Biosensors and Bioelectronics 68 (2015) 654–659
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A DNA machine-based fluorescence amplification strategy for sensitive detection of uracil-DNA glycosylase activity Yushu Wu a, Lei Wang b, Jing Zhu a, Wei Jiang a,n a Key Laboratory for Colloid and Interface Chemistry of Education Ministry, School of Chemistry and Chemical Engineering, Shandong University, 250100 Jinan, PR China b School of Pharmaceutical Sciences, Shandong University, 250012 Jinan, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 29 November 2014 Received in revised form 28 January 2015 Accepted 29 January 2015 Available online 30 January 2015
Sensitive detection of uracil-DNA glycosylase (UDG) activity is critical for function study of UDG and clinical diagnosis. Here, we developed a novel fluorescent strategy for sensitive detection of UDG activity based on the signal amplification by a label-free and enzyme-free DNA machine. A double-strand DNA (dsDNA) probe P1–P2 with uracil bases and trigger sequence was designed for UDG recognition and signal transduction. Two hairpin probes H1 and H2 which were partially complementary were employed to construct the label-free and enzyme-free DNA machine. Under the action of UDG, uracil bases were removed from the P1-P2 dsDNA probe, and then a strand P2ʹ with abasic sites was released. Subsequently, the liberated P2ʹ activated the DNA machine and generated numerous H1–H2 complexes containing G-quadruplex (G4) structures in the end. Finally, the G4 structures could bind with N-methylmesoporphyrin IX (NMM) to form G4-NMM complexes with the enhanced fluorescence responses. This strategy could detect UDG activity as low as 0.00044 U/mL. In addition, the strategy was also applied for the analysis of UDG activity in HeLa cells lysate with low effect of cellular components. Moreover, this strategy was successfully applied for assaying the inhibition of UDG using uracil glycosylase inhibitor (UGI). This strategy provided a potential tool for sensitive quantification of UDG activity in UDG functional study and clinical diagnosis. & 2015 Elsevier B.V. All rights reserved.
Keywords: Uracil-DNA glycosylase activity DNA machine Fluorescence amplification Label-free Enzyme-free
1. Introduction Uracil-DNA glycosylase (UDG) is a DNA repair enzyme that plays crucial roles in maintaining cellular genome integrity. It is able to remove uracil lesions from DNA through catalyzing the hydrolysis of the N-glycosidic bond that links the uracil to the deoxyribose (Kunkel and Erie, 2005; Stivers and Jiang, 2003). Abnormal UDG activity would suppress the cellular responses to uracil lesions and directly relate to various diseases, including human immunodeficiency (Imai et al., 2003), lymphoma (Sousa et al., 2007) and Bloom syndrome (Seal et al., 1988). It is revealed that the activity of UDG has become a promising biomarker for these diseases, and detection of UDG activity represents a candidate tool for function study of UDG and clinical diagnosis (Ono et al., 2013). The commonly-used methods for UDG activity detection are based on gel electrophoresis (Prorok et al., 2013; de Souza-Pinto et al., 2004), electrochemical assays (McWilliams et al., 2014) and n
Corresponding author. Fax: þ86 531 88564464. E-mail address: [email protected] (W. Jiang).
http://dx.doi.org/10.1016/j.bios.2015.01.069 0956-5663/& 2015 Elsevier B.V. All rights reserved.
colorimetric assays (Liu et al., 2014). Except these methods, fluorescence-based methods are attractive due to the advantages of safety, simplicity and sensitivity. Many fluorescent UDG activity assays have been designed based on the uracil-containing DNA probes for target recognition and signal output, which achieved simple detection of UDG activity (Ono et al., 2013; Hu et al., 2011; Ono et al., 2012). However, the sensitivities of the reported fluorescent assays are not entirely satisfactory, so the signal amplification approaches have been introduced into the fluorescent UDG activity assays. Yu's group proposed an autocatalytic DNAzyme amplification method for more sensitive fluorescent assay of UDG activity, resulting in a low detection limit of 0.002 U/mL (Zhang et al., 2012). Additionally, Lu and his colleagues demonstrated an amplified fluorescent strategy for UDG activity quantification through the UDG-dependent deactivation or activation of the DNAzyme, bringing a low detection limit of 0.0034 U/mL (Xiang and Lu, 2012). While good performance has been realized through the above-mentioned amplified fluorescent UDG assays, these assays depend on either double labeled fluorescent probe, or/and protein enzyme. For the double labeled fluorescent probe that is labeled with a fluorophore and a quencher covalently, the
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quencher may not completely quench the fluorescence of the donors, leading to the high background and limiting the further improvement of detection sensitivity (Zhou et al., 2011). In addition, the protein enzyme is sensitive to reaction conditions and easy to be inactive, which disturbs the signal amplification capability resulting in the problem of the poor repeatability. Therefore, further efforts are needed in the development of new fluorescent strategies for label-free, enzyme-free and sensitive detection of UDG activity. DNA machines are supramolecular nucleic acid structures that perform machine-like functions at the molecular level in the presence of the appropriate triggers (Liu et al., 2014). The uses of DNA machines as switches (Qi et al., 2013), walkers (Liu et al., 2013), or amplified sensors (Wang et al., 2012; Wang et al., 2011) have been discussed. Specifically, since DNA machines can perform amplification autonomously and effectively only under simple operations, they have been utilized for amplified analysis of nucleic acids (Lu et al., 2012; Zhuang et al., 2014; Freeman et al., 2011; Li et al., 2014; Xu et al., 2015; Li et al., 2015), small molecules (Zhang et al., 2010; Zhang et al., 2011) and metal ions (Wang et al., 2012; Li et al., 2008; He et al., 2014). Herein, a sensitive fluorescence amplification strategy based on a label-free and enzyme-free DNA machine was described for UDG activity measurement. We employed a double-strand DNA (dsDNA) probe containing uracil bases and trigger sequence to recognize UDG target with the releasing of trigger strand, which could activate the label-free and enzyme-free DNA machine, generating amplified fluorescent signal. Due to the unique designs of both the dsDNA probe and the specific DNA machine, background reduction and signal amplification were successfully achieved, which resulted in a low detection limit (0.00044 U/mL). Moreover, the strategy was also applied for UDG activity analysis in HeLa cells lysate with low effect of cellular components. The proposed strategy offered a potential tool for sensitive quantification of UDG activity in UDG functional study and clinical diagnosis.
