ChemComm View Article Online

Published on 17 March 2014. Downloaded by Temple University on 27/10/2014 03:48:32.

COMMUNICATION

View Journal | View Issue

Cite this: Chem. Commun., 2014, 50, 4733

Sensitive detection of polynucleotide kinase using rolling circle amplification-induced chemiluminescence†

Received 12th January 2014, Accepted 17th March 2014

Wei Tang,‡ Guichi Zhu‡ and Chun-yang Zhang*

DOI: 10.1039/c4cc00256c www.rsc.org/chemcomm

We develop a new method for the sensitive detection of polynucleotide kinase (PNK) using rolling circle amplification-induced chemiluminescence. This method exhibits high sensitivity with a detection limit of 2.20  104 U mL1, which is superior to most reported approaches. Moreover, this method can be used to screen both the inhibitors and the activators of PNK, and can be further applied for real sample analysis.

Polynucleotide kinase (PNK) is an important enzyme with 50 -kinase and 30 -phosphatase activities, and can catalyze the nucleic acid termini conversion of 50 -OH/30 -PO4 into 50 -PO4/30 -OH.1 PNK plays a critical role through the regulation of the 50 -phosphate terminal in many cellular events including DNA recombination,2 DNA/RNA repair,3 mRNA processing and degradation.4,5 Especially, the mutations of PNK might be associated with some severe neurological diseases, such as microcephaly and seizures,6 and the aberrant PNK activity might lead to some human genetic disorders, such as the Bloom syndrome, Werner syndrome, and Rothmund–Thomson syndrome.7 Consequently, the development of sensitive methods for PNK activity assay is highly desirable in biological research and clinical diagnostics. The conventional methods for a PNK activity assay include radioisotope labeling,1,5,8 autoradiography,9a and polyacrylamide gel electrophoresis.9b Although these methods are well established, most of them suffer from radioactive contamination and are hazardous to human health, which limit their practical applications. To overcome these drawbacks, alternative methods have been developed, such as fluorescent,10 electrochemical,11 and colorimetric assays.12 Among these approaches, the fluorescent method is most widely used due to its excellent specificity. However, it always requires costly fluorescent-labeled probes and easily suffers from the photobleaching.13a The electrochemical method exhibits high sensitivity and fast response, but it needs both Single-Molecule Detection and Imaging Laboratory, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China. E-mail: [email protected]; Fax: +86-755-86392299; Tel: +86-755-86392211 † Electronic supplementary information (ESI) available: Details of experimental procedures and additional figures. See DOI: 10.1039/c4cc00256c ‡ These authors contributed equally.

This journal is © The Royal Society of Chemistry 2014

complex immobilization and separation steps.13b The colorimetric method can be carried out using simple and cost-effective instruments. Nevertheless, it is hard to reach a satisfactory sensitivity.13c Thus the development of a low-cost, convenient and highly sensitive method to detect PNK activity still remains a great challenge. Herein, we develop a sensitive and simple method for PNK activity assay using rolling circle amplification (RCA)-induced chemiluminescence. The RCA amplification is an isothermal enzymatic process capable of generating very long single stranded DNA with tandem repeats,14 and has been widely applied for the detection of nucleic acids15 and proteins.16 In this research, we employ the PNK-mediated RCA reaction for the DNAzyme generation and DNAzyme-driven chemiluminescence for the signal readout. The DNAzyme is a special nucleic acid sequence, which can fold into a G-quadruplex structure with the assistance of hemin to produce a strong chemiluminescence signal.17 Taking advantage of both the RCA amplification and the DNAzyme-induced signal enhancement, the proposed method can sensitively measure PNK activity with a detection limit of 2.20  104 U mL1, and can be applied for screening both the inhibitors and the activators of PNK as well as for real sample analysis. As shown in Scheme 1, the proposed method consists of three principal processes: (1) phosphorylation and ligation reaction, (2) RCA reaction, and (3) chemiluminescence detection. The sequence of substrate DNA is 50 -G ACA CAT TTT TCC CAA C C  AAC  A  C G A G T CCC GCC CTA CCC ATT TTT TTA CCC ATC CCG CCC AAC CCT TTT 0 TCA  C C C  C T  C A  A T C C  G A  C-3  , the underlined and italic regions indicate the binding site of padlock probe and the complementary site of DNAzyme, respectively. The sequence of padlock probe is 50 -TAC GTC GGT CGG ATC GGG AGT GTG T-30 . In the first step, the 50 end of single stranded DNA substrate is phosphorylated through the catalysis of PNK. Then the phosphorylated DNA substrate hybridizes with the padlock probe, forming a closed circular DNA with the assistance of DNA ligase. In the second step, the circular DNA and the padlock probe are used as the template and the primer for RCA reaction, respectively. The product of the RCA reaction is a long repetitive single-stranded DNA with the function of DNAzymes. In the third step, the DNAzymes fold into a series of G-quadruplex

Chem. Commun., 2014, 50, 4733--4735 | 4733

View Article Online

Published on 17 March 2014. Downloaded by Temple University on 27/10/2014 03:48:32.

