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Toehold-mediated strand displacement reaction-dependent fluorescent strategy for sensitive detection of uracil-DNA glycosylase activity
Author’s Accepted Manuscript Toehold-mediated strand displacement reactiondependent fluorescent strategy for sensitive detection of uracil-DNA glycosylase activity Yushu Wu, Lei Wang, Wei Jiang www.elsevier.com/locate/bios
PII: DOI: Reference:
S0956-5663(16)31069-7 http://dx.doi.org/10.1016/j.bios.2016.10.053 BIOS9275
To appear in: Biosensors and Bioelectronic Received date: 30 July 2016 Revised date: 9 October 2016 Accepted date: 19 October 2016 Cite this article as: Yushu Wu, Lei Wang and Wei Jiang, Toehold-mediated strand displacement reaction-dependent fluorescent strategy for sensitive detection of uracil-DNA glycosylase activity, Biosensors and Bioelectronic, http://dx.doi.org/10.1016/j.bios.2016.10.053 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Toehold-mediated strand displacement reaction-dependent fluorescent strategy for sensitive detection of uracil-DNA glycosylase activity
Yushu Wu,a Lei Wang,b Wei Jianga,*
a
Key Laboratory for Colloid and Interface Chemistry of Education Ministry,
School of Chemistry and Chemical Engineering, Shandong University, 250100 Jinan, P.R. China b
School of Pharmaceutical Sciences, Shandong University, 250012 Jinan, P.R. China
Corresponding author: Tel: 86-531-88363888; fax: 86-531-88564464. E-mail: [email protected] Abstract Sensitive detection of uracil-DNA glycosylase (UDG) activity is beneficial for evaluating the repairing process of DNA lesions. Here, toehold-mediated strand displacement reaction (TSDR)-dependent fluorescent strategy was constructed for sensitive detection of UDG activity. A single-stranded DNA (ssDNA) probe with two uracil bases and a trigger sequence were designed. A hairpin probe with toehold domain was designed, and a reporter probe was also designed. Under the action of UDG, two uracil bases were removed from ssDNA probe, generating apurinic/apyrimidinic (AP)
sites. Then, the AP sites could inhibit the TSDR between ssDNA probe and hairpin probe, leaving the trigger sequence in ssDNA probe still free. Subsequently, the trigger sequence was annealed with the reporter probe, initiating the polymerization and nicking amplification reaction. As a result, numerous G-quadruplex (G4) structures were formed, which could bind with N-methyl-mesoporphyrin IX (NMM) to generate enhanced fluorescent signal. In the absence of UDG, the ssDNA probe could hybridize with the toehold domain of the hairpin probe to initiate TSDR, blocking the trigger sequence, and then the subsequent amplification reaction would not occur. The proposed strategy was successfully implemented for detecting UDG activity with a detection limit of 2.7 × 10-5 U/mL. Moreover, the strategy could distinguish UDG well from other interference enzymes. Furthermore, the strategy was also applied for detecting UDG activity in HeLa cells lysate with low effect of cellular components. These results indicated that the proposed strategy offered a promising tool for sensitive quantification of UDG activity in UDG-related function study and disease prognosis. Keywords: Uracil-DNA glycosylase activity; Apurinic/apyrimidinic site; Mismatch; Toehold-mediated strand displacement reaction; Fluorescent strategy.