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2. Experimental section 2.1. Reagents and apparatus The DNA oligonucleotides used in this work were synthesized and purified by Sangon Inc. (Shanghai, China) and the sequences of the oligonucleotides were listed in Table S1. Uracil-DNA glycosylase (UDG), uracil glycosylase inhibitor (UGI) and 8-oxoguanine DNA glycosylase (hOGG1) were obtained from New England Biolabs Ltd. (Beijing, China). One unit of UGI activity was defined as the amount of protein required to inhibit one unit of E.coli UDG in 1 h at 37 °C in a total reaction volume of 50 mL. One unit of UDG was the amount of enzyme which would catalyze the release of 60 pmol of uracil per minute from double stranded, uracil-containing DNA. Exonuclease I (Exo I) and Exonuclease III (Exo III) were purchased from Thermo Fisher Scientific Ltd. (China). N-methylmesoporphyrin IX (NMM) was purchased from Frontier Scientific Inc. (Logan, Utah, USA). The NMM stock solution was prepared in dimethyl sulfoxide (DMSO) and stored in the dark at 20 °C. All other chemicals were of analytical grade and used as received. All solutions were prepared using the ultrapure water that was obtained from a Millipore Milli-Q water purification system ( 4 18.25 M Ω). Fluorescence measurements were performed using a Hitachi F-7000 fluorescence spectrometer (Hitachi. Ltd., Japan). The excitation wavelength was 399 nm with a recording emission range of 570–700 nm. The fluorescence intensity at 618 nm was used to evaluate the performance of the proposed strategy. The slits of both excitation and emission were 10 nm and the photomultiplier tube voltage was 700 V. 2.2. Assay for UDG activity and inhibition To prepare the double-strand DNA (dsDNA) probe (P1–P2) and hairpin structures, a mixture of P1 and P2, H1 and H2 were all denatured at 95 °C for 10 min, respectively. And then they were all cooled slowly to room temperature in a hybridization buffer (10 mM NaCl, 2.0 mM MgCl2, pH 8.0). For assaying UDG activity, the prepared dsDNA probe P1-P2 and various amounts of UDG
Scheme 1. Schematic illustration of the fluorescence amplification strategy based on a label-free and enzyme-free DNA machine for sensitive detection of UDG activity. U and A denote the uracil and adenine deoxyribonucleotides. Figures marked with n are complementary to the corresponding unmarked figures. In P1–P2 probe, the black segment is the uracil-containing DNA sequence, the red segment is the trigger sequence that can trigger downstream DNA machine, and the purple segment is the inhibition strand. In H1 probe, the red segment is complementary to the trigger sequence in P1-P2 probe, and the blue segment is the sequence that can open H2 probe. In H2 probe, the blue segment is complementary to the blue segment in H1 probe, the red segment is complementary to the red segment in H1 probe, and the green segment is the G-rich sequence.
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were added to the reaction buffer (20 mM Tris–HCl, 1.0 mM EDTA, 1.0 mM DTT, 10 mM NaCl, 2.0 mM MgCl2, pH 8.0) to give a total volume of 50 mL. The mixture was incubated at 37 °C for 90 min to allow the base cleavage reaction to take place. Then H1, H2 and 10 mL 10 TNaK buffer (200 mM Tris, 1.4 M NaCl, 50 mM KCl, pH 7.5) were added into the above solution with a final volume of 100 mL. The amplification reaction of the DNA machine was carried out at 37 °C for 45 min. At last, NMM was added to the resulting product and the mixture was incubated at 37 °C for 30 min. The control experiment was carried out under the same condition without adding UDG. For assaying the inhibition of UDG, 250 nM prepared dsDNA probe P1–P2 was incubated with 1 U/mL UDG and various concentrations of UGI. The mixture was heated at 37 °C for 90 min. Then H1, H2 and 10 mL 10 TNaK buffer (200 mM Tris, 1.4 M NaCl, 50 mM KCl, pH 7.5) were added into the mixture with a final volume of 100 mL, and the final concentrations of H1 and H2 were 450 nM and 675 nM, respectively. The amplification reaction of the DNA machine was carried out at 37 °C for 45 min. At last, NMM was added to the resulting product and the mixture was incubated at 37 °C for 30 min. All experiments were repeated three times. 2.3. Gel electrophoresis to investigate the dissociation of the dsDNA probe The indicated concentrations of P1–P2 and UDG were kept at 37 °C for 90 min to obtain the UDG-treated product. And we also analyzed P1, P2 and the dsDNA probe P1–P2 in the absence of UDG, respectively. Samples were mixed with loading buffer and loaded into the 15% polyacrylamide gels. The electrophoresis was run at a constant current of 30 mA for about 2 h, using 1 TBE (89 mM Tris, 89 mM Boric Acid, 2.0 mM EDTA, pH 8.3) as running buffer. The gels were stained with ethidium bromide for 5 min, destained in distilled water for 5 min, and then photographed under UV imaging system (Bio-RAD Laboratories Inc. USA). 2.4. Preparation of HeLa cells lysate Approximately 1 108 HeLa cells samples were pelleted by centrifugation (5 min, 3000 rpm, 4 °C) and resuspended in 20 mL of lysis buffer (10 mM Tris–HCl, pH 7.0) on ice using a sonicator (four pulses at 200 W for 30 s with a tapered microtip). The mixture solution was then centrifuged at 12,000 rpm for 30 min at 4 °C to remove insoluble material. The resulting supernatant was collected and filtered through a 0.45 mm filter membranes, yielding crude lysate.
3. Results and discussion 3.1. The principle of the fluorescence amplification strategy based on a label-free and enzyme-free DNA machine for UDG activity assay Scheme 1 outlined the principal design of the proposed strategy for UDG activity assay. The nucleic acid strands utilized here were rationally designed to obtain a satisfactory result. Among them, P1 was an inhibition strand. In addition, P2 was a chimeric conjugate of the uracil-containing DNA sequence (in black) and trigger sequence (in red). The single-strand DNA (ssDNA) P2 which contained both the uracil bases and trigger sequence was partly hybridized with the inhibition strand P1 to form P1–P2 dsDNA probe. The dsDNA probe showed a relatively high melting temperature, making the probe a stable hybrid and blocking the trigger sequence from hybridization with a hairpin probe. Two hairpin probes H1 and H2 which were partially complementary were designed to construct the label-free and enzyme-free DNA
machine according to the trigger sequence. To avoid sophisticated chemical labeling, a G-quadruplex (G4) sequence was grafted at the end of H2 (domains 4 and 5). When H2 was in its hairpin structure, domain 4 was hidden in the stem of H2. Therefore, G4 conformation cannot form at this time. Under the action of UDG, uracil bases could be removed from the deoxyriboses phosphate backbone of P2, generating abasic (AP) sites and leading to a lower melting temperature of the P1–P2 dsDNA probe, which resulted in the liberation of P2ʹ from P1–P2. The liberated P2ʹ could then initiate the signal amplification process of the designed DNA machine. Particularly, domain 1 in trigger strand P2’ was exposed and could hybridize to the accessible toehold 1n in hairpin H1, which initiated the branch migration and exposed 3, 4, 1 of hairpin H1. The resultant full H1–P2ʹ hybridization opened H1 and exposed a new toehold, domain 3, which could nucleate at the 3n of hairpin H2. As a result, domains 3, 2, 1, 4 and 5 of hairpin H2 were fully accessible via branch migration. Since the H1–H2 complex was more stable than H1–P2ʹ hybridization, the accessible domains 3, 2, 1 of hairpin H2 could displace P2ʹ, forming H1–H2 complex and releasing domains 4 and 5 in H2. The liberated domains 4 and 5 in H2 were able to fold into a G4 conformation in the presence of monovalent ions. Then, the released P2ʹ could trigger the next reaction cycle to consecutively generate H1–H2 complexes containing G4 structures. Finally, NMM which had a pronounced structural selectivity for G4 structures but not for dsDNA or ssDNA forms (Hu et al., 2011) was added to the reaction system to form G4-NMM complex generating label-free fluorescence signal. In this way, one released trigger strand can create increased fluorescence signal for the sensitive assay of UDG activity through the amplification process of the label-free and enzyme-free DNA machine. 