Electrochemical detection of hepatitis C virus based on site-specific DNA cleavage of BamHI endonuclease

Abstract

We have developed a new electrochemical approach for qualitative and quantitative detection of hepatitis C virus (HCV) based on the site-specific DNA cleavage of BamHI endonuclease.

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Hepatitis C virus (HCV), a single-stranded RNA virus containing about 10 000 nucleotide bases ,¹ is considered to be a major causative agent of chronic hepatitis and progressive liver fibrosis leading to cirrhosis and hepatocellular carcinoma.² The monitoring of HCV RNA in serum or plasma is used for diagnosing or confirming active infections and for assessing patient response to therapy.^3,4 There has been considerable interest in developing reliable and simple methods for detecting and quantifying HCV.⁴ Those approaches have involved various nucleic acid amplification methodologies,⁵electrochemiluminescence,⁶surface plasmon resonance,⁷ a piezoelectric genosensor,¹ and a biosensor based on fluorescence detection,⁸ and electrochemical detection (a DNAprobe labeled with horseradish peroxidase (HRP) and electrochemical detection of the reduction of I₂ generated by HRP in the presence of H₂O₂)⁹etc. All those methods, however, are considered to be time-consuming and laborious, and require sophisticated and expensive instruments.

Protein–DNA interactions play important roles in life processes, such as DNA replication, transcription, recombination, damage, and repair.^10–14 The restriction endonuclease is a well-studied class of DNA-binding proteins and has been one important tool in the development of modern molecular biology.^10,15,16 However, to our best knowledge, there has been no report on the DNA analysis based on the cleavage of endonuclease. We have developed a new approach for qualitative and quantitative HCV detection based on site-specific DNA cleavage of BamHI endonuclease. The developed method exhibits the advantages of ease of performance, good specificity and selectivity, and the ability to perform real-time monitoring.

The detection of HCV using synthetic oligonucleotides is illustrated in Fig. 1. The thionine-labeled probeDNA was self-assembled on the surface of a gold electrode and hybridized with target cDNA, which is a 21-mer oligonucleotide related to HCV. The detection is based on the variation of the voltammetric signal of thionine before and after digestion with BamHI endonuclease, which acetate is a site-specific endonuclease with a molecular mass of about 22 kDa.¹⁷BamHI recognizes the duplex symmetrical sequence 5′-GGATCC-3′ and catalyzes double-stranded cleavage between the guanines in the presence of Mg²⁺.¹⁸ After digestion by BamHI, the DNA hybrid was cleaved at a specific site and the electrochemical signal of thionine was decreased or disappeared. The extent of the decrease was related to the concentration of target cDNA in solution, which forms the basis of the quantitative detection of target cDNA.

Fig. 1 Illustration of the site-specific cleavage of BamHI endonuclease using thionine as an electrochemical indicator.

The 21-base sequence of HCV RNA (base location: 8245–8265) was reverse transcribed into complementary DNA (cDNA). The base sequences of the synthetic oligonucleotides are as follows. Thionine-labeled probe (S1): 5′-SH-TGG GGA TCC CGT ATG ATA CCC-thionine-3′, target cDNA (S2): 5′-GGG TAT CAT ACG GGA TCC CCA-3′, and two-base mismatched cDNA (S3): 5′-GGG TAT CAT ACG CGT TCC CCA-3′. Immobilization of S1 was performed by immersing the cleaned gold electrode (2 mm in diameter, CH Instruments) in 20 μL of S1 solution (1 μM) and incubating for 48 h at 4 °C (step a, Fig. 1). Then the modified electrode was thoroughly rinsed with Tris-HClbuffer (pH 7.6) and water in turn to remove the weakly adsorbed S1. Hybridization was conducted at 37 °C by immersing the S1-immobilized gold electrode in 2 mL of Tris-HClbuffer (pH 7.6) containing the cDNA (S2), or two-base mismatched cDNA (S3) for 2 h (step b, Fig. 1). BamHI cleavage was performed by incubating the DNA hybrid modified gold electrode in Tris-HCl (pH 7.6), containing 20 U μL⁻¹BamHI, 10 mM MgCl₂, and 50 mM NaCl, at 37 °C for 3 h (step c, Fig. 1). After treatment, the electrode was washed and transferred into acetatebuffer (0.1 M, pH 5) to record the electrochemical response.