1. Introduction Uracil-DNA glycosylase (UDG) is a protein enzyme which is involved in the repairing process of DNA lesions (Stivers and Jiang, 2003; Savva et al., 1995). It can specifically recognize and remove the uracil leision from DNA, leaving an apurinic/apyrimidinic (AP) site in DNA to trigger the downstream repairing process (Kunkel and Erie, 2005; Lindahl et al., 1976; Lindahl et al., 1979; Lindahl et al., 1982; Sancar et al., 1996). In this regard, UDG plays indispensable roles in maintaining genomic integrity. Abnormal UDG activity would disturb the repairing process and induce the gene mutation, which results in various diseases including human immunodeficiency (Imai et al., 2003), lymphoma (Sousa et al., 2007) and Bloom syndrome (Seal et al., 1988). Therefore, detection of UDG activity is beneficial for evaluating the repairing process of DNA lesions in function study and disease prognosis (Ono et al., 2013). The activity of UDG is commonly measured by gel-based methods (de Souza-Pinto et al., 2004; Prorok et al., 2013), electrochemical methods (McWilliams et al., 2014) and colorimetric methods (Liu et al., 2014; Nie et al., 2015). In addition to such methods, fluorescent methods have drawn wide attention due to the advantages of safety, simplicity and sensitivity. In these fluorescent methods, DNA probes containing multiple uracil bases are generally used for the recognition of UDG (Hu et al., 2011; Zhang et al., 2012; Leung et al., 2013; Lee et al., 2015; Wu et al., 2015). Multiple uracil bases in the DNA probe are removed by UDG to generate AP sites. Since the AP site is a type of mismatch (Fang et al., 2015), the number of base pairs in the DNA probe is reduced and
the conformation of the DNA probe will change, leading to the release of primer sequence or signal probe. However, when the activity of UDG is low, the multiple uracil bases in each DNA probe could not be removed completely and the conformation of probe could not change completely, which reduce the efficiency of signal transduction. Thus, such methods are not efficient for detecting UDG with low activity, which limits their applications. The toehold-mediated strand displacement reaction (TSDR) contains two main processes: the hybridization process between the toehold domain and the fuel strand, as well as the branch migration process (Zhang and Winfree, 2009; Zhang et al., 2010). In the TSDR, the mismatched base at complementary domain of toehold can inhibit the branch migration process since the rate of dissociation from toehold is much higher than that of branch migration (Subramanian et al., 2011; Wang et al., 2012; Zhu et al., 2014; Gao et al., 2014). Here, the TSDR-dependent fluorescent strategy was constructed for sensitive detection of UDG activity. Under the action of UDG, two uracil bases in the single-stranded DNA (ssDNA) probe were removed to generate AP sites. Then, the AP sites, which served as mismatched bases, could inhibit the TSDR between ssDNA probe and hairpin probe, leaving the free trigger sequence in the ssDNA. Subsequently, the trigger sequence could initiate the polymerization and nicking amplification reaction, which generated numerous G-quadruplex (G4) structures. Finally, the G4 structures could bind with N-methyl-mesoporphyrin IX (NMM) to generate enhanced fluorescent signal. In the absence of UDG, the ssDNA probe could hybridize with the toehold domain of hairpin probe to initiate the TSDR, resulting in the blocking of the trigger sequence.
Accordingly, the subsequent amplification reaction would not occur. The proposed strategy could detect UDG activity with a detection limit of 2.7 × 10-5 U/mL. Additionally, the strategy could distinguish UDG well from other interference enzymes. Furthermore, the strategy was also applied for detecting UDG activity in HeLa cells lysate with low effect of cellular components. These results indicated that the proposed strategy offered a promising tool for sensitive quantification of UDG activity in UDG-related function study and disease prognosis.
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. UDG, human 8-oxoguanine DNA glycosylase (hOGG1), human alkyladenine DNA glycosylase (hAAG), DNase I, uracil glycosylase inhibitor (UGI), Vent (exo-) polymerase, Nt.BstNBI nicking enzyme, 10 × Thermopol buffer and 10 × NEBuffer 3 were all obtained from New England Biolabs Ltd. (Beijing, China). dNTPs were obtained from Fermentas (Beijing, China). NMM was purchased from Frontier Scientific Inc. (Logan, Utah, USA). The NMM solution was prepared in dimethyl sulfoxide (DMSO). The ultrapure water used in this work was obtained from a Millipore Milli-Q water purification system (> 18.25 MΩ•cm). The Hitachi F-7000 fluorescence spectrometer (Hitachi. Ltd., Japan) was used for the fluorescence measurements. The excitation wavelength was 399 nm. And the emission
spectra were collected from 560 nm to 700 nm. The fluorescence intensity at 618 nm was used for evaluating the performance of the proposed strategy. Both the slit widths for the excitation and emission were 10 nm. The photomultiplier tube voltage was 700 V. 2.2 Detection of UDG activity To obtain the hairpin structures, the hairpin probe (named TP) and the reporter probe (named RP) were denatured at 90 ºC for 5 min and cooled slowly to room temperature, respectively. 5.0 µL 1.5 µM ssDNA probe (named UP) and various concentrations of UDG were added to 1 × Thermopol buffer (20 mM Tris-HCl, pH 8.8, 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% TritonX-100) to give a total volume of 20 µL. The mixture was incubated at 37 ºC for 60 min to perform the UDG recognition reaction. Subsequently, 5.0 µL 4.5 µM TP and 1.0 µL 10 × Thermopol buffer (200 mM Tris-HCl, pH 8.8, 100 mM KCl, 100 mM (NH4)2SO4, 20 mM MgSO4, 1% TritonX-100) were added into the above solution with a final volume of 30 µL. The TSDR was carried out at 37 ºC for 120 min. Then, 300 nM RP, 0.16 U/μL Vent (exo-) polymerase, 0.40 U/μL Nt.BstNBI nicking enzyme, 0.40 mM dNTPs and 0.5 × NEB buffer 3˄25 mM Tris-HCl, pH 7.9, 50 mM NaCl, 5.0 mM MgCl2, 1.0 mM DTT˅were added to give a total volume of 50 µL. The mixture was incubated at 55 ºC for 90 min to perform the polymerization and nicking amplification reaction. Finally, KCl (160 mM) and NMM (4.0 µM) were added to the mixture. After incubating at 37 ºC for 30 min, the fluorescence intensity of the mixture was recorded on Hitachi F-7000 fluorescence spectrometer. 2.3 Evaluation of UDG activity inhibition To evaluate the inhibition of UDG activity, different concentrations of UGI were
added to the sensing system before the addition of 1.0 U/mL UDG. The following procedures were similar to those in the detection of UDG activity. After the TSDR as well as the polymerization and nicking amplification reaction, the fluorescence intensity of the sensing system was recorded on Hitachi F-7000 fluorescence spectrometer. 2.4 Preparation of HeLa cells lysate Approximately 1 × 108 HeLa cells samples were pelleted by centrifugation (5 min, 3000 rpm, 4 ºC) and resuspended in 100 µL 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 ºC to remove insoluble material. The resulting supernatant was collected and filtered through a 0.45 µm filter membranes, yielding crude lysate.
