Sensitive Electrochemical Determination of Unlabeled MutS Protein

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Anal. Chem. 2004, 76, 5930-5936

Sensitive Electrochemical Determination of Unlabeled MutS Protein and Detection of Point Mutations in DNA Emil Palecˇek,*,† Michal Masarˇı´k,† Rene Kizek,†,‡ Dirk Kuhlmeier,§ Jo 1 rg Hassmann,§ and § Ju 1 rgen Schu 1 lein

Institute of Biophysics, Academy of Sciences of the Czech Republic, Kralovopolska 135, CZ-612 65 Brno, Czech Republic, and november AG, Gesellschaft fu¨r Molekulare Medizin, Ulrich-Schalk-Strasse 3, D-91056 Erlangen, Germany

MutS protein plays an important role in the DNA repair system in prokaryotic and eukaryotic cells; it recognizes unpaired and mispaired bases in duplex DNA and can be used for detection of point mutations in vitro. We have shown that small amounts of this protein can be detected electrochemically at mercury and carbon electrodes without any labeling. Using constant current stripping analysis (CPSA) and mercury electrodes, tens of attomoles of this protein can be detected. The sensitivity of the determination at carbon electrodes is by more than 3 orders of magnitude lower. Using biotinylated DNA duplexes attached to magnetic beads, single-base mismatches and insertion/deletions were recognized by MutS. Picogram amounts of this protein were detected by CPSA after MutS releasing from the beads. DNA mismatch repair plays a critical role in genomic integrity and replication fidelity in both prokaryotes and eukaryotes (reviewed in ref 1). MutS protein is a member of the ABC ATPase superfamily and an important part of the DNA repair system. This protein recognizes unpaired and mispaired bases in duplex DNA and initiates DNA mismatch repair. The crystal structure of MutS and its complexes with substrate DNAs were reported.2,3 Mutations in human MutS genes result in hereditary predisposition to colorectal cancer and sporadic tumors.3 Many genetic disorders are caused by mutations in different genome sites, and commonly, single-nucleotide polymorphisms (SNPs) in the human genome are detected and intensively studied. Reliable screening tools securing fast and inexpensive detection of mismatch base pairs in human DNA are therefore sought after. Several methods for identification of SNPs were developed to circumvent the time-consuming direct sequencing methods. Most of them are based on DNA hybridization with optical (reviewed * Corresponding author: (e-mail) [email protected]; (phone) 420541517177; (fax) 420 541211293. † Academy of Sciences of the Czech Republic. ‡ Present address: Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University of Agriculture and Forestry, Zemedelska 1, 613 00 Brno, Czech Republic. § Gesellschaft fu ¨ r Molekulare Medizin. (1) Jiricny, J. Curr. Biol. 2000, 10, R788-R790. (2) Obmolova, G.; Ban, C.; Hsieh, P.; Yang, W. Nature 2000, 407, 703-710. (3) Lamers, M. H.; Perrakis, A.; Enzlin, J. H.; Winterwerp, H. H.; de Wind, N.; Sixma, T. K. Nature 2000, 407, 711-717.