Although single-scanning square wave voltammetry (SWV) and differential pulse voltammetry (DPV) are usually two methods used for recording the response of DNAbiosensors ,^12–14,19cyclic voltammetry is used in this work since the interaction modes between probe molecule and DNA can be obtained from the analysis of cyclic voltammetric peak potentials and currents.²⁰ The anodic and cathodic peaks can also be studied simultaneously by using cyclic voltammetry. The cyclic voltammogram of the S1-modified gold electrode in 0.1 M acetatebuffer (pH 5) demonstrates a pair of well-defined redox peaks with the anodic and cathodic peak potentials of ca. 0 and −28 mV (vs. SCE, at 50 mV s⁻¹), respectively (Fig. 2a), which were in accordance with the characteristic redox features of thionine.²¹ After the S1-immobilized gold electrode was hybridized with S2, the cyclic voltammogram also showed a pair of redox peaks (Fig. 2b). The positions and the heights of the redox peaks are almost the same as those shown in Fig. 2a, suggesting that hybridization of S1 with S2 does not affect the redox features of thionine. Fig. 2d shows the cyclic voltammogram of the S1/S2 hybrid-modified gold electrode after 3 h treatment with BamHI. It is obvious that the response of thionine has almost disappeared. As is known, BamHI is a site-specific endonuclease. When the S1/S2 hybrid was treated with BamHI, the dsDNA was cleaved at a specific site (5′-G/GATCC-3′), and the thionine was removed from the electrode surface. Therefore, no redox peak of thionine is observed in Fig. 2d.

Fig. 2 Cyclic voltammograms of S1 (a) and S1/S2 (b) modified gold electrodes in 0.1 M acetatebuffer (pH 5). (c) and (d) are the cyclic voltammograms for S1/S3-modified (c) and S1/S2-modified (d) electrodes, respectively, after digestion with BamHI for 3 h. The inset shows the dependence of the peak current of the S1/S2-modified electrode in 0.1 M acetatebuffer (pH 5) after digestion with BamHI for 3 h on the concentration of S2.

To confirm the disappearance of the redox peak in Fig. 2d resulted directly from the cleavage of the endonuclease, two-base mismatched cDNA (S3) was selected to assay the cleavage specificity of BamHI endonuclease. The S1-immobilized gold electrode was hybridized with S3 for 2 h, and then treated in enzymatic cleavage mixture for 3 h. After it was rinsed with copious amounts of water, the electrode was dipped into acetatebuffer (pH 5) and the cyclic voltammogram was recorded. The redox peaks of thionine can be still observed (Fig. 2c). Moreover, the peak currents remain almost invariant compared with those presented in Fig. 2a, suggesting that BamHI has no effect on the S1/S3 hybrid because the hybrid does not contain the specific recognition sequence for BamHI. Therefore, the signal disappearance in Fig. 2d is a direct consequence of the enzymatic cleavage of the specific recognition sequence for BamHI. The results demonstrate that the interaction model of DNA–BamHI endonuclease can be used for qualitative HCV detection.

BamHI–DNA interaction was studied in real-time, which was monitored by detecting the decrease of the voltammetric signal of thionine with increasing digestion time. After the S1/S2 hybrid modified gold electrode was immersed in the cleavage mixture, the peak current of thionine decreased rapidly, indicating that the cleavage occurred. The peak current undergoes a rapid decrease in the first 30 min, and starts to level off after 60 min (not shown here). The cleavage will be finished at about 2 h since the height of the redox peak of thionine decreases to almost zero.

This interaction model can also be applied to quantitatively detect the specific cDNA sequence related to HCV. To assess the analytical performance of the proposed model, the thionine-labeled S1 modified gold electrode was hybridized with various concentrations of target cDNA (S2). The S1/S2 hybrid was then treated with BamHI cleavage mixture, and finally, the voltammetric response of thionine was recorded. The result indicates that the peak currents decrease with increasing concentration of S2 in the hybridization solution. The value of Δi, measured as the difference between the peak currents of the S1/S2 hybrid-modified electrodes before and after cleavage, increases linearly with S2 at lower concentration and then levels off at higher concentration (inset in Fig. 2). When the concentration of S2 is high, for example the concentration of S2 is higher than 5 μM, almost all the thionine-labeled S1 immobilized on the surface of the gold electrode is hybridized with S2 and subsequently cleaved by BamHI. Therefore, almost all thionine molecules immobilized on the gold electrode are removed after digestion with BamHI. Thus, the value of Δi remains practically constant even though the concentration of S2 is further increased and a plateau is obtained at high concentration of S2 (inset of Fig. 2). The value of Δi has a linear relationship with the concentration of S2 ranging from 0.1 to 2.5 μM, with a correlation coefficient of 0.999. The linear range would be useful in clinical analysis since the normal concentration of HCV RNA in serum or plasma analyzed in the clinic is usually around 1.5–2.0 μM.^3,4 The limit of the detection is estimated to be (0.02 ± 0.005) μM at an S/N (signal/noise) ratio of 3. These results demonstrate the great potential for practical application of the proposed model for the qualitative detection and quantitative analysis of HCV.

