBP probing by EISwas developed based on TATA box site-specific binding. The EISdetection of TBP has a sensitivity of 0.8 nM with excellent selectiv-ity. Moreover, EIS provides an alternative route to study the effectof a variety of interferences (TFOs, anticancer drugs, etc.) on TBP

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Fig. 3. (A) Plot of lnRet/Ret,eq with disassociation time for TBP from DNA I duplexes,or DNA I duplexes in presence of complementary or mismatched TFOs, Ret,eq:impedance at equilibrium point. (B) Impedance responses versus time for TBPbinding to DNA I duplexes or daunomycin treated DNA I duplexes. TBP concentra-tion: 80 nM.

2104 H. Chang, J. Li / Electrochemistry Communications 11 (2009) 2101–2104

binding to TATA box, which is very helpful in the understanding ofbiological function of TBP.

Acknowledgments

This work was financially supported by the National NaturalScience Foundation of China (No. 20675044), National Basic Re-search Program of China (Nos. 2007CB310500 and 28-AZC0901)and China Postdoctoral Science Foundation (No. 20080430349).

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