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in ref 4) and recently also with electrochemical detection (reviewed in refs 5-11). Several papers have been published using MutS protein to detect mismatched bases in DNA.12-16 Binding of MutS to its DNA binding sites was detected in various ways, including quartz crystal microbalance,16 protection of DNA from exonucleolytic cleavage,15 or detection of radioactively or fluorescence-labeled MutS protein.12 To our knowledge, no attempt has been made to detect the DNA MutS binding by electrochemical methods. Electrochemical analysis of proteins was successfully applied in bioanalysis for several decades in the middle of the 20th century.17 Later electrochemists turned their attention to direct electrochemistry of a limited number of redox-active centercontaining proteins (reviewed in ref 18) undergoing fast electrontransfer electrode processes, and the potentialities of the electrochemical methods as tools for protein analysis in biochemistry and biomedicine were neglected. The possibilities of electrochemical analysis of unlabeled proteins not containing any redox center for direct electrochemistry are limited to oxidation of tyrosine and tryptophan residues at carbon electrodes19,20 (and references therein) or reactions involving cystine/cysteine residues such as the formation and reduction of Hg-S bonds, reduction of the disulfidic group,21 or the catalytic hydrogen evolution in cobalt(4) Heller, M. J. Annu. Rev. Biomed. Eng. 2002, 4, 129-153. (5) Wang, J. Anal. Chim. Acta 2003, 500, 247-257. (6) Wang, J. Chem. Eur. J. 1999, 5, 1681-1685. (7) Umek, R. M.; Lin, S. W.; Vielmetter, J.; Terbrueggen, R. H.; Irvine, B.; Yu, C. J.; Kayyem, J. F.; Yowanto, H.; Blackburn, G. F.; Farkas, D. H.; Chen, Y. P. J. Mol. Diagn. 2001, 3, 74-84. (8) Popovich, N. D.; Thorp, H. H. Interface 2002, 11, 30-34. (9) Palecek, E.; Fojta, M.; Jelen, F. Bioelectrochemistry 2002, 56, 85-90. (10) Palecek, E. Talanta 2002, 56, 809-819. (11) Palecek, E.; Fojta, M. Anal. Chem. 2001, 73, 74A-83A. (12) Behrensdorf, H. A.; Pignot, M.; Windhab, N.; Kappel, A. Nucleic Acids Res. 2002, 30, e64. (13) Bi, L. J.; Zhou, Y. F.; Zhang, X. E.; Deng, J. Y.; Zhang, Z. P.; Xie, B.; Zhang, C. G. Anal. Chem. 2003, 75, 4113-4119. (14) Biswas, I.; Hsieh, P. J. Biol. Chem. 1996, 271, 5040-5048. (15) Sachadyn, P.; Stanislawska, A.; Kur, J. Nucleic Acids Res. 2000, 28, e36. (16) Su, X. D.; Robelek, R.; Wu, Y. J.; Wang, G. Y.; Knoll, W. Anal. Chem. 2004, 76, 489-494. (17) Brezina, M.; Zuman, P. Polarography in Medicine, Biochemistry and Pharmacy; Interscience: New York, 1958. (18) Armstrong, F. A. In Bioelectrochemistry; Wilson, G. S., Ed.; Wiley-VCH: Weinheim, 2002; Vol. 9, pp 11-29. (19) Palecek, E.; Jelen, F.; Teijeiro, C. Anal. Chim. Acta 1993, 273, 175-186. (20) Tomschik, M.; Havran, L.; Fojta, M.; Palecek, E. Electroanalysis 1998, 10, 403. 10.1021/ac049474x CCC: $27.50

© 2004 American Chemical Society Published on Web 08/27/2004

Table 1. 30-Mer DNA Duplexes Used for MutS Binding

containing solutions at mercury electrodes (Brdicka reaction, reviewed in ref 22) and to ac voltammetric studies of protein adsorption/desorption behavior. The so-called dc polarographic pre-sodium wave of proteins (obtained with mercury dropping electrodes) (reviewed in refs 17, 22, and 23) was little used for analytical purposes because its shape was poorly developed due to its highly negative potential, close to the background discharge. Using the constant current chronopotentiometric stripping analysis (CPSA), we recently observed a well-developed peak at nanomolar peptide concentrations at a hanging mercury drop electrode (HMDE).20 This peak, which is due to the catalytic hydrogen evolution, appeared at highly negative potentials, but in the CP mode, it was well separated from the background discharge; it was denominated as peak H. Using this peak, femtomoles of peptides and proteins have been recently determined.20,24-26 In this paper, we have shown that MutS is strongly adsorbed at the HMDE, and using the CPSA peak H, this protein can be determined at concentrations below 1 ng/mL. With the so-called adsorptive transfer stripping technique, the analysis can easily be performed in several-microliter samples, making it possible to determine tens of attomoles of the protein. The sensitivity of MutS analysis at carbon electrodes is substantially lower, enabling determination of tens of femtomoles of the protein. We attached biotinylated DNA to streptavidin-coated magnetic beads and detected MutS protein after its specific binding to DNA singlebase mismatches using CPSA peak H. EXPERIMENTAL SECTION Materials. Characteristics of the MutS (Epicenter Technologies Corp.) from Thermus aquaticus are given in Figure S1 (Supporting Information). Deoxyoligonucleotides purchased from Faculty of Science, Masaryk University (Brno, Czech Republic) are shown in Table 1. Dynabeads M-280 streptavidin (DB-STV) and magnetic particle concentrator MPC-S were from DynalBio(21) Havran, L.; Billova, S.; Palecek, E. Electroanalysis, in press. (22) Palecek, E. In Topics in Bioelectrochemistry and Bioenergetics; Milazzo, G., Ed.; J. Wiley: London, 1983; Vol. 5, pp 65-155. (23) Heyrovsky, J.; Kuta, J. Principles of Polarography, 1st ed.; Czechoslovak Academy of Science: Prague, 1965. (24) Tomschik, M.; Havran, L.; Palecek, E.; Heyrovsky, M. Electroanalysis 2000, 12, 274-279. (25) Kizek, R.; Trnkova, L.; Palecek, E. Anal. Chem. 2001, 73, 4801-4807. (26) Masarik, M.; Stobiecka, A.; Kizek, R.; Jelen, F.; Pechan, Z.; Hoyer, W.; Jovin, T. M.; Subramaniam, V.; Palecek, E. Electroanalysis, in press.