3. Results and discussion 3.1 The principle of the TSDR-dependent fluorescent strategy for UDG activity assay Scheme 1 was the principle of the TSDR-dependent fluorescent strategy for detecting UDG activity. A ssDNA probe (named UP) with two uracil bases and a trigger sequence was designed. A hairpin probe (named TP) with toehold domain was designed. And a reporter probe (named RP) was also designed. Under the action of UDG, the uracil bases were removed from UP, generating AP sites. The obtained probe with AP sites was denoted UP’. The UP’ could not initiate TSDR, leaving the trigger sequence in UP’ still free. Subsequently, the trigger sequence was annealed with RP, initiating the strand displacement amplification (SDA) reaction, namely, the polymerization and nicking
amplification reaction. Finally, numerous G4 structures were formed, which could bind with NMM to generate enhanced fluorescent signal. However, in the absence of UDG, UP could hybridize with the toehold domain of TP to initiate the TSDR, resulting in the blocking of the trigger sequence. Accordingly, the subsequent amplification reaction would not occur. Thus, the proposed strategy could be used for the detection of UDG activity.
Scheme 1 is here
3.2 Feasibility research of the developed strategy in UDG activity assay As shown in Fig. 1, fluorescence emission spectra were used to investigate the viability of the TSDR-dependent fluorescent strategy for detecting UDG activity. The system without UP or RP showed very weak fluorescence intensity (Fig. 1, curve c and curve d), suggesting the necessity of UP and RP for the strategy. The control experiment without UDG exhibited weak fluorescence intensity (Fig. 1, curve b). However, the fluorescence intensity had significant enhancement upon the addition of UDG to the system containing UP, TP and RP (Fig. 1, curve a). Thus, these results demonstrated that the proposed strategy could be applied to detect UDG activity.
Fig. 1 is here
3.3 Optimization of the reaction conditions
To obtain the best performance of the sensing system, the sequence of toehold domain in the hairpin probe TP was first optimized. By altering the number of G/C bases in toehold domain from 4 to 0, five different hairpin probes TP (TP1 to TP5) were obtained. Fig. S1 showed the dependence of the net signal ΔF (ΔF = F - F0, where F and F0 were fluorescence intensities of the system in the presence and absence of UDG, respectively) on the sequence of toehold domain in TP. It was observed that the value of ΔF gradually increased from TP1 to TP3 and reached a plateau at TP4. The result indicated that the hairpin probe TP4 with 1 G/C base in toehold domain ensured a high net signal and could be beneficial for sensitive detection of the target. Therefore, TP4 was chosen for the following investigations. Furthermore, the performance of the sensing system was also influenced by other factors, including the concentrations of TP, RP and NMM. Therefore, the effects of these factors on the system were investigated, respectively. The concentration of TP played an important role in the efficiency of TSDR between UP and TP, so we optimized the TP concentration. The concentrations of TP from C1 to C6 were 50.0 nM, 450 nM, 750 nM, 1.20 µM, 1.50 µM and 1.80 µM, respectively. As presented in Fig. S2, the background fluorescence in the control systems gradually decreased with the increasing concentration of TP. However, in the presence of UDG, the fluorescence intensity also decreased gradually with the increase of TP concentration. The reason was that the high concentration of TP could improve the efficiency of TSDR. When the TP concentration was high, the TSDR between UP and TP would still occur even in the presence of UDG. Accordingly, the trigger sequence in UP was blocked and