Communication

Scheme 1 Schematic illustration of PNK activity assay using rolling circle amplification-induced chemiluminescence.

structures with the assistance of hemin molecules, generating a high chemiluminescence signal in the presence of hydrogen peroxide (H2O2) and luminol. However, in the absence of PNK, neither phosphorylation nor RCA reaction can be initiated and no chemiluminescence enhancement is observed. To confirm the necessity of PNK, the padlock probe, the DNA ligase and the polymerase for RCA reaction, we performed an agarose gel electrophoresis experiment with SYBR gold as the fluorescent indicator. As shown in Fig. 1A, no distinct band is observed when only three reactants are present (Fig. 1A, lanes 1, 2, 3 and 4), indicating no occurrence of the RCA reaction. When all four reactants are present, a series of DNA bands with high molecular weight are observed (Fig. 1A, lane 5), suggesting the occurrence of the RCA reaction. This result is further confirmed by the chemiluminescence assay of the amplification products of the RCA reaction. As shown in Fig. 1B, a high chemiluminescence signal is observed only in the presence of all four reactants (Fig. 1B, column 5), whereas no significant chemiluminescence signal is detected in the presence of only three reactants (Fig. 1B, columns 1–4). These results indicate that the RCA reaction can be carried out only in the presence of PNK, the padlock probe, the DNA ligase and the polymerase, and the products of RCA reaction can be folded into a series of G-quadruplex structures for the chemiluminescence assay (Scheme 1).

Fig. 1 (A) Electrophoretic analysis of the amplification products. Lane m is the DNA marker. The experimental conditions for lanes 1–5 are indicated above. The products were separated by 0.8% agarose gel electrophoresis and stained by SYBR gold. (B) Chemiluminescence analysis of the amplification products. The 10 U mL1 PNK, 2 U DNA ligase, 2 U polymerase and 136 nM padlock probe are used in the experiments. Error bars show the standard deviation of three experiments.

4734 | Chem. Commun., 2014, 50, 4733--4735

ChemComm

Fig. 2 (A) Variance of the chemiluminescence intensity with the concentration of PNK. The concentration of PNK is 0.0001, 0.0003, 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, 100 U mL1, respectively. (B) The chemiluminescence intensity is log-linear correlation with the concentration of PNK in the range from 0.01 U mL1 to 3 U mL1. Error bars show the standard deviation of three experiments.

To evaluate the sensitivity of the proposed method, we measured the PNK activity at various concentrations under the optimal experimental conditions (see ESI,† Fig. S1 and S2). Fig. 2A shows the variance of the chemiluminescence intensity with the concentration of PNK. Notably, in logarithmic scales the chemiluminescence intensity exhibits a linear correlation with the concentration of PNK over a range of 2 orders of magnitude from 0.01 U mL1 to 3 U mL1 (Fig. 2B). The regression equation is I = 7.45  105 + 1.99  105 log10 C with a correlation coefficient of 0.984, where I and C are the chemiluminescence intensity and the concentration of PNK (U mL1), respectively. The limit of detection is calculated to be 2.20  104 U mL1 by evaluating the average signal of the blank plus three times the standard deviation.17b The sensitivity of the proposed method has improved by as much as 3 orders of magnitude as compared with that of the nanoparticle-based fluorescent assay (0.49 U mL1),18 and 2 orders of magnitude as compared with that of the electrochemical assay (0.01 U mL1)11 and that of the colorimetric assay (0.06 U mL1).12 The capability of the proposed method to screen the inhibitor and the activator of PNK was evaluated by using adenosine 50 -diphosphate sodium salt (ADP) and spermine as the inhibitor and the activator, respectively. ADP is a noncompetitive inhibitor of PNK due to its inhibition role in the phosphorylation reaction.1,19 As shown in Fig. 3A, the relative activity of PNK decreases with the increase of ADP concentration. The IC50 value of ADP is calculated to be 1.32 mM, which is consistent with the reported value.20 Spermine plays a key role in the regulation of eukaryotic cells and the protection from DNA damage, and has been used as an activator of PNK.21 As shown in Fig. 3B, PNK activity is greatly stimulated by the increasing spermine concentration with 2-fold enhancement at the concentration of 0.5 mM, which is consistent with the reported results.19 These results indicate that the proposed method can be applied for screening both the inhibitors and the activators of PNK. We further employed the proposed method to measure the PNK activity in the human embryonic kidney cell line (HEK293T). In this research, we used hydrogen peroxide (H2O2) to stimulate the PNK activity.22 H2O2 is considered to be one of the main causes for DNA double-strand breaks, and can induce DNA repair along with an aberrant activity of PNK in the cells.23 After H2O2 treatment and nucleoprotein extraction, we analyzed the PNK protein levels and

This journal is © The Royal Society of Chemistry 2014

View Article Online

ChemComm

Communication

Published on 17 March 2014. Downloaded by Temple University on 27/10/2014 03:48:32.

This work was supported by the National Natural Science Foundation of China (Grant No. 21325523), the Award for the Hundred Talent Program of the Chinese Academy of Sciences, and the Fund for Shenzhen Engineering Laboratory of Singlemolecule Detection and Instrument Development [Grant No. (2012) 433].

Fig. 3 (A) Inhibition effects of ADP on the phosphorylation. The assays were performed in the reaction buffer containing 0.02 U mL1 PNK under the optimized experimental conditions. Inset shows the structure of ADP. (B) Activation effects of spermine on the phosphorylation. The PNK concentration is 0.01 U mL1. Inset shows the structure of spermine. Error bars show the standard deviation of three experiments.

Fig. 4 (A) Western blot assay of PNK protein in HEK293T cells without H2O2 treatment (lane 1) and with 0.2 mM H2O2 treatment (lane 2). Tubulin and PNK were detected with specific antibodies, respectively. Tubulin was used as a reference for the protein levels; (B) chemiluminescence assay of PNK activity in HEK293T cells without H2O2 treatment (column 1) and with 0.2 mM H2O2 treatment (column 2). Error bars show the standard deviation of three experiments.

PNK activities. As shown in Fig. 4A, H2O2 treatment induces a high PNK protein level (Fig. 4A, lane 2) in comparison with the control group without H2O2 treatment (Fig. 4A, lane 1). We further performed the chemiluminescence assay to detect the PNK activity in HEK293T cells (Fig. 4B). The relative activity of PNK in HEK293T cells with H2O2 treatment (Fig. 4B, column 2) is as much as 2-fold higher than that in the control group without H2O2 treatment (Fig. 4B, column 1). Notably, this is the first report of the direct detection of PNK activity in the unspiked cell samples rather than in the spiked samples with the addition of PNK into serum10c and cell extracts.18,24 In addition, the proposed method shows an excellent selectivity for PNK with no response to nonspecific adenylate kinase and pyruvate kinase (see ESI,† Fig. S3). In conclusion, we have developed a new method for the sensitive detection of PNK activity using RCA-induced chemiluminescence. Taking advantage of the high amplification efficiency of RCA and signal enhancement of chemiluminescence, the proposed method can sensitively measure PNK activity with a detection limit of 2.20  104 U mL1, which is superior to most reported approaches.10a,b,11,12,18,20,24,25 Moreover, the proposed method can be used to screen the inhibitors and the activators of PNK. Importantly, the proposed method can directly measure the PNK activity in the unspiked cell samples, holding great promise for further applications in biological research and clinical diagnostics.

This journal is © The Royal Society of Chemistry 2014

Notes and references 1 J. R. Lillehaug and K. Kleppe, Biochemistry, 1975, 14, 1221. 2 F. Karimi-Busheri, G. Daly, P. Robins, B. Canas, D. J. Pappin, J. Sgouros, G. G. Miller, H. Fakhrai, E. M. Davis and M. M. Le Beau, J. Biol. Chem., 1999, 274, 24187. 3 L. K. Wang, C. D. Lima and S. Shuman, EMBO J., 2002, 21, 3873. ˇ. Strazdaite ˇieliene ˙-Z ˙, A. Zajanc ˇkauskaite ˙, L. Kaliniene ˙, R. Mesˇkys 4 Z ˙, Arch. Virol., 2014, 159, 327. and L. Truncaite 5 S. Durand, G. Richard, F. Bontems and M. Uzan, Proc. Natl. Acad. Sci. U. S. A., 2012, 109, 7073. 6 (a) J. Shen, E. C. Gilmore, C. A. Marshall, M. Haddadin, J. J. Reynolds, W. Eyaid, A. Bodell, B. Barry, D. Gleason, K. Allen, V. S. Ganesh, B. S. Chang, A. Grix, R. S. Hill, M. Topcu, K. W. Caldecott, A. J. Barkovich and C. A. Walsh, Nat. Genet., 2010, 42, 245; (b) J. J. Reynolds, A. K. Walker, E. C. Gilmore, C. A. Walsh and K. W. Caldecott, Nucleic Acids Res., 2012, 40, 6608. 7 (a) S. Sharma, K. Doherty and R. Brosh, Biochem. J., 2006, 398, 319; (b) R. M. Brosh Jr and V. A. Bohr, Nucleic Acids Res., 2007, 35, 7527. 8 M. Amitsur, R. Levitz and G. Kaufmann, EMBO J., 1987, 6, 2499. 9 (a) D. H. Phillips and V. M. Arlt, Nat. Protoc., 2007, 2, 2772; (b) F. Karimi-Busheri, J. Lee, M. Weinfeld and A. E. Tomkinson, Nucleic Acids Res., 1998, 26, 4395. 10 (a) C. Song and M. Zhao, Anal. Chem., 2009, 81, 1383; (b) C. Ma, H. Fang, K. Wang, K. Xia, H. Chen, H. He and W. Zeng, Anal. Biochem., 2013, 443, 166; (c) F. Chen, Y. Zhao, L. Qi and C. Fan, Biosens. Bioelectron., 2013, 47, 218; (d) Z. Tang, K. Wang, W. Tan, C. Ma, J. Li, L. Liu, Q. Guo and X. Meng, Nucleic Acids Res., 2005, 33, e97; (e) T. Hou, X. Wang, X. Liu, T. Lu, S. Liu and F. Li, Anal. Chem., 2014, 86, 884. 11 Y. Peng, J. Jiang and R. Yu, RSC Adv., 2013, 3, 18128. 12 C. Jiang, C. Yan, J. Jiang and R. Yu, Anal. Chim. Acta, 2013, 766, 88. 13 (a) W. Shen, H. M. Deng and Z. Q. Gao, J. Am. Chem. Soc., 2012, 134, 14678; (b) Q. Su, D. Xing and X. Zhou, Biosens. Bioelectron., 2010, 25, 1615; (c) C. Lodeiro, J. L. Capelo, J. C. Mejuto, E. Oliveira, ˜ ez, Chem. Soc. Rev., 2010, H. M. Santos, B. Pedras and C. Nun 39, 2948. 14 S. A. McManus and Y. Li, J. Am. Chem. Soc., 2013, 135, 7181. 15 Y. Tian, Y. He and C. Mao, ChemBioChem, 2006, 7, 1862. 16 L. Yang, C. W. Fung, E. J. Cho and A. D. Ellington, Anal. Chem., 2007, 79, 3320. 17 (a) H. Q. Wang, W. Y. Liu, Z. Wu, L. J. Tang, X. M. Xu, R. Q. Yu and J. H. Jiang, Anal. Chem., 2011, 83, 1883; (b) Y. P. Zeng, J. Hu, Y. Long and C. Y. Zhang, Anal. Chem., 2013, 85, 6143. 18 L. Zhang, J. Zhao, H. Zhang, J. Jiang and R. Yu, Biosens. Bioelectron., 2013, 44, 6. 19 C. Ma and E. S. Yeung, Anal. Bioanal. Chem., 2010, 397, 2279. 20 L. Lin, Y. Liu, J. Yan, X. Wang and J. Li, Anal. Chem., 2012, 85, 334. 21 J. R. Lillehaug and K. Kleppe, Biochemistry, 1975, 14, 1225. 22 N. Driessens, S. Versteyhe, C. Ghaddhab, A. Burniat, X. De Deken, J. Van Sande, J. E. Dumont, F. Miot and B. Corvilain, Endocr.–Relat. Cancer, 2009, 16, 845. 23 (a) T. Izumi, L. R. Wiederhold, G. Roy, R. Roy, A. Jaiswal, K. K. Bhakat, S. Mitra and T. K. Hazra, Toxicology, 2003, 193, 43; (b) F. Altieri, C. Grillo, M. Maceroni and S. Chichiarelli, Antioxid. Redox Signaling, 2008, 10, 891. 24 (a) L. Lin, Y. Liu, X. Zhao and J. Li, Anal. Chem., 2011, 83, 8396; (b) H. Jiao, B. Wang, J. Chen, D. Liao, W. Li and C. Yu, Chem. Commun., 2012, 48, 7862. 25 (a) Y. Wang, X. He, K. Wang, X. Ni, J. Su and Z. Chen, Biosens. Bioelectron., 2012, 32, 213; (b) G. Wang, X. He, G. Xu, L. Chen, Y. Zhu, X. Zhang and L. Wang, Biosens. Bioelectron., 2012, 43, 125.

Chem. Commun., 2014, 50, 4733--4735 | 4735

Sensitive detection of polynucleotide kinase using rolling circle amplification-induced chemiluminescence.

We develop a new method for the sensitive detection of polynucleotide kinase (PNK) using rolling circle amplification-induced chemiluminescence. This ...
1MB Sizes 0 Downloads 4 Views