Figure 1. Schematic illustration describing the CPSA equipment with a mercury electrode and adsorptive transfer technique. (A) Simple scheme of adsorptive transfer technique. MutS protein was adsorbed at the electrode from a 5-µL drop at open current circuit. After the accumulation time (tA), the electrode was washed and immersed into the background electrolyte to perform the electrochemical measurements. AE, auxiliary electrode; WE, working electrode; RE, reference electrode. (B) Basic scheme of the electrical circuit used for polarization of the electrode by direct current (dc); (c) electrolytic cell with electrodes. (1) Dependence of potential (E) on time (t). The delay in potential change signals the electrode process. A derivatized curve is measured.24

tech ASA (Oslo, Norway). Components of the supporting electrolytes were analytical grade reagents purchased from SigmaAldrich. Tris(2-carboxyethyl)phosphine hydrochloride was purchased from Molecular Probes (Eugene, OR). All solutions were prepared using ACS water (Sigma Aldrich). Apparatus. CPSA was performed using the Autolab electrochemical instrument (EcoChemie, Utrecht, The Netherlands) connected with the VA-Stand 663 (Metrohm, Herisau, Switzerland). The software GPES 4.4 supplied by EcoChemie was used for smoothing and baseline correction. A standard cell with three electrodes was used. The working electrode was a HMDE with a drop area of 0.4 mm2 or a carbon paste electrode (CPE). CPE was made of 70% graphite powder (Aldrich) and 30% mineral oil (Sigma; free of DNase, RNase, and protease). This carbon paste was housed in a Teflon body, having a 2.5-mm-diameter disk surface. Prior to measurements, the electrode surface was renewed by polishing with wet filter paper. The reference electrode was the Ag/AgCl/3 M KCl electrode, and a platinum wire was used as an auxiliary electrode. Electrochemical measurements were performed at room temperature if not stated otherwise. An adsorptive transfer procedure was performed as described: 27 MutS protein was adsorbed at the electrode from a 5- (HMDE) or 6-µL (CPE) drop at open current circuit (Figure 1). After the (27) Palecek, E.; Jelen, F.; Teijeiro, C.; Fucik, V.; Jovin, T. M. Anal. Chim. Acta 1993, 273, 175-186.