the subsequent amplification reaction would not be initiated. Thus, 450 nM was chosen as the optimized TP concentration owing to the maximum value of the net signal ΔF. Due to that the concentration of RP could significantly affect the amplification efficiency, the RP concentration was further investigated. The RP concentration was varied from 150 nM to 375 nM. As shown in Fig. S3, the value of ΔF gradually increased with the increasing concentration of RP from 150 nM to 300 nM and reached a plateau beyond 300 nM. Thus, 300 nM of RP was employed in the sensing system. The fluorescence signal of the sensing system depended on the amount of NMM bound to G4 structures, so the concentration of NMM was further optimized. The NMM concentration was varied from 1.00 µM to 10.0 µM. As depicted in Fig. S4, the value of ΔF reached its maximum with 4.00 µM and then decreased at higher concentration. Therefore, 4.00 µM of NMM was used in the subsequent experiment. 3.4 Analytical performance of the developed strategy To study the analytical performance of the developed strategy, a series of samples with various concentrations of UDG were measured under the optimal conditions. Fig. 2A showed the fluorescence emission spectra of the sensing system. As can be seen, the fluorescence intensity gradually increased as the UDG concentration varied from 0 to 1.0 U/mL, which indicated that more and more G4 structures were formed. As shown in Fig. 2B, the corresponding calibration curve between the net signal ΔF and UDG concentration was obtained. Notably, the inset of Fig. 2B clearly showed that the net signal ΔF exhibited a good linear relationship with UDG concentration in the range from 0.00020 U/mL to 0.0080 U/mL. This linear relationship was described as ΔF = 30.13 +
9.936 × 104 C with a correlation coefficient of R2 = 0.9993, where C represented the concentration of UDG. And the detection limit for UDG activity was calculated to be 2.7 × 10-5 U/mL in terms of the 3δ rule. As shown in Table S2, the detection limit of this strategy was lower than that of the previously reported UDG activity assay (Hu et al., 2011; Zhang et al., 2012; Leung et al., 2013; Lee et al., 2015; Wu et al., 2015). In this strategy, two strand displacement reactions were involved. The first was the TSDR, the second was the SDA reaction, namely, the polymerization and nicking amplification reaction. Due to the AP sites generated from the removal of only two uracil bases could inhibit the TSDR, the highly efficient signal transduction was achieved. Moreover, due to the high signal amplification capability of SDA, the detection sensitivity was further improved. Therefore, this strategy offered a promising tool for sensitive quantification of UDG activity in UDG-related function study and disease prognosis.
Fig. 2 is here
The precision and reproducibility of the developed strategy were investigated due to they were vital parameters to evaluate the performance of a sensing system. To investigate the precision, three repetitive experiments for the samples were performed on the same day. According to the experimental results, the relative standard deviations (RSD) were 4.1%, 3.7%, 3.1% at 0.001 U/mL, 0.004 U/mL and 0.006 U/mL of UDG, respectively. In addition, to investigate the repeatability, three repetitive experiments for the samples were performed on three different days. The RSD for samples containing the
above concentrations were 5.8%, 5.9% and 5.5%, respectively. The results displayed that the precision and reproducibility of the developed strategy were acceptable. Since the selectivity was an important factor to assess the utility of the sensing system in biological samples, the selectivity of the proposed strategy for UDG activity was further investigated. The nucleases including hOGG1, hAAG and DNase I were selected as interference enzymes to assess the selectivity of this proposed strategy. And all enzymes had the same concentration of 1.0 U/mL. The results were presented in Fig. 3. In the presence of UDG, a high relative fluorescence response was obtained. In contrast, in the presence of hOGG1, hAAG or DNase I, the relative fluorescence response was very low and comparable to that in the blank solution. Moreover, when UDG was spiked in the mixed sample consisting of UDG, hOGG1, hAAG and DNase I, the relative fluorescence response was as high as that in the system containing UDG only. The results suggested that the strategy we proposed here had high selectivity toward UDG and could distinguish UDG in the mixed sample containing other interference enzymes. The high selectivity of our strategy was mainly due to the specific site recognition of UDG toward its substrate.