The developed method was applied to HCV detection in real samples from patient sera. The sera samples were supplied by a hospital. The real samples containing HCV RNA were subjected to reverse transcriptase reaction, providing cDNA. The obtained amplicons were initially evaluated using the standard qualitative Amplicor® HCV Test (microwell format with spectrophotometric detection, Amplicor® HCV Test Kit, version 2.0, Roche Molecular Diag. Co., USA) and further tested for hybridization with the S1probe and cleavage by BamHI using the developed method. The results demonstrate that the samples characterized as positive in the Amplicor® test are able to hybridize with S1 and the voltammetric signal of thionine decreases significantly after cleavage by BamHI. However, those samples appearing as negative in the Amplicor® assay cannot hybridize with S1 and the voltammetric peak current of thionine remains almost unchanged after cleavage by BamHI. These results indicate the great potential for practical application of the proposed method for the detection of HCV in real clinical samples.

In summary, we have developed a new approach for the qualitative and quantitative detection of HCV based on site-specific DNA cleavage of BamHI endonuclease. The major advantages of this enzymatic cleavage assay are its good specificity, ease of performance, and the ability to perform real-time monitoring. The developed protocol can be taken as a general method of DNA detection and is expected to be applicable to other types of DNA analysis.

We thank the National Natural Science Foundation of China (20673057, 20773067, 20833006), and the Program for New Century Excellent Talents in University (NET-06-0508) for support of this work.

Notes and references

1. P. Skládal, C. S. Riccardi, H. Yamanaka and P. I. Costa, J. Virol. Methods, 2004, 117, 145.
2. N. Fujita, S. Horiike, R. Sugimoto, H. Tanaka, M. Iwasa, Y. Kobayashi, K. Hasegawa, N. Ma, S. Kawanishi, Y. Adachi and M. Kaito, Free Radical Biol. Med., 2007, 42, 353.
3. T. Umemura, R. Y.-H. Wang, C. Schechterly, J. W.-K. Shih, K. Kiyosawa and H. J. Alter, Hepatology, 2006, 43, 91.
4. H. Le Guillou-Guillemette and F. Lunel-Fabiani, Detection and Quantification of Serum or Plasma HCV RNA: Mini Review of Commercially Available Assays, in Hepatitis C: Methods and Protocols, ed. H. Tang, Humana Press, Totowa, NJ, 2nd edn, 2009, vol. 510, pp. 3–14.
5. S. C. Lee, A. Antony, N. Lee, J. Leibow, J. Q. Yang, S. Soviero, K. Gutekunst and M. Rosenstraus, J. Clin. Microbiol., 2000, 38, 4171.
6. A. Guichon, H. Chiaprelli, A. Martinez, C. Rodrigues, A. Trento, J. C. Russi and G. Carballal, J. Clin. Virol., 2004, 29, 84.
7. P. M. Richalet-Secordel, F. Poisson and M. H. V. Van Regenmortel, Clin. Diagn. Virol., 1996, 5, 111.
8. G. Bildan, M. Billon, T. Livache, G. Mathis, A. Roget and L. M. Torres-Rodriguez, Synth. Met., 1999, 102, 1363.
9. C. S. Riccardi, K. Dahmouche, C. V. Santilli, P. I. Costa and H. Yamanaka, Talanta, 2006, 70, 637.
10. G. Nardone, J. George and J. G. Chirikjian, J. Biol. Chem., 1986, 261, 12128.
11. B. Li, Y. Du, H. Wei and S. Dong, Chem. Commun., 2007, 3780.
12. S. Krishnan, E. G. Hvastkovs, B. Bajrami, I. Jansson, J. B. Schenkman and J. F. Rusling, Chem. Commun., 2007, 1713.
13. M. So, J. B. Schenkman and J. F. Rusling, Chem. Commun., 2008, 4354.
14. D. O. Hull, B. Bajrmi, I. Jansson, J. B. Schenkman and J. F. Rusling, Anal. Chem., 2009, 81, 716.
15. M. Fojta, T. Kubičárová and E. Paleček, Electroanalysis, 1999, 11, 1005.
16. Y. Jin, W. Lu, J. Hu, X. Yao and J. Li, Electrochem. Commun., 2007, 9, 1086.
17. B. Hinsch and M.-R. Kula, Nucleic Acids Res., 1980, 8, 623.
18. L. E. Englerl, P. Sapienzal, L. F. Dorner, R. Kucera, I. Schildkraut and L. Jen-Jacobson, J. Mol. Biol., 2001, 307, 619.
19. Y. Zhang, Y. Wang, H. Wang, J.-H. Jiang, G.-L. Shen, R.-Q. Yu and J. Li, Anal. Chem., 2009 DOI:10.1021/ac802512d.
20. M. T. Carter, M. Rodriguez and A. J. Bard, J. Am. Chem. Soc., 1989, 111, 8901.
21. Z. Dai, J. Chen, F. Yan and H. Ju, Cancer Detect. Prev., 2005, 29, 233.

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