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accumulation time (tA), the electrode was washed and immersed into the background electrolyte to perform the electrochemical measurements. Both the adsorption of MutS and the subsequent CPSA were carried out using 0.2 M sodium phosphate, pH 7.0 (HMDE), or 0.2 M sodium acetate, pH 5.0 (CPE), if not stated otherwise. The nucleic acid concentrations were determined spectrophotometrically using a HP 8452 spectrophotometer. For incubation of MutS at elevated temperatures, Thermomixer, Eppendorf was used. Separation of DNA-MutS Complex on Magnetic Beads. A 100-µL sample of the biotinylated oligonucleotide (ODN-biot; 100 µg/mL, Table 1) was mixed with the same volume of fully complementary or mismatched ODN at equimolar concentration in 50 mM sodium phosphate pH 7.0, 0.3 M NaCl. Solutions were heated to 80 °C for 10 min followed by slow cooling to room temperature for 1 h. Aliquots of 20 µL of streptavidin-coated magnetic beads (DB-STV; DynalBiotech ASA)were washed twice in the 100 µL of hybridization buffer. Biotinylated perfect duplex or mismatched duplex (20 µL; 1 µg/mL) were added to the DB-STV in the hybridization buffer. Alternatively the DNA duplex was prepared by attaching single-stranded ODN-biot to DB-STV followed by hybridization with a complementary ODN in the solution. The former procedure yielded higher amounts of MutS specifically bound to mismatched DNA duplexes; it was therefore used in this paper. The resulting mixture was placed on a shaker for 30 min at room temperature. DB-STVs with captured ODNbiot duplexes (dsODN-biot) were washed three times (100 µL of hybridization buffer). A 50-µL aliquot of PBS (0.14 M NaCl + 3 mM KCl + 4 mM Na2PO4, pH 7.4) and 5% dried milk were then added to the DB-STV-dsODN-biot and shaken for 15 min to block the free DB-STV surface. DB-STV-dsODN-biot were then washed three times in 100 µL of PBS. To 20 µL of DB-STV-dsODN-biot, 1 µg of MutS in 20 µL of PBS (50 µg/mL), with 1 mM phosphine was added and the mixture was shaken at 50 °C if not stated otherwise. DB-STV-dsODN-biot were washed three times with PBS. MutS was released from DB-STV-dsODN-biot in 20 µL of 1 M NaClO4 by heating at 85 °C for 15 min; 5-µL aliquotes were taken for the AdTS electrochemical measurements. MutS Binding to Base Mismatches and Insertion/Deletions in DNA. Thermostable MutS (T. aquaticus) is known to bind optimally to mismatched DNA at elevated temperatures.14 We tested MutS binding to DNA containing GT mismatch at different temperatures from 4 to 80 °C, and using peak H, we observed optimum binding at temperatures around 50 °C (not shown). In our further measurements, we incubated MutS with DNA at 50 °C. Biotinylated ODN 30-mer duplexes were attached to DB-STV and incubated with 500 ng of MutS to recognize perfectly matched duplexes from those containing single-base mismatch or insertion/deletion mutations (Figure 4). After washing the unbound MutS away, MutS was dissociated from DB-STV bound DNA by treatment with 20 µL of 1 M NaClO4 at 85 °C for 15 min. The released MutS was determined by CPSA using peak H. Adsorption of MutS to the electrode at room temperature from 1 M NaClO4 or from 0.2 M sodium phosphate after heating the solution to 70, 80, or 85 °C induced only small differences in the height of peak H (not shown). We prepared a new calibration curve with MutS pretreated as described above using incubations at 50 and 85 °C. This calibration was used for determination of 5932

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MutS dissociated from the magnetic beads; it did not substantially differ from that in Figure 3A. RESULTS AND DISCUSSION Electrochemical Analysis of MutS Protein. Earlier we showed that biomacromolecules such as nucleic acids and proteins adsorb strongly and irreversibly at mercury and some carbon electrodes, CPE and pyrolytic graphite electrodes.20,28 We proposed29 a medium exchange method, in which the biomacromolecule is first adsorbed at the electrode (usually from 3- to 10-µL drop of solution), the electrode is then washed and immersed in an electrolytic cell (containing only a blank background electrolyte) where the electrochemical measurement is performed (reviewed in refs 30 and 31). This method was denominated as adsorptive transfer stripping voltammetry (AdTSV). In this paper, we used AdTS in combination with either square wave voltammetry (SWV) or constant current chronopotentiometry (CP) as we wished to analyze minimum volumes and low concentrations of MutS. In agreement with very low content of cysteine [a single cysteine (Cys) residue (0.1%) per monomeric molecule, Figure S1], MutS protein at a relatively high concentration of 50 µg/mL and accumulation time (tA) of 60 s produced no typical voltammetric Brdicka signal 17, 22 at the HMDE (not shown). Under the same conditions, other proteins with higher Cys content, such as bovine serum albumin, produced well-developed signals. We were therefore limited to studies of tyrosine (Tyr) oxidation at carbon electrodes and MutS-catalyzed hydrogen evolution at mercury electrodes. Tyrosine Oxidation at Carbon Electrodes. At a concentration of 25 µg/mL and a relatively short tA of 90 s in 0.2 M sodium acetate (pH 5.0), MutS yielded at ∼+0.8 V a square wave voltammetric oxidation signal at a CPE; this signal turned into a well-developed peak after baseline correction (Figure 2). As expected, no separated tryptophan (Trp) peak appeared because the high Tyr peak probably overlapped a much smaller signal of Trp due to low Trp and high Tyr contents (2 Trp and 28 Tyr residues per monomeric molecule, Figure S1). We measured dependence of oxidation peak on MutS concentration, and we were able to detect MutS down to 500 ng/mL, i.e., 3 ng in 6 µL, corresponding to 33 fmol (Figure 2). CPSA Peak H at Mercury Electrodes. At a MutS concentration of 100 ng/mL and a relatively short tA (120 s), we obtained a narrow well-developed AdT CPSA peak H at ∼-1.7 V in 0.2 M sodium phosphate, pH 7.0 (Figure 3). We measured dependence of this peak on pH in the range from 6.3 to 8.0. Between pH 6.3 and 7.0, peak height depended little on pH; at higher pH's this peak gradually decreased, reaching ∼55% of its original value at pH 8.0 (not shown). We tested three different buffers, such as 0.2 M ammonium phosphate, 0.2 M NH4OH-NH4Cl, and 0.2 M sodium borate, both for MutS adsorption and for the electrochemical detection, but we observed no dramatic effect of buffer composition on the height of the MutS peak H (not shown). We (28) Cai, X.; Rivas, G.; Farias, M. A. P.; Shiraishi, H.; Wang, J.; Palecek, E. Anal. Chim. Acta 1996, 332, 49-57. (29) Palecek, E.; Postbieglova´, I. J. Electroanal. Chem. 1986, 214, 359-371. (30) Palecek, E. Electroanalysis 1996, 8, 7-14. (31) Palecek, E.; Fojta, M.; Jelen, F.; Vetterl, V. In The encyclopaedia of electrochemistry; Bard, A. J., Stratsmann, M., Eds.; Wiley-VCH: 2002; Vol. 9, pp 365-430.