Fig. 3 is here
In order to investigate the practical applicability of the developed strategy, this strategy was further used for the detection of UDG activity in HeLa cells lysate. As shown in Fig. 4, when the lysis buffer instead of UDG was added to the sensing system,
the fluorescence intensity was very low. In contrast, when 1.0 µL of HeLa cells lysate instead of UDG was added into the sensing system with a final concentration of 1.0 × 104 cells/µL, an obvious increase in the fluorescence intensity was observed. Furthermore, when UGI, an inhibitor of UDG, was added to the sensing system containing HeLa cells lysate, the fluorescence intensity was very low. These results indicated that the fluorescence enhancement was derived from UDG activity rather than any other component in the lysate. Taken together, the developed strategy could be tolerant to the cellular components and held a great potential for detecting UDG activity in complex biological samples.
Fig. 4 is here
3.5 Evaluation of UDG activity inhibition Since the inhibition of UDG activity could enhance the therapeutic efficacy of DNA-damaging chemotherapeutic agents (Bulgar et al., 2012; Moeller et al., 2010; Priet et al., 2005), it was attractive to evaluation of the inhibition of UDG activity. Here, the capability of this strategy for detecting the inhibition of UDG activity was also evaluated. UGI, which was selected as a model inhibitor, could form a specific complex with UDG at a 1:1 stoichiometry (Kaushal et al., 2008). As presented in Fig. 5, the relative fluorescence intensity gradually decreased with the increasing concentration of UGI. The result demonstrated that the proposed strategy could be used to detect the inhibition of UDG activity and had a great potential to be used as a platform to screen UDG inhibitors.
Fig. 5 is here
4. Conclusions In summary, the TSDR-dependent fluorescent strategy was developed for sensitive detection of UDG activity. This strategy contained two strand displacement reactions: the first was the TSDR, and the second was the SDA reaction, namely, the polymerization and nicking amplification reaction. Upon the removal of uracil bases by UDG, the generated AP sites, which served as mismatched bases, could inhibit the first strand displacement reaction TSDR, leaving the trigger sequence in the ssDNA probe still free. Then, the free trigger sequence could initiate the second strand displacement reaction SDA. Due to the AP sites generated from the removal of only two uracil bases could inhibit the TSDR, the highly efficient signal transduction was achieved. And due to the high signal amplification capability of SDA, the detection sensitivity was further improved. The detection limit of this strategy was as low as 2.7 × 10-5 U/mL, which was better than that of the previously reported UDG activity assay (Table S2). Additionally, the strategy could distinguish UDG well from other interference enzymes. Furthermore, the strategy was also applied for detecting UDG activity in HeLa cells lysate with low effect of cellular components. These results indicated that the proposed strategy offered a promising tool for sensitive quantification of UDG activity in UDG-related function study and disease prognosis.
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Figure Captions
Scheme 1. Schematic presentation of the TSDR-dependent fluorescent strategy for detecting UDG activity.
Fig. 1. Fluorescence emission spectra for the sensing system containing: (a) UP, UDG, TP, RP, Vent (exo-) polymerase and Nt.BstNBI nicking enzyme; (b) UP, TP, RP, Vent (exo-) polymerase and Nt.BstNBI nicking enzyme; (c) UDG, TP, RP, Vent (exo-) polymerase and Nt.BstNBI nicking enzyme; (d) UP, UDG, TP, Vent (exo-) polymerase and Nt.BstNBI nicking enzyme.
Fig. 2. (A) Fluorescence emission spectra of the sensing system with various concentrations of UDG. (B) Calibration curve between the net signal ΔF and UDG
concentration. Inset: the linear relationship between the net signal ΔF and UDG concentration ranging from 0.00020 U/mL to 0.0080 U/mL. Error bars show the standard deviations of the results from three independent experiments.
Fig. 3. Relative fluorescence responses of the sensing system towards UDG against other nucleases: (1) blank; (2) UDG; (3) mixed sample consisting of UDG, hOGG1, hAAG and DNase I; (4) hOGG1; (5) hAAG; (6) DNase I. Error bars show the standard deviations of the results from three independent experiments.
Fig. 4. Fluorescence emission spectra in the absence and presence of HeLa cells lysate, and the inhibitory effect of 2.0 U/mL UGI on the UDG activity in the HeLa cells lysate.
Fig. 5. Relative fluorescence intensity of the sensing system in the presence of UGI at increasing concentration (0 U/mL, 0.020 U/mL, 0.10 U/mL, 0.20 U/mL, 0.60 U/mL, 1.0 U/mL, 2.0 U/mL). The concentration of UDG is 1.0 U/mL. Error bars show the standard deviations of the results from three independent experiments.
A fluorescence strategy for sensitive detection of UDG activity was proposed.
The strategy depended on the toehold-mediated strand displacement reaction (TSDR).
AP sites generated from the removal of two uracil bases by UDG inhibit the TSDR.
The highly efficient signal transduction was achieved.