3.2. Feasibility research As shown in Fig. 1, fluorescence emission spectra were used to investigate the viability of our approach. The system only containing H2 showed very weak fluorescence intensity (Fig. 1A, curve a), suggesting the formation of G4 conformation was difficult when the sequence was partly hidden in the stem of H2. The system containing H1 and H2 exhibited slightly enhanced fluorescence intensity (Fig. 1A, curve b), indicating the interaction of H1 and H2 was weak without target. When P1–P2 was added into the H1, H2 system, the fluorescence intensity exhibited further enhancement (Fig. 1A, curve c), which indicated the interaction of H1 and H2 was facilitated by P1–P2 resulting in background fluorescence. However, the fluorescence intensity had significant enhancement upon addition of UDG to the system containing P1–P2, H1 and H2 (Fig. 1A, curve d). This suggested that UDG could trigger the self-assembly of H1 and H2 by removing uracil bases in dsDNA probe P1–P2 with the releasing of trigger strand P2ʹ. Polyacrylamide gel electrophoresis (PAGE) was also used to verify the dissociation of the dsDNA probe. After the UDG treatment, the dsDNA probe was dissociated into discrete bands, as shown in Fig. 1B. Thus, these results demonstrated that the proposed strategy could be adopted for UDG activity detection. 3.3. Optimization of the reaction conditions The reaction conditions played important roles in the assay, such as the probe concentration and the reaction time. Therefore, the reaction conditions were optimized in order to achieve the best results. The optimal system should give low background signal, as well as a great fluorescence enhancement on the addition of UDG. It was widely accepted that the concentration of the probe had an important effect on the sensing process. In our study, P1 was the inhibition probe, P2 was the conjugate probe containing both
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Fig. 1. (A) Fluorescence emission spectra of the system: (a) H2 alone, (b) H1 þ H2, (c) P1–P2 þ H1þ H2, and (d) P1–P2 þ UDG (1.0 U/mL)þ H1þ H2. (B) Nondenaturing polyacrylamide gel (15%) electrophoresis analysis of the dissociation of dsDNA probe P1–P2 under the action of UDG. Lane 1 contains 5.0 mM P1. Lane 2 contains 1.0 mM P2. Lane 3 contains 1.0 mM P1–P2 dsDNA probe without UDG. Lane 4 contains 1.0 mM P1–P2 dsDNA probe with 10 U/mL UDG.
the uracil bases and trigger sequence, H1 and H2 were amplification probes which were employed to construct the DNA machine. We investigated the concentrations of these probes when the concentration of UDG was 1.0 U/mL. The low hybridization efficiency of P1 and P2 would result in high background signal. In order to achieve the good suppressive effect as well as the low background signal, we first optimized the concentration of P1. We fixed the concentration of P2 as 250 nM and changed the concentration of P1, which caused the changing in the hybridization proportion of P1 to P2. As shown in Fig. S1, the net signal ΔF (Δ F¼F–F0, where F and F0 were fluorescence intensities of the system in the presence and absence of UDG) was no longer increasing when the concentration of P1 was greater than 1.00 μM. Therefore, we chose 1.25 μM as the optimal concentration of P1. Furthermore, we also optimized the concentrations of other probes. As shown in Figs. S2–S4, the ΔF reached the maximum when the concentration of P2, H1 and H2 were 250 nM, 450 nM and 675 nM, respectively. The sensing process was also influenced by the reaction time.
The uracil cleavage reaction time and amplification reaction time were set as 90 min and 45 min for the subsequent experiments based on the optimization experiment results (Figs. S5 and S6). 3.4. Analytical performance of the developed strategy in UDG activity assay Under the optimal experimental conditions, we investigated the fluorescence responses of the as-proposed strategy to different concentrations of UDG. As depicted in Fig. 2A, the fluorescence intensity was enhanced as the concentration of UDG increased from 0 to 1.0 U/mL, suggesting that the formation of the fluorescent G4-NMM complex was highly dependent on the concentration of UDG. The calibration curve for detection of UDG activity was shown in Fig. 2B. As illustrated in the inset of Fig. 2B, the net signal ΔF exhibited a linear correlation to the activity of UDG ranging from 0.0025 to 0.020 U/mL with an R2 of 0.999. According to the 3δ rule, the detection limit for UDG was as low as
Fig. 2. (A) Fluorescence responses to the different concentrations of UDG. (B) The calibration curve of ΔF versus the UDG concentration. The inset shows the linear responses at low UDG concentrations. Error bars represent the standard deviations of the results from three independent experiments. Condition: CP1 ¼1.25 μM, CP2 ¼250 nM, CH1 ¼ 450 nM, and CH2 ¼675 nM, uracil cleavage reaction time 90 min, amplification reaction time 45 min.
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Fig. 3. Relative fluorescence intensity of the reaction systems with the addition of UDG (1.0 U/mL) or 10-fold excess of Exo I, Exo III and hOGG1 (10 U/mL). Error bars represent the standard deviations of the results from three independent experiments. Condition: CP1 ¼1.25 μM, CP2 ¼ 250 nM, CH1 ¼450 nM, and CH2 ¼ 675 nM.
Fig. 4. Fluorescence emission spectra in the absence and presence of 60% HeLa cells lysate, and the inhibitory effect of 2.0 U/mL UGI on the UDG activity in the HeLa cells lysate. Condition: CP1 ¼ 1.25 μM, CP2 ¼250 nM, CH1 ¼450 nM, CH2 ¼675 nM.
clinical diagnosis. 0.00044 U/mL, which was lower than most reported fluorescent amplification strategies for UDG activity detection (Zhang et al., 2012; Xiang and Lu, 2012). The high sensitivity of our method was most probably attributed to the signal amplification of the designed DNA machine, as well as the background reduction of the unique dsDNA probe. Hence, the proposed strategy held great promise as an effective tool for quantification of UDG activity. To further evaluate the selectivity of this strategy, we studied its fluorescence responses towards UDG against other nucleases including Exo I, Exo III and another glycosylase hOGG1. As shown in Fig. 3, only UDG caused significant relative fluorescence intensity, whereas Exo I, Exo III or hOGG1 gave low relative intensity which was comparable to that in the blank solution. This result indicated that the proposed strategy exhibited good selectivity to active UDG. As important parameters to assess a strategy in practical application, the precision and reproducibility of the proposed strategy were investigated. To evaluate the precision of the proposed strategy, a series of three repetitive experiments of target samples were carried out on the same day. The relative standard deviations (RSD) achieved for the samples containing 0.0025 U/mL, 0.010 U/mL and 0.020 U/mL of UDG were 4.4%, 3.7% and 3.7%, respectively. Similarly, a series of three repetitive experiments of target samples were conducted on three different days to demonstrate the repeatability of the strategy. The RSD achieved for samples containing different UDG concentrations (0.0025 U/mL, 0.010 U/mL and 0.020 U/mL) were 5.0%, 4.2% and 4.0%, respectively. These results indicated that the as-proposed strategy possessed acceptable precision and reproducibility. To demonstrate the applicability of the proposed strategy for detection of UDG activity, it was also used for the analysis of UDG activity in HeLa cells lysate. As shown in Fig. 4, lysis buffer could only induce very low fluorescence signal. In contrast, the fluorescence signal of the system had significant enhancement in the assay of 60% (the volume fraction of HeLa cells lysate in reaction system) HeLa cells lysate due to the presence of UDG activity. To confirm that the fluorescence enhancement was induced by UDG rather than any other component in the lysate, UGI was also added to the cell lysate, resulting in no appreciable fluorescence enhancement. The results revealed that the developed strategy could be tolerant toward the cellular components and would become a candidate tool for UDG activity assay in function study of UDG and
3.5. Assaying the inhibition of UDG The utility of this DNA machine-based fluorescence amplification strategy for assaying the inhibition of UDG was also studied. Herein, UGI was selected as a model inhibitor for this study. UGI can form a tight and physiologically irreversible complex with UDG in 1:1 M stoichiometry (Kaushal et al., 2008). As shown in Fig. 5, the relative intensity of fluorescence in the system was diminished in the presence of UGI in a dose-dependent manner. The result demonstrated that the proposed strategy can be used to assay the inhibition of UDG.
4. Conclusions In conclusion, we developed a novel fluorescent amplification strategy based on a label-free and enzyme-free DNA machine for
Fig. 5. Relative fluorescence intensity of the system in the presence of increasing concentrations of UGI (0, 0.10, 0.20, 0.40, 0.80, 1.0 and 2.0 U/mL). Error bars represent the standard deviations of the results from three independent experiments.
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sensitive detection of UDG activity. This strategy employed a dsDNA probe to release a trigger strand after being recognized by UDG, and the released trigger strand could initiate the amplification of the DNA machine. The designed dsDNA probe containing uracil bases and trigger sequence exhibited good suppressive effect on the trigger sequence with high hybridization efficiency, resulting in low background. Additionally, the label-free design did not need covalent attachment of fluorophore or quencher to DNA, preventing participation of fluorescent labels in the background. Moreover, the signal amplification capability of the designed DNA machine was used to further improve the detection sensitivity. This strategy could detect UDG activity as low as 0.00044 U/mL, which was lower than most reported fluorescent amplification strategies for UDG activity detection. Furthermore, the enzymefree design made the signal amplification capability of the DNA machine independent on the vulnerable enzyme, leading to a good repeatability. Finally, the strategy was also applied for analysis of UDG activity in HeLa cells lysate with low effect of cellular components. The results revealed that the developed strategy provided a potential tool for sensitive quantification of UDG activity in UDG functional study and clinical diagnosis.
Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant nos. 21175081, 21175082, 21375078 and 21475077).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2015.01.069.
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References de Souza-Pinto, N.C., Harris, C.C., Bohr, V.A., 2004. Oncogene 23, 6559–6568. Freeman, R., Liu, X.Q., Willner, I., 2011. Nano Lett. 11, 4456–4461. He, J.L., Zhu, S.L., Wu, P., Li, P.P., Li, T., Cao, Z., 2014. Biosens. Bioelectron. 60, 112–117. Hu, D., Huang, Z.Z., Pu, F., Ren, J.S., Qu, X.G., 2011. Chem. Eur. J 17, 1635–1641. Imai, K., Slupphaug, G., Lee, W.-I., Revy, P., Nonoyama, S., Catalan, N., Yel, L., Forveille, M., Kavli, B., Krokan, H.E., Ochs, H.D., Fischer, A., Durandy, A., 2003. Nat. Immunol. 4, 1023–1028. Kaushal, P.S., Talawar, R.K., Krishna, P.D., Varshney, U., Vijayan, M., 2008. Acta Crystallogr. D Biol. Crystallogr. 64, 551–560. Kunkel, T.A., Erie, D.A., 2005. Annu. Rev. Biochem. 74, 681–710. Li, D., Wieckowska, A., Willner, I., 2008. Angew. Chem. 120, 3991–3995. Li, H.L., Ren, J.T., Liu, Y.Q., Wang, E.K., 2014. Chem. Commun. 50, 704–706. Li, X.H., Gan, L.J., Ou, Q.S., Zhang, X., Cai, S.X., Wu, D.Z., Chen, M., Xia, Y.K., Chen, J.H., Yang, B., 2015. Biosens. Bioelectron. 66, 399–404. Liu, X.J., Chen, M.Q., Hou, T., Wang, X.Z., Liu, S.F., Li, F., 2014. Biosens. Bioelectron. 54, 598–602. Liu, X.Q., Lu, C.-H., Willner, I., 2014. Acc. Chem. Res. 47, 1673–1680. Liu, X.Q., Niazov-Elkan, A., Wang, F., Willner, I., 2013. Nano Lett. 13, 219–225. Lu, C.H., Wang, F., Willner, I., 2012. J. Am. Chem. Soc. 134, 10651–10658. McWilliams, M.A., Anka, F.H., Balkus, K.J., Slinker, J.D., 2014. Biosens. Bioelectron. 54, 541–546. Ono, T., Edwards, S.K., Wang, S., Jiang, W., Kool, E.T., 2013. Nucleic Acids Res. 41, 127–138. Ono, T., Wang, S.L., Koo, C.K., Engstrom, L., David, S.S., Kool, E.T., 2012. Angew. Chem. 51, 1689–1692. Prorok, P., Alili, D., Saint-Pierre, C., Gasparutto, D., Zharkov, D.O., Ishchenko, A.A., Tudek, B., Saparbaev, M.K., 2013. Proc. Natl. Acad. Sci. USA 110, 3695–3703. Qi, X.J., Lu, C.H., Liu, X.Q., Shimron, S., Yang, H.H., Willner, I., 2013. Nano Lett. 13, 4920–4924. Seal, G., Brech, K., Karp, S.J., Cool, B.L., Sirover, M.A., 1988. Proc. Natl. Acad. Sci. USA 85, 2339–2343. Sousa, M.M., Krokan, H.E., Slupphaug, G., 2007. Mol. Aspects Med. 28, 276–306. Stivers, J.T., Jiang, Y.L., 2003. Chem. Rev. 103, 2729–2759. Wang, F., Elbaz, J., Teller, C., Willner, I., 2011. Angew. Chem. 50, 295–299. Wang, F., Elbaz, J., Willner, I., 2012. J. Am. Chem. Soc. 134, 5504–5507. Wang, F., Orbach, R., Willner, I., 2012. Chem. Eur. J 18, 16030–16036. Xiang, Y., Lu, Y., 2012. Anal. Chem. 84, 9981–9987. Xu, J.G., Dong, H.Y., Shen, W.Y., He, S.D., Li, H.L., Lu, Y.S., Wu, Z.S., Jia, L., 2015. Biosens. Bioelectron. 66, 504–511. Zhang, H., Fang, C.C., Zhang, S.S., 2010. Chem. Eur. J 16, 12434–12439. Zhang, H., Fang, C.C., Zhang, S.S., 2011. Chem. Eur. J 17, 7531–7537. Zhang, L.L., Zhao, J.J., Jiang, J.H., Yu, R.Q., 2012. Chem. Commun. 48, 8820–8822. Zhou, Z.X., Du, Y., Dong, S.J., 2011. Anal. Chem. 83, 5122–5127. Zhuang, J.Y., Lai, W.Q., Chen, G., Tang, D.P., 2014. Chem. Commun. 50, 2935–2938.
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A DNA machine-based fluorescence amplification strategy for sensitive detection of uracil-DNA glycosylase activity Yushu Wu a, Lei Wang b, Jing Zhu a, Wei Jiang a,n a Key Laboratory for Colloid and Interface Chemistry of Education Ministry, School of Chemistry and Chemical Engineering, Shandong University, 250100 Jinan, PR China b School of Pharmaceutical Sciences, Shandong University, 250012 Jinan, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 29 November 2014 Received in revised form 28 January 2015 Accepted 29 January 2015 Available online 30 January 2015
Sensitive detection of uracil-DNA glycosylase (UDG) activity is critical for function study of UDG and clinical diagnosis. Here, we developed a novel fluorescent strategy for sensitive detection of UDG activity based on the signal amplification by a label-free and enzyme-free DNA machine. A double-strand DNA (dsDNA) probe P1–P2 with uracil bases and trigger sequence was designed for UDG recognition and signal transduction. Two hairpin probes H1 and H2 which were partially complementary were employed to construct the label-free and enzyme-free DNA machine. Under the action of UDG, uracil bases were removed from the P1-P2 dsDNA probe, and then a strand P2ʹ with abasic sites was released. Subsequently, the liberated P2ʹ activated the DNA machine and generated numerous H1–H2 complexes containing G-quadruplex (G4) structures in the end. Finally, the G4 structures could bind with N-methylmesoporphyrin IX (NMM) to form G4-NMM complexes with the enhanced fluorescence responses. This strategy could detect UDG activity as low as 0.00044 U/mL. In addition, the strategy was also applied for the analysis of UDG activity in HeLa cells lysate with low effect of cellular components. Moreover, this strategy was successfully applied for assaying the inhibition of UDG using uracil glycosylase inhibitor (UGI). This strategy provided a potential tool for sensitive quantification of UDG activity in UDG functional study and clinical diagnosis. & 2015 Elsevier B.V. All rights reserved.
Keywords: Uracil-DNA glycosylase activity DNA machine Fluorescence amplification Label-free Enzyme-free
1. Introduction Uracil-DNA glycosylase (UDG) is a DNA repair enzyme that plays crucial roles in maintaining cellular genome integrity. It is able to remove uracil lesions from DNA through catalyzing the hydrolysis of the N-glycosidic bond that links the uracil to the deoxyribose (Kunkel and Erie, 2005; Stivers and Jiang, 2003). Abnormal UDG activity would suppress the cellular responses to uracil lesions and directly relate to various diseases, including human immunodeficiency (Imai et al., 2003), lymphoma (Sousa et al., 2007) and Bloom syndrome (Seal et al., 1988). It is revealed that the activity of UDG has become a promising biomarker for these diseases, and detection of UDG activity represents a candidate tool for function study of UDG and clinical diagnosis (Ono et al., 2013). The commonly-used methods for UDG activity detection are based on gel electrophoresis (Prorok et al., 2013; de Souza-Pinto et al., 2004), electrochemical assays (McWilliams et al., 2014) and n
Corresponding author. Fax: þ86 531 88564464. E-mail address: [email protected] (W. Jiang).
http://dx.doi.org/10.1016/j.bios.2015.01.069 0956-5663/& 2015 Elsevier B.V. All rights reserved.
colorimetric assays (Liu et al., 2014). Except these methods, fluorescence-based methods are attractive due to the advantages of safety, simplicity and sensitivity. Many fluorescent UDG activity assays have been designed based on the uracil-containing DNA probes for target recognition and signal output, which achieved simple detection of UDG activity (Ono et al., 2013; Hu et al., 2011; Ono et al., 2012). However, the sensitivities of the reported fluorescent assays are not entirely satisfactory, so the signal amplification approaches have been introduced into the fluorescent UDG activity assays. Yu's group proposed an autocatalytic DNAzyme amplification method for more sensitive fluorescent assay of UDG activity, resulting in a low detection limit of 0.002 U/mL (Zhang et al., 2012). Additionally, Lu and his colleagues demonstrated an amplified fluorescent strategy for UDG activity quantification through the UDG-dependent deactivation or activation of the DNAzyme, bringing a low detection limit of 0.0034 U/mL (Xiang and Lu, 2012). While good performance has been realized through the above-mentioned amplified fluorescent UDG assays, these assays depend on either double labeled fluorescent probe, or/and protein enzyme. For the double labeled fluorescent probe that is labeled with a fluorophore and a quencher covalently, the
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quencher may not completely quench the fluorescence of the donors, leading to the high background and limiting the further improvement of detection sensitivity (Zhou et al., 2011). In addition, the protein enzyme is sensitive to reaction conditions and easy to be inactive, which disturbs the signal amplification capability resulting in the problem of the poor repeatability. Therefore, further efforts are needed in the development of new fluorescent strategies for label-free, enzyme-free and sensitive detection of UDG activity. DNA machines are supramolecular nucleic acid structures that perform machine-like functions at the molecular level in the presence of the appropriate triggers (Liu et al., 2014). The uses of DNA machines as switches (Qi et al., 2013), walkers (Liu et al., 2013), or amplified sensors (Wang et al., 2012; Wang et al., 2011) have been discussed. Specifically, since DNA machines can perform amplification autonomously and effectively only under simple operations, they have been utilized for amplified analysis of nucleic acids (Lu et al., 2012; Zhuang et al., 2014; Freeman et al., 2011; Li et al., 2014; Xu et al., 2015; Li et al., 2015), small molecules (Zhang et al., 2010; Zhang et al., 2011) and metal ions (Wang et al., 2012; Li et al., 2008; He et al., 2014). Herein, a sensitive fluorescence amplification strategy based on a label-free and enzyme-free DNA machine was described for UDG activity measurement. We employed a double-strand DNA (dsDNA) probe containing uracil bases and trigger sequence to recognize UDG target with the releasing of trigger strand, which could activate the label-free and enzyme-free DNA machine, generating amplified fluorescent signal. Due to the unique designs of both the dsDNA probe and the specific DNA machine, background reduction and signal amplification were successfully achieved, which resulted in a low detection limit (0.00044 U/mL). Moreover, the strategy was also applied for UDG activity analysis in HeLa cells lysate with low effect of cellular components. The proposed strategy offered a potential tool for sensitive quantification of UDG activity in UDG functional study and clinical diagnosis.
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2. Experimental section 2.1. Reagents and apparatus The DNA oligonucleotides used in this work were synthesized and purified by Sangon Inc. (Shanghai, China) and the sequences of the oligonucleotides were listed in Table S1. Uracil-DNA glycosylase (UDG), uracil glycosylase inhibitor (UGI) and 8-oxoguanine DNA glycosylase (hOGG1) were obtained from New England Biolabs Ltd. (Beijing, China). One unit of UGI activity was defined as the amount of protein required to inhibit one unit of E.coli UDG in 1 h at 37 °C in a total reaction volume of 50 mL. One unit of UDG was the amount of enzyme which would catalyze the release of 60 pmol of uracil per minute from double stranded, uracil-containing DNA. Exonuclease I (Exo I) and Exonuclease III (Exo III) were purchased from Thermo Fisher Scientific Ltd. (China). N-methylmesoporphyrin IX (NMM) was purchased from Frontier Scientific Inc. (Logan, Utah, USA). The NMM stock solution was prepared in dimethyl sulfoxide (DMSO) and stored in the dark at 20 °C. All other chemicals were of analytical grade and used as received. All solutions were prepared using the ultrapure water that was obtained from a Millipore Milli-Q water purification system ( 4 18.25 M Ω). Fluorescence measurements were performed using a Hitachi F-7000 fluorescence spectrometer (Hitachi. Ltd., Japan). The excitation wavelength was 399 nm with a recording emission range of 570–700 nm. The fluorescence intensity at 618 nm was used to evaluate the performance of the proposed strategy. The slits of both excitation and emission were 10 nm and the photomultiplier tube voltage was 700 V. 2.2. Assay for UDG activity and inhibition To prepare the double-strand DNA (dsDNA) probe (P1–P2) and hairpin structures, a mixture of P1 and P2, H1 and H2 were all denatured at 95 °C for 10 min, respectively. And then they were all cooled slowly to room temperature in a hybridization buffer (10 mM NaCl, 2.0 mM MgCl2, pH 8.0). For assaying UDG activity, the prepared dsDNA probe P1-P2 and various amounts of UDG
Scheme 1. Schematic illustration of the fluorescence amplification strategy based on a label-free and enzyme-free DNA machine for sensitive detection of UDG activity. U and A denote the uracil and adenine deoxyribonucleotides. Figures marked with n are complementary to the corresponding unmarked figures. In P1–P2 probe, the black segment is the uracil-containing DNA sequence, the red segment is the trigger sequence that can trigger downstream DNA machine, and the purple segment is the inhibition strand. In H1 probe, the red segment is complementary to the trigger sequence in P1-P2 probe, and the blue segment is the sequence that can open H2 probe. In H2 probe, the blue segment is complementary to the blue segment in H1 probe, the red segment is complementary to the red segment in H1 probe, and the green segment is the G-rich sequence.
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were added to the reaction buffer (20 mM Tris–HCl, 1.0 mM EDTA, 1.0 mM DTT, 10 mM NaCl, 2.0 mM MgCl2, pH 8.0) to give a total volume of 50 mL. The mixture was incubated at 37 °C for 90 min to allow the base cleavage reaction to take place. Then H1, H2 and 10 mL 10 TNaK buffer (200 mM Tris, 1.4 M NaCl, 50 mM KCl, pH 7.5) were added into the above solution with a final volume of 100 mL. The amplification reaction of the DNA machine was carried out at 37 °C for 45 min. At last, NMM was added to the resulting product and the mixture was incubated at 37 °C for 30 min. The control experiment was carried out under the same condition without adding UDG. For assaying the inhibition of UDG, 250 nM prepared dsDNA probe P1–P2 was incubated with 1 U/mL UDG and various concentrations of UGI. The mixture was heated at 37 °C for 90 min. Then H1, H2 and 10 mL 10 TNaK buffer (200 mM Tris, 1.4 M NaCl, 50 mM KCl, pH 7.5) were added into the mixture with a final volume of 100 mL, and the final concentrations of H1 and H2 were 450 nM and 675 nM, respectively. The amplification reaction of the DNA machine was carried out at 37 °C for 45 min. At last, NMM was added to the resulting product and the mixture was incubated at 37 °C for 30 min. All experiments were repeated three times. 2.3. Gel electrophoresis to investigate the dissociation of the dsDNA probe The indicated concentrations of P1–P2 and UDG were kept at 37 °C for 90 min to obtain the UDG-treated product. And we also analyzed P1, P2 and the dsDNA probe P1–P2 in the absence of UDG, respectively. Samples were mixed with loading buffer and loaded into the 15% polyacrylamide gels. The electrophoresis was run at a constant current of 30 mA for about 2 h, using 1 TBE (89 mM Tris, 89 mM Boric Acid, 2.0 mM EDTA, pH 8.3) as running buffer. The gels were stained with ethidium bromide for 5 min, destained in distilled water for 5 min, and then photographed under UV imaging system (Bio-RAD Laboratories Inc. USA). 2.4. Preparation of HeLa cells lysate Approximately 1 108 HeLa cells samples were pelleted by centrifugation (5 min, 3000 rpm, 4 °C) and resuspended in 20 mL of lysis buffer (10 mM Tris–HCl, pH 7.0) on ice using a sonicator (four pulses at 200 W for 30 s with a tapered microtip). The mixture solution was then centrifuged at 12,000 rpm for 30 min at 4 °C to remove insoluble material. The resulting supernatant was collected and filtered through a 0.45 mm filter membranes, yielding crude lysate.
3. Results and discussion 3.1. The principle of the fluorescence amplification strategy based on a label-free and enzyme-free DNA machine for UDG activity assay Scheme 1 outlined the principal design of the proposed strategy for UDG activity assay. The nucleic acid strands utilized here were rationally designed to obtain a satisfactory result. Among them, P1 was an inhibition strand. In addition, P2 was a chimeric conjugate of the uracil-containing DNA sequence (in black) and trigger sequence (in red). The single-strand DNA (ssDNA) P2 which contained both the uracil bases and trigger sequence was partly hybridized with the inhibition strand P1 to form P1–P2 dsDNA probe. The dsDNA probe showed a relatively high melting temperature, making the probe a stable hybrid and blocking the trigger sequence from hybridization with a hairpin probe. Two hairpin probes H1 and H2 which were partially complementary were designed to construct the label-free and enzyme-free DNA
machine according to the trigger sequence. To avoid sophisticated chemical labeling, a G-quadruplex (G4) sequence was grafted at the end of H2 (domains 4 and 5). When H2 was in its hairpin structure, domain 4 was hidden in the stem of H2. Therefore, G4 conformation cannot form at this time. Under the action of UDG, uracil bases could be removed from the deoxyriboses phosphate backbone of P2, generating abasic (AP) sites and leading to a lower melting temperature of the P1–P2 dsDNA probe, which resulted in the liberation of P2ʹ from P1–P2. The liberated P2ʹ could then initiate the signal amplification process of the designed DNA machine. Particularly, domain 1 in trigger strand P2’ was exposed and could hybridize to the accessible toehold 1n in hairpin H1, which initiated the branch migration and exposed 3, 4, 1 of hairpin H1. The resultant full H1–P2ʹ hybridization opened H1 and exposed a new toehold, domain 3, which could nucleate at the 3n of hairpin H2. As a result, domains 3, 2, 1, 4 and 5 of hairpin H2 were fully accessible via branch migration. Since the H1–H2 complex was more stable than H1–P2ʹ hybridization, the accessible domains 3, 2, 1 of hairpin H2 could displace P2ʹ, forming H1–H2 complex and releasing domains 4 and 5 in H2. The liberated domains 4 and 5 in H2 were able to fold into a G4 conformation in the presence of monovalent ions. Then, the released P2ʹ could trigger the next reaction cycle to consecutively generate H1–H2 complexes containing G4 structures. Finally, NMM which had a pronounced structural selectivity for G4 structures but not for dsDNA or ssDNA forms (Hu et al., 2011) was added to the reaction system to form G4-NMM complex generating label-free fluorescence signal. In this way, one released trigger strand can create increased fluorescence signal for the sensitive assay of UDG activity through the amplification process of the label-free and enzyme-free DNA machine. 3.2. Feasibility research As shown in Fig. 1, fluorescence emission spectra were used to investigate the viability of our approach. The system only containing H2 showed very weak fluorescence intensity (Fig. 1A, curve a), suggesting the formation of G4 conformation was difficult when the sequence was partly hidden in the stem of H2. The system containing H1 and H2 exhibited slightly enhanced fluorescence intensity (Fig. 1A, curve b), indicating the interaction of H1 and H2 was weak without target. When P1–P2 was added into the H1, H2 system, the fluorescence intensity exhibited further enhancement (Fig. 1A, curve c), which indicated the interaction of H1 and H2 was facilitated by P1–P2 resulting in background fluorescence. However, the fluorescence intensity had significant enhancement upon addition of UDG to the system containing P1–P2, H1 and H2 (Fig. 1A, curve d). This suggested that UDG could trigger the self-assembly of H1 and H2 by removing uracil bases in dsDNA probe P1–P2 with the releasing of trigger strand P2ʹ. Polyacrylamide gel electrophoresis (PAGE) was also used to verify the dissociation of the dsDNA probe. After the UDG treatment, the dsDNA probe was dissociated into discrete bands, as shown in Fig. 1B. Thus, these results demonstrated that the proposed strategy could be adopted for UDG activity detection. 3.3. Optimization of the reaction conditions The reaction conditions played important roles in the assay, such as the probe concentration and the reaction time. Therefore, the reaction conditions were optimized in order to achieve the best results. The optimal system should give low background signal, as well as a great fluorescence enhancement on the addition of UDG. It was widely accepted that the concentration of the probe had an important effect on the sensing process. In our study, P1 was the inhibition probe, P2 was the conjugate probe containing both
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Fig. 1. (A) Fluorescence emission spectra of the system: (a) H2 alone, (b) H1 þ H2, (c) P1–P2 þ H1þ H2, and (d) P1–P2 þ UDG (1.0 U/mL)þ H1þ H2. (B) Nondenaturing polyacrylamide gel (15%) electrophoresis analysis of the dissociation of dsDNA probe P1–P2 under the action of UDG. Lane 1 contains 5.0 mM P1. Lane 2 contains 1.0 mM P2. Lane 3 contains 1.0 mM P1–P2 dsDNA probe without UDG. Lane 4 contains 1.0 mM P1–P2 dsDNA probe with 10 U/mL UDG.
the uracil bases and trigger sequence, H1 and H2 were amplification probes which were employed to construct the DNA machine. We investigated the concentrations of these probes when the concentration of UDG was 1.0 U/mL. The low hybridization efficiency of P1 and P2 would result in high background signal. In order to achieve the good suppressive effect as well as the low background signal, we first optimized the concentration of P1. We fixed the concentration of P2 as 250 nM and changed the concentration of P1, which caused the changing in the hybridization proportion of P1 to P2. As shown in Fig. S1, the net signal ΔF (Δ F¼F–F0, where F and F0 were fluorescence intensities of the system in the presence and absence of UDG) was no longer increasing when the concentration of P1 was greater than 1.00 μM. Therefore, we chose 1.25 μM as the optimal concentration of P1. Furthermore, we also optimized the concentrations of other probes. As shown in Figs. S2–S4, the ΔF reached the maximum when the concentration of P2, H1 and H2 were 250 nM, 450 nM and 675 nM, respectively. The sensing process was also influenced by the reaction time.
The uracil cleavage reaction time and amplification reaction time were set as 90 min and 45 min for the subsequent experiments based on the optimization experiment results (Figs. S5 and S6). 3.4. Analytical performance of the developed strategy in UDG activity assay Under the optimal experimental conditions, we investigated the fluorescence responses of the as-proposed strategy to different concentrations of UDG. As depicted in Fig. 2A, the fluorescence intensity was enhanced as the concentration of UDG increased from 0 to 1.0 U/mL, suggesting that the formation of the fluorescent G4-NMM complex was highly dependent on the concentration of UDG. The calibration curve for detection of UDG activity was shown in Fig. 2B. As illustrated in the inset of Fig. 2B, the net signal ΔF exhibited a linear correlation to the activity of UDG ranging from 0.0025 to 0.020 U/mL with an R2 of 0.999. According to the 3δ rule, the detection limit for UDG was as low as
Fig. 2. (A) Fluorescence responses to the different concentrations of UDG. (B) The calibration curve of ΔF versus the UDG concentration. The inset shows the linear responses at low UDG concentrations. Error bars represent the standard deviations of the results from three independent experiments. Condition: CP1 ¼1.25 μM, CP2 ¼250 nM, CH1 ¼ 450 nM, and CH2 ¼675 nM, uracil cleavage reaction time 90 min, amplification reaction time 45 min.
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Fig. 3. Relative fluorescence intensity of the reaction systems with the addition of UDG (1.0 U/mL) or 10-fold excess of Exo I, Exo III and hOGG1 (10 U/mL). Error bars represent the standard deviations of the results from three independent experiments. Condition: CP1 ¼1.25 μM, CP2 ¼ 250 nM, CH1 ¼450 nM, and CH2 ¼ 675 nM.
Fig. 4. Fluorescence emission spectra in the absence and presence of 60% HeLa cells lysate, and the inhibitory effect of 2.0 U/mL UGI on the UDG activity in the HeLa cells lysate. Condition: CP1 ¼ 1.25 μM, CP2 ¼250 nM, CH1 ¼450 nM, CH2 ¼675 nM.
clinical diagnosis. 0.00044 U/mL, which was lower than most reported fluorescent amplification strategies for UDG activity detection (Zhang et al., 2012; Xiang and Lu, 2012). The high sensitivity of our method was most probably attributed to the signal amplification of the designed DNA machine, as well as the background reduction of the unique dsDNA probe. Hence, the proposed strategy held great promise as an effective tool for quantification of UDG activity. To further evaluate the selectivity of this strategy, we studied its fluorescence responses towards UDG against other nucleases including Exo I, Exo III and another glycosylase hOGG1. As shown in Fig. 3, only UDG caused significant relative fluorescence intensity, whereas Exo I, Exo III or hOGG1 gave low relative intensity which was comparable to that in the blank solution. This result indicated that the proposed strategy exhibited good selectivity to active UDG. As important parameters to assess a strategy in practical application, the precision and reproducibility of the proposed strategy were investigated. To evaluate the precision of the proposed strategy, a series of three repetitive experiments of target samples were carried out on the same day. The relative standard deviations (RSD) achieved for the samples containing 0.0025 U/mL, 0.010 U/mL and 0.020 U/mL of UDG were 4.4%, 3.7% and 3.7%, respectively. Similarly, a series of three repetitive experiments of target samples were conducted on three different days to demonstrate the repeatability of the strategy. The RSD achieved for samples containing different UDG concentrations (0.0025 U/mL, 0.010 U/mL and 0.020 U/mL) were 5.0%, 4.2% and 4.0%, respectively. These results indicated that the as-proposed strategy possessed acceptable precision and reproducibility. To demonstrate the applicability of the proposed strategy for detection of UDG activity, it was also used for the analysis of UDG activity in HeLa cells lysate. As shown in Fig. 4, lysis buffer could only induce very low fluorescence signal. In contrast, the fluorescence signal of the system had significant enhancement in the assay of 60% (the volume fraction of HeLa cells lysate in reaction system) HeLa cells lysate due to the presence of UDG activity. To confirm that the fluorescence enhancement was induced by UDG rather than any other component in the lysate, UGI was also added to the cell lysate, resulting in no appreciable fluorescence enhancement. The results revealed that the developed strategy could be tolerant toward the cellular components and would become a candidate tool for UDG activity assay in function study of UDG and
3.5. Assaying the inhibition of UDG The utility of this DNA machine-based fluorescence amplification strategy for assaying the inhibition of UDG was also studied. Herein, UGI was selected as a model inhibitor for this study. UGI can form a tight and physiologically irreversible complex with UDG in 1:1 M stoichiometry (Kaushal et al., 2008). As shown in Fig. 5, the relative intensity of fluorescence in the system was diminished in the presence of UGI in a dose-dependent manner. The result demonstrated that the proposed strategy can be used to assay the inhibition of UDG.
4. Conclusions In conclusion, we developed a novel fluorescent amplification strategy based on a label-free and enzyme-free DNA machine for
Fig. 5. Relative fluorescence intensity of the system in the presence of increasing concentrations of UGI (0, 0.10, 0.20, 0.40, 0.80, 1.0 and 2.0 U/mL). Error bars represent the standard deviations of the results from three independent experiments.
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sensitive detection of UDG activity. This strategy employed a dsDNA probe to release a trigger strand after being recognized by UDG, and the released trigger strand could initiate the amplification of the DNA machine. The designed dsDNA probe containing uracil bases and trigger sequence exhibited good suppressive effect on the trigger sequence with high hybridization efficiency, resulting in low background. Additionally, the label-free design did not need covalent attachment of fluorophore or quencher to DNA, preventing participation of fluorescent labels in the background. Moreover, the signal amplification capability of the designed DNA machine was used to further improve the detection sensitivity. This strategy could detect UDG activity as low as 0.00044 U/mL, which was lower than most reported fluorescent amplification strategies for UDG activity detection. Furthermore, the enzymefree design made the signal amplification capability of the DNA machine independent on the vulnerable enzyme, leading to a good repeatability. Finally, the strategy was also applied for analysis of UDG activity in HeLa cells lysate with low effect of cellular components. The results revealed that the developed strategy provided a potential tool for sensitive quantification of UDG activity in UDG functional study and clinical diagnosis.
Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant nos. 21175081, 21175082, 21375078 and 21475077).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2015.01.069.
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References de Souza-Pinto, N.C., Harris, C.C., Bohr, V.A., 2004. Oncogene 23, 6559–6568. Freeman, R., Liu, X.Q., Willner, I., 2011. Nano Lett. 11, 4456–4461. He, J.L., Zhu, S.L., Wu, P., Li, P.P., Li, T., Cao, Z., 2014. Biosens. Bioelectron. 60, 112–117. Hu, D., Huang, Z.Z., Pu, F., Ren, J.S., Qu, X.G., 2011. Chem. Eur. J 17, 1635–1641. Imai, K., Slupphaug, G., Lee, W.-I., Revy, P., Nonoyama, S., Catalan, N., Yel, L., Forveille, M., Kavli, B., Krokan, H.E., Ochs, H.D., Fischer, A., Durandy, A., 2003. Nat. Immunol. 4, 1023–1028. Kaushal, P.S., Talawar, R.K., Krishna, P.D., Varshney, U., Vijayan, M., 2008. Acta Crystallogr. D Biol. Crystallogr. 64, 551–560. Kunkel, T.A., Erie, D.A., 2005. Annu. Rev. Biochem. 74, 681–710. Li, D., Wieckowska, A., Willner, I., 2008. Angew. Chem. 120, 3991–3995. Li, H.L., Ren, J.T., Liu, Y.Q., Wang, E.K., 2014. Chem. Commun. 50, 704–706. Li, X.H., Gan, L.J., Ou, Q.S., Zhang, X., Cai, S.X., Wu, D.Z., Chen, M., Xia, Y.K., Chen, J.H., Yang, B., 2015. Biosens. Bioelectron. 66, 399–404. Liu, X.J., Chen, M.Q., Hou, T., Wang, X.Z., Liu, S.F., Li, F., 2014. Biosens. Bioelectron. 54, 598–602. Liu, X.Q., Lu, C.-H., Willner, I., 2014. Acc. Chem. Res. 47, 1673–1680. Liu, X.Q., Niazov-Elkan, A., Wang, F., Willner, I., 2013. Nano Lett. 13, 219–225. Lu, C.H., Wang, F., Willner, I., 2012. J. Am. Chem. Soc. 134, 10651–10658. McWilliams, M.A., Anka, F.H., Balkus, K.J., Slinker, J.D., 2014. Biosens. Bioelectron. 54, 541–546. Ono, T., Edwards, S.K., Wang, S., Jiang, W., Kool, E.T., 2013. Nucleic Acids Res. 41, 127–138. Ono, T., Wang, S.L., Koo, C.K., Engstrom, L., David, S.S., Kool, E.T., 2012. Angew. Chem. 51, 1689–1692. Prorok, P., Alili, D., Saint-Pierre, C., Gasparutto, D., Zharkov, D.O., Ishchenko, A.A., Tudek, B., Saparbaev, M.K., 2013. Proc. Natl. Acad. Sci. USA 110, 3695–3703. Qi, X.J., Lu, C.H., Liu, X.Q., Shimron, S., Yang, H.H., Willner, I., 2013. Nano Lett. 13, 4920–4924. Seal, G., Brech, K., Karp, S.J., Cool, B.L., Sirover, M.A., 1988. Proc. Natl. Acad. Sci. USA 85, 2339–2343. Sousa, M.M., Krokan, H.E., Slupphaug, G., 2007. Mol. Aspects Med. 28, 276–306. Stivers, J.T., Jiang, Y.L., 2003. Chem. Rev. 103, 2729–2759. Wang, F., Elbaz, J., Teller, C., Willner, I., 2011. Angew. Chem. 50, 295–299. Wang, F., Elbaz, J., Willner, I., 2012. J. Am. Chem. Soc. 134, 5504–5507. Wang, F., Orbach, R., Willner, I., 2012. Chem. Eur. J 18, 16030–16036. Xiang, Y., Lu, Y., 2012. Anal. Chem. 84, 9981–9987. Xu, J.G., Dong, H.Y., Shen, W.Y., He, S.D., Li, H.L., Lu, Y.S., Wu, Z.S., Jia, L., 2015. Biosens. Bioelectron. 66, 504–511. Zhang, H., Fang, C.C., Zhang, S.S., 2010. Chem. Eur. J 16, 12434–12439. Zhang, H., Fang, C.C., Zhang, S.S., 2011. Chem. Eur. J 17, 7531–7537. Zhang, L.L., Zhao, J.J., Jiang, J.H., Yu, R.Q., 2012. Chem. Commun. 48, 8820–8822. Zhou, Z.X., Du, Y., Dong, S.J., 2011. Anal. Chem. 83, 5122–5127. Zhuang, J.Y., Lai, W.Q., Chen, G., Tang, D.P., 2014. Chem. Commun. 50, 2935–2938.