Figure 2. AdTS SWV of MutS protein on CPE. (A) SW voltammogram of 280 nM MutS protein (25 µg/mL; ∼2 pmol) at an accumulation time, tA 90 s. (B) Dependence of the SWV peak height on the concentration of MutS at tA 60 s; inset, low concentration range. The protein was adsorbed from a 6-µL drop on CPE at open current circuit for an accumulation time tA 90 s. The electrode was washed and immersed into the blank background electrolyte (not containing MutS) and the voltammogram recorded. Background electrolyte, 0.2 M sodium acetate, pH 5.0; tA 60 s. Instrument settings: frequency 200 Hz, step potential 5 mV, amplitude 25 mV, initial potential 0.1 V, and end potential 1.1 V.

Figure 3. AdT CPSA of MutS protein on a HMDE. (A) Dependence of the peak H height on concentration of MutS; inset, low concentration range. tA ) 120 s. (B) (1) Dependence of the height and potential (Ep) of MutS peak H on accumulation time (tA). Concentration of MutS was 10 ng/mL. (2). Chronopotentiogram (peak H) of MutS at a concentration of 0.5 ng/mL; tA 300 s. MutS was adsorbed to HMDE from 0.2 M sodium phosphate, pH 7.0; the same buffer was used as background electrolyte for electrochemical measurements. Stripping current Istr was -5 µA.

therefore performed our further measurements in sodium phosphate (pH 7.0), which we had used before in the analysis of other proteins.26 The height of peak H decreased with the stripping current (Istr) in a usual way, showing the optimum at -5 µA (not shown); this Istr value was used in most of our further AdT CPS measurements. Between 0.5 and 10 ng/mL at tA 120 s, the height of peak H increased linearly with MutS concentration with a regression line of y ) 1522.2x + 2647.7 (R2 ) 0.915), followed by a less steep (but almost linear) increase at higher MutS concentrations up to 100 ng/mL. The dependence of peak H height on tA at a concentration of 10 ng/mL had a similar shape (Figure 3). At a MutS concentration of 500 pg/mL and tA 300 s, a well-developed and symmetric peak H was obtained without any baseline

correction (Figure 3B). This concentration of MutS was determined at tA 120 s, with a standard deviation of 8.8% (n ) 8). Even at much lower concentrations, a well-developed peak H appeared, but the accuracy of the measurement decreased significantly probably due to the adsorption of MutS on the glass of the HMDE capillary. Because we found concentrations of ∼1 ng/mL highly satisfactory for our purposes (i.e., ∼5 pg in a 5-µL drop or ∼28 amol of the monomeric MutS), we did not attempt to further increase the sensitivity of the MutS determination. We tested the effect of temperature on the peak height (during the adsorption and during the electrochemical measurement) and we observed the highest signal at temperatures between 20 and 25 °C, i.e., close to room temperature (not shown). Analytical Chemistry, Vol. 76, No. 19, October 1, 2004

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Figure 4. Schematic representation of MutS binding to mismatched DNA attached to magnetic beads, folllowed by (a) separation of unbound MutS, (b) dissociation of DNA-bound MutS from the beads, and (c) electrochemical determination of MutS either at carbon or at mercury electrodes.

Hydrogen Evolution Catalyzed by MutS Protein at Mercury Electrodes. Using the mercury electrodes and peak H, we obtained sensitivity by 3-4 orders magnitude higher as compared to carbon electrodes. No baseline correction was used for peak H; it can be expected that the sensitivity of the determination can be increased after its further optimization. Peak H is due to catalytic hydrogen evolution.24 Reduction of protons occurs at mercury electrodes with the highest overvoltage of all metal electrodes. Proteins adsorbed at the mercury surface may reduce this overvoltage, giving rise to electrochemical signals much higher than any signals due to usual diffusion-controlled processes20,30,32 The protein has to bind a proton, which enters the electrode reaction. Detailed schemes of the catalytic hydrogen evolution have been proposed by several authors.23,32,33 In principle, they postulated that the electron uptake by protonated catalyst BH+ resulted in production of a radical BH•, followed by evolution of H2, as well as by regeneration and protonation of the catalyst via reaction with the acid component of the buffer. Protonated amino groups were supposed to be involved in the protein catalytic reaction.17,22,23 In this work, we have shown that picomolar concentrations of MutS can be determined in a 5-µL volume at moderate accumulation time (Figure 3B), corresponding to attomoles or picograms of the protein. This is probably not the highest sensitivity of this method. Our preliminary results suggest that optimization of the conditions, including changes in pH of the background electrolyte, will allow detection of even lower (32) Banica, F. In Encyclopedia of Analytical Chemistry; Meyers, R., Ed.; Wiley: New York, 2000; pp 11115-11144. (33) Mairanovskii, S. G. Catalytic and kinetics waves in polarography; Plenum Press: New York, 1968.

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MutS concentration. High sensitivity of the MutS determination by peak H might be caused by high content of basic amino acids [82 Arg residues (10.1%) and 25 Lys (3.1%)], representing a source of protonated amino groups. Detection of Insertion/Deletions in DNA Using MutS Protein. We used CPSA to measure binding of MutS to a 30-mer heteroduplex with a single GT mismatch as shown in Figure 4. We tested the relation between the amount of added MutS and the height of peak H. This peak increased almost linearly up to 2 µg of MutS; addition of 4 µg of this protein resulted in only very small increase of peak H. Using the carbon electrodes and the highest amount of MutS, we obtained only a very poor signal of MutS after its release from the GT mismatch containing DNA duplex (not shown). We therefore continued the experiments only with HMDE, which yielded a well-developed CPSA peak even with 500 ng of added MutS (Figure 5B). The peak produced by DB-STV alone (without DNA and MutS) representing the background, corresponded to ∼1.5% of the above peak H (Figure 5B). The signal of a perfect duplex was only slightly higher. We also tested duplexes with T insertion alone and two duplexes with T insertions plus a GT mismatch: in one of them, GT pair and T insertion were located side by side (ODN C in Table 1) while in the other one (ODN D) they were separated by 19 base pairs. The intensity of peak H decreased in an order GT plus T insertion (distantly located) > GT > GT plus T insertion (side by side) > T insertion (Figure 5), suggesting that presence of T insertions decreased the MutS binding efficiency if located next to the GT mismatch (as compared to the GT mismatch-containing duplex), in agreement with previously published data.12,34,35 On the other

Figure 5. Electrochemical detection of single-base mismatches and insertions in DNA. DNA duplex (Table 1) were treated with MutS, as shown in Figure 4, and the protein was detected by CPSA at HMDE. (A) Bar graph showing the relative heights of peak H after MutS binding to various types of DNA duplexes. (1) DB-STV alone; (2) perfect duplex DNA (A in Table 1); (3) T insertion containing DNA duplex (E in Table 1); (4) G:T mismatch containing DNA duplex (B in Table 1); (5) G:T mismatch and T insertion side by side containing DNA duplex (C in Table 1); (6) G:T mismatch and T insertion distantly located containing DNA duplex (D in Table 1) the peak height of this DNA was taken as 100%. (B) Chronopotentiograms of MutS protein measured after DB-STV procedure. (]) corresponds to peak of MutS protein binding to G:T mismatch, (s) corresponds to peak of MutS protein binding to perfect duplex, and the thin line corresponds to background electrolyte. Other detailes in Experimental and in Figures 2A and 3.