The strategy could detect UDG activity as low as 2.7 × 10-5 U/mL.
Scheme 1
Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
PII: DOI: Reference:
S0956-5663(16)31069-7 http://dx.doi.org/10.1016/j.bios.2016.10.053 BIOS9275
To appear in: Biosensors and Bioelectronic Received date: 30 July 2016 Revised date: 9 October 2016 Accepted date: 19 October 2016 Cite this article as: Yushu Wu, Lei Wang and Wei Jiang, Toehold-mediated strand displacement reaction-dependent fluorescent strategy for sensitive detection of uracil-DNA glycosylase activity, Biosensors and Bioelectronic, http://dx.doi.org/10.1016/j.bios.2016.10.053 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Toehold-mediated strand displacement reaction-dependent fluorescent strategy for sensitive detection of uracil-DNA glycosylase activity
Yushu Wu,a Lei Wang,b Wei Jianga,*
a
Key Laboratory for Colloid and Interface Chemistry of Education Ministry,
School of Chemistry and Chemical Engineering, Shandong University, 250100 Jinan, P.R. China b
School of Pharmaceutical Sciences, Shandong University, 250012 Jinan, P.R. China
Corresponding author: Tel: 86-531-88363888; fax: 86-531-88564464. E-mail: [email protected] Abstract Sensitive detection of uracil-DNA glycosylase (UDG) activity is beneficial for evaluating the repairing process of DNA lesions. Here, toehold-mediated strand displacement reaction (TSDR)-dependent fluorescent strategy was constructed for sensitive detection of UDG activity. A single-stranded DNA (ssDNA) probe with two uracil bases and a trigger sequence were designed. A hairpin probe with toehold domain was designed, and a reporter probe was also designed. Under the action of UDG, two uracil bases were removed from ssDNA probe, generating apurinic/apyrimidinic (AP)
sites. Then, the AP sites could inhibit the TSDR between ssDNA probe and hairpin probe, leaving the trigger sequence in ssDNA probe still free. Subsequently, the trigger sequence was annealed with the reporter probe, initiating the polymerization and nicking amplification reaction. As a result, numerous G-quadruplex (G4) structures were formed, which could bind with N-methyl-mesoporphyrin IX (NMM) to generate enhanced fluorescent signal. In the absence of UDG, the ssDNA probe could hybridize with the toehold domain of the hairpin probe to initiate TSDR, blocking the trigger sequence, and then the subsequent amplification reaction would not occur. The proposed strategy was successfully implemented for detecting UDG activity with a detection limit of 2.7 × 10-5 U/mL. Moreover, the strategy could distinguish UDG well from other interference enzymes. Furthermore, the strategy was also applied for detecting UDG activity in HeLa cells lysate with low effect of cellular components. These results indicated that the proposed strategy offered a promising tool for sensitive quantification of UDG activity in UDG-related function study and disease prognosis. Keywords: Uracil-DNA glycosylase activity; Apurinic/apyrimidinic site; Mismatch; Toehold-mediated strand displacement reaction; Fluorescent strategy.
1. Introduction Uracil-DNA glycosylase (UDG) is a protein enzyme which is involved in the repairing process of DNA lesions (Stivers and Jiang, 2003; Savva et al., 1995). It can specifically recognize and remove the uracil leision from DNA, leaving an apurinic/apyrimidinic (AP) site in DNA to trigger the downstream repairing process (Kunkel and Erie, 2005; Lindahl et al., 1976; Lindahl et al., 1979; Lindahl et al., 1982; Sancar et al., 1996). In this regard, UDG plays indispensable roles in maintaining genomic integrity. Abnormal UDG activity would disturb the repairing process and induce the gene mutation, which results in various diseases including human immunodeficiency (Imai et al., 2003), lymphoma (Sousa et al., 2007) and Bloom syndrome (Seal et al., 1988). Therefore, detection of UDG activity is beneficial for evaluating the repairing process of DNA lesions in function study and disease prognosis (Ono et al., 2013). The activity of UDG is commonly measured by gel-based methods (de Souza-Pinto et al., 2004; Prorok et al., 2013), electrochemical methods (McWilliams et al., 2014) and colorimetric methods (Liu et al., 2014; Nie et al., 2015). In addition to such methods, fluorescent methods have drawn wide attention due to the advantages of safety, simplicity and sensitivity. In these fluorescent methods, DNA probes containing multiple uracil bases are generally used for the recognition of UDG (Hu et al., 2011; Zhang et al., 2012; Leung et al., 2013; Lee et al., 2015; Wu et al., 2015). Multiple uracil bases in the DNA probe are removed by UDG to generate AP sites. Since the AP site is a type of mismatch (Fang et al., 2015), the number of base pairs in the DNA probe is reduced and