hand, such a decrease was not observed in the ODN D (Table 1) where structural anomalies were distantly located. In fact, in this ODN a larger amount of bound MutS was detected (Figure 5A) suggesting that both the GT mismatch and the T insertion were recognized by MutS in ODN D. Potentialities of Carbon Electrodes. In this paper, we have shown that the DNA mismatch binding MutS protein can be determined electrochemically at carbon electrodes at concentrations down to hundreds of nanograms per milliliter. In this way, tens of femtomoles of MutS can be determined in 6 µL of MutS samples. Relatively good sensitivity of the protein determination at the carbon electrode is connected with the presence of 28 Tyr residues (3.5%) in MutS. However, this sensitivity appeared insufficient for mismatch detection in our experiments (Figure 4). Larger volumes of DB-STV and higher amounts of MutS protein would be necessary to obtain adequate MutS responses at carbon electrodes. Our preliminary experiments suggest that the sensitivity of MutS determination might be inreased by optimizing the experimental conditions. For example, partial hydrolysis or controlled denaturation of the protein (after its dissociation from DNA), making more Tyr residues accessible for the electrode process, may increase the sensitivity of MutS determination. Detection of Point Mutations. Recently a method for the de novo discovery of genetic variations, including SNPs on microelectronic chip devices, has been developed.12 In this method, a (34) Biswas, I.; Hsieh, P. J. Biol. Chem. 1997, 272, 13355-13364. (35) Joshi, A.; Rao, B. J. J. Biosci. 2001, 26, 595-606.

fluorescence-labeled MutS protein was used. Here we have shown that MutS can be determined electrochemically without any protein labeling. Disposable carbon (e.g., screen-printed) electrodes can be easily used in microarray systems. Mercury electrodes, which offer substantially higher sensitivities, are less suitable for such systems, but it seems they can be replaced by solid dental amalgam electrodes36,37 offering potential windows similar to that of liquid mercury. Our recent results obtained with cathodic stripping voltammetry of DNA bases38 and catalytic hydrogen evolution signals produced by osmium-labeled DNA (Yosypchuk, Palecek, et al., unpublished) show that the liquid mercury drop electrodes can be fully replaced by the solid amalgam electrodes. The first electrochemical detection of point mutation was achieved in 199639 by means of a relatively simple technique, using peptide nucleic acid as a probe, carbon electrodes, and a simple redox indicator. Since that time, a number of techniques have been developed (e.g., refs 8, 31, and 40-43) suitable for the (36) Yosypchuk, B.; Novotny, L. Crit. Rev. Anal. Chem. 2002, 32, 141-151. (37) Yosypchuk, B.; Novotny, L. Electroanalysis 2002, 14, 1733-1738. (38) Jelen, F.; Yosypchuk, B.; Kourilova, A.; Novotny, L.; Palecek, E. Anal. Chem. 2002, 74, 4788-4793. (39) Wang, J.; Palecek, E.; Nielsen, P. E.; Rivas, G.; Cai, X. H.; Shiraishi, H.; Dontha, N.; Luo, D. B.; Farias, P. A. M. J. Am. Chem. Soc. 1996, 118, 76677670. (40) Boon, E. M.; Ceres, D. M.; Drummond, T. G.; Hill, M. G.; Barton, J. K. Nat. Biotechnol. 2000, 18, 1096-1100. (41) Caruana, D. J.; Heller, A. J. Am. Chem. Soc. 1999, 121, 769-774. (42) Wang, J.; Rivas, G.; Cai, X. H.; Chicharro, M.; Parrado, C.; Dontha, N.; Begleiter, A.; Mowat, M.; Palecek, E.; Nielsen, P. E. Anal. Chim. Acta 1997, 344, 111-118.

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detection of point mutations. Most of them are based on different stabilities of mismatched duplexes versus perfect duplexes, and no one uses MutS. This lack of MutS electrochemical papers can be caused by (a) problems with sensitive determination of proteins not containing groups undergoing fast electron transfer and (b) difficulties with protein detection by so-called single-surface techniques, which have been mostly used in the electrochemical DNA hybridization sensors.6,8,9,11 Another interesting approach in studies of DNA-protein interactions, based on dsDNA-mediated charge transport, was developed by Boon et al.44 They showed that binding of a baseflipping enzyme, MHhaI, to DNA greatly decreases the signal of DNA-bound daunomycin, suggesting that this protein binding disturbs the integrity of the base stack because of base flipping.45 This approach was used to detect single-base mismatch using another DNA repair enzyme (MutY) binding to 8-oxoG:A and G:A mismatches.44 However, no data consistent with the base flipping as a method for mismatch recognition by MutY were found. On the other hand, the same group has recently shown that cluster, contained in MutY, can be utilized in the detection of MutYDNA interactions.46 Although not detectable in the absence of DNA, the DNA-bound MutY displays on cyclic voltammograms a reversible couple at gold electrodes. This method requires enzymes with a prosthetic group capable of undergoing a redox process under certain conditions. Our method based on protein determination by means of peak H (Figures 4 and 5) is not limited to a relatively small group of proteins and may probably be applicable to most of the proteins (all proteins and peptides, which have been tested so far, produced peak H under suitable conditions). Moreover, the MutS concentration used in our experiments was by several orders of magnitude lower than that of MutY (800 µM).46 (43) Rajski, S. R.; Jackson, B. A.; Barton, J. K. Mutat. Res. 2000, 447, 49-72. (44) Boon, E. M.; Pope, M. A.; Williams, S. D.; David, S. S.; Barton, J. K. Biochemistry 2002, 41, 8464-8470. (45) Rajski, S. R.; Kumar, S.; Roberts, R. J.; Barton, J. K. J. Am. Chem. Soc. 1999, 121, 5615-5616. (46) Boon, E. M.; Livingston, A. L.; Chmiel, N. H.; David, S. S.; Barton, J. K. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 12543-12547. (47) Wang, J.; Liu, G. D.; Zhu, Q. Y. Anal. Chem. 2003, 75, 6218-6222. (48) Wang, J.; Polsky, R.; Merkoci, A.; Turner, K. L. Langmuir 2003, 19, 989991. (49) Fojta, M.; Havran, L.; Kizek, R.; Billova, S.; Palecek, E. Biosens. Bioelectron., in press. (50) Fojta, M.; Havran, L.; Vojtiskova, M.; Palecek, E. J. Am. Chem. Soc. 2004, 126, 6532-6533.

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CONCLUSION In this paper, we have shown that MutS protein is electroactive at mercury and carbon electrodes and can be detected by CPSA at picomolar concentrations, corresponding to tens of attomoles in 5-µL samples measured by AdT CPSA. To take advantage of the high sensitivity of the AdT CPS determination of MutS, we used the so-called double-surface technique (DST), in which the DNA hybridization is performed at one surface and electrochemical detection at another surface, the detection electrode.9,38 Using DST, the sensitivity of the DNA hybridization was greatly increased47,48 and electrochemical determination (without any radioactive or fluorescence labeling) of the lengths of long repetitive sequences, such as nucleotide triplet expansions in human neurodegenerative diseases, became possible.49,50 Here we have applied DST to determine the protein specifically bound to DNA. We have shown that single-base mismatches and insertion/ deletions in DNA duplexes attached to magnetic beads are detectable by means of CPSA at mercury electrodes. Most probably the same analysis will be possible also with carbon or solid amalgam electrodes after optimizing the conditions of the protein determination. The amounts of DNA and MutS protein necessary for the analysis can be decreased by orders of magnitude through electrode miniaturization. Highly sensitive label-free determination of MutS opens the door to development of DNA chips for a high-throughput electrochemical determination of point mutation in genomic DNAs and support the recent tendencies to complement optical detection4 in DNA hybridization sensors by simpler and less expensive electrochemical detection.68,11 Moreover, the method described in this paper represents a new approach in the analysis of DNA (or RNA)-protein interactions applicable to a large number of NA-binding proteins. ACKNOWLEDGMENT The authors express their gratitude to Dr. M. Fojta for critical reading of the manuscript. This work was supported by november AG, Erlangen, and by grants from the Academy of Sciences of the Czech Republic Z 5004920 and S5004355 (to E.P.). SUPPORTING INFORMATION AVAILABLE Characteristics, including amino acid sequence of MutS protein of T. aquaticus. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review April 6, 2004. Accepted July 23, 2004. AC049474X