the conformation of the DNA probe will change, leading to the release of primer sequence or signal probe. However, when the activity of UDG is low, the multiple uracil bases in each DNA probe could not be removed completely and the conformation of probe could not change completely, which reduce the efficiency of signal transduction. Thus, such methods are not efficient for detecting UDG with low activity, which limits their applications. The toehold-mediated strand displacement reaction (TSDR) contains two main processes: the hybridization process between the toehold domain and the fuel strand, as well as the branch migration process (Zhang and Winfree, 2009; Zhang et al., 2010). In the TSDR, the mismatched base at complementary domain of toehold can inhibit the branch migration process since the rate of dissociation from toehold is much higher than that of branch migration (Subramanian et al., 2011; Wang et al., 2012; Zhu et al., 2014; Gao et al., 2014). Here, the TSDR-dependent fluorescent strategy was constructed for sensitive detection of UDG activity. Under the action of UDG, two uracil bases in the single-stranded DNA (ssDNA) probe were removed to generate AP sites. Then, the AP sites, which served as mismatched bases, could inhibit the TSDR between ssDNA probe and hairpin probe, leaving the free trigger sequence in the ssDNA. Subsequently, the trigger sequence could initiate the polymerization and nicking amplification reaction, which generated numerous G-quadruplex (G4) structures. Finally, the G4 structures could bind with N-methyl-mesoporphyrin IX (NMM) to generate enhanced fluorescent signal. In the absence of UDG, the ssDNA probe could hybridize with the toehold domain of hairpin probe to initiate the TSDR, resulting in the blocking of the trigger sequence.
Accordingly, the subsequent amplification reaction would not occur. The proposed strategy could detect UDG activity with a detection limit of 2.7 × 10-5 U/mL. Additionally, the strategy could distinguish UDG well from other interference enzymes. Furthermore, the strategy was also applied for detecting UDG activity in HeLa cells lysate with low effect of cellular components. These results indicated that the proposed strategy offered a promising tool for sensitive quantification of UDG activity in UDG-related function study and disease prognosis.
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. UDG, human 8-oxoguanine DNA glycosylase (hOGG1), human alkyladenine DNA glycosylase (hAAG), DNase I, uracil glycosylase inhibitor (UGI), Vent (exo-) polymerase, Nt.BstNBI nicking enzyme, 10 × Thermopol buffer and 10 × NEBuffer 3 were all obtained from New England Biolabs Ltd. (Beijing, China). dNTPs were obtained from Fermentas (Beijing, China). NMM was purchased from Frontier Scientific Inc. (Logan, Utah, USA). The NMM solution was prepared in dimethyl sulfoxide (DMSO). The ultrapure water used in this work was obtained from a Millipore Milli-Q water purification system (> 18.25 MΩ•cm). The Hitachi F-7000 fluorescence spectrometer (Hitachi. Ltd., Japan) was used for the fluorescence measurements. The excitation wavelength was 399 nm. And the emission
spectra were collected from 560 nm to 700 nm. The fluorescence intensity at 618 nm was used for evaluating the performance of the proposed strategy. Both the slit widths for the excitation and emission were 10 nm. The photomultiplier tube voltage was 700 V. 2.2 Detection of UDG activity To obtain the hairpin structures, the hairpin probe (named TP) and the reporter probe (named RP) were denatured at 90 ºC for 5 min and cooled slowly to room temperature, respectively. 5.0 µL 1.5 µM ssDNA probe (named UP) and various concentrations of UDG were added to 1 × Thermopol buffer (20 mM Tris-HCl, pH 8.8, 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% TritonX-100) to give a total volume of 20 µL. The mixture was incubated at 37 ºC for 60 min to perform the UDG recognition reaction. Subsequently, 5.0 µL 4.5 µM TP and 1.0 µL 10 × Thermopol buffer (200 mM Tris-HCl, pH 8.8, 100 mM KCl, 100 mM (NH4)2SO4, 20 mM MgSO4, 1% TritonX-100) were added into the above solution with a final volume of 30 µL. The TSDR was carried out at 37 ºC for 120 min. Then, 300 nM RP, 0.16 U/μL Vent (exo-) polymerase, 0.40 U/μL Nt.BstNBI nicking enzyme, 0.40 mM dNTPs and 0.5 × NEB buffer 3˄25 mM Tris-HCl, pH 7.9, 50 mM NaCl, 5.0 mM MgCl2, 1.0 mM DTT˅were added to give a total volume of 50 µL. The mixture was incubated at 55 ºC for 90 min to perform the polymerization and nicking amplification reaction. Finally, KCl (160 mM) and NMM (4.0 µM) were added to the mixture. After incubating at 37 ºC for 30 min, the fluorescence intensity of the mixture was recorded on Hitachi F-7000 fluorescence spectrometer. 2.3 Evaluation of UDG activity inhibition To evaluate the inhibition of UDG activity, different concentrations of UGI were
added to the sensing system before the addition of 1.0 U/mL UDG. The following procedures were similar to those in the detection of UDG activity. After the TSDR as well as the polymerization and nicking amplification reaction, the fluorescence intensity of the sensing system was recorded on Hitachi F-7000 fluorescence spectrometer. 2.4 Preparation of HeLa cells lysate Approximately 1 × 108 HeLa cells samples were pelleted by centrifugation (5 min, 3000 rpm, 4 ºC) and resuspended in 100 µL 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 ºC to remove insoluble material. The resulting supernatant was collected and filtered through a 0.45 µm filter membranes, yielding crude lysate.
3. Results and discussion 3.1 The principle of the TSDR-dependent fluorescent strategy for UDG activity assay Scheme 1 was the principle of the TSDR-dependent fluorescent strategy for detecting UDG activity. A ssDNA probe (named UP) with two uracil bases and a trigger sequence was designed. A hairpin probe (named TP) with toehold domain was designed. And a reporter probe (named RP) was also designed. Under the action of UDG, the uracil bases were removed from UP, generating AP sites. The obtained probe with AP sites was denoted UP’. The UP’ could not initiate TSDR, leaving the trigger sequence in UP’ still free. Subsequently, the trigger sequence was annealed with RP, initiating the strand displacement amplification (SDA) reaction, namely, the polymerization and nicking
amplification reaction. Finally, numerous G4 structures were formed, which could bind with NMM to generate enhanced fluorescent signal. However, in the absence of UDG, UP could hybridize with the toehold domain of TP to initiate the TSDR, resulting in the blocking of the trigger sequence. Accordingly, the subsequent amplification reaction would not occur. Thus, the proposed strategy could be used for the detection of UDG activity.
Scheme 1 is here
3.2 Feasibility research of the developed strategy in UDG activity assay As shown in Fig. 1, fluorescence emission spectra were used to investigate the viability of the TSDR-dependent fluorescent strategy for detecting UDG activity. The system without UP or RP showed very weak fluorescence intensity (Fig. 1, curve c and curve d), suggesting the necessity of UP and RP for the strategy. The control experiment without UDG exhibited weak fluorescence intensity (Fig. 1, curve b). However, the fluorescence intensity had significant enhancement upon the addition of UDG to the system containing UP, TP and RP (Fig. 1, curve a). Thus, these results demonstrated that the proposed strategy could be applied to detect UDG activity.
Fig. 1 is here
3.3 Optimization of the reaction conditions
To obtain the best performance of the sensing system, the sequence of toehold domain in the hairpin probe TP was first optimized. By altering the number of G/C bases in toehold domain from 4 to 0, five different hairpin probes TP (TP1 to TP5) were obtained. Fig. S1 showed the dependence of the net signal ΔF (ΔF = F - F0, where F and F0 were fluorescence intensities of the system in the presence and absence of UDG, respectively) on the sequence of toehold domain in TP. It was observed that the value of ΔF gradually increased from TP1 to TP3 and reached a plateau at TP4. The result indicated that the hairpin probe TP4 with 1 G/C base in toehold domain ensured a high net signal and could be beneficial for sensitive detection of the target. Therefore, TP4 was chosen for the following investigations. Furthermore, the performance of the sensing system was also influenced by other factors, including the concentrations of TP, RP and NMM. Therefore, the effects of these factors on the system were investigated, respectively. The concentration of TP played an important role in the efficiency of TSDR between UP and TP, so we optimized the TP concentration. The concentrations of TP from C1 to C6 were 50.0 nM, 450 nM, 750 nM, 1.20 µM, 1.50 µM and 1.80 µM, respectively. As presented in Fig. S2, the background fluorescence in the control systems gradually decreased with the increasing concentration of TP. However, in the presence of UDG, the fluorescence intensity also decreased gradually with the increase of TP concentration. The reason was that the high concentration of TP could improve the efficiency of TSDR. When the TP concentration was high, the TSDR between UP and TP would still occur even in the presence of UDG. Accordingly, the trigger sequence in UP was blocked and
the subsequent amplification reaction would not be initiated. Thus, 450 nM was chosen as the optimized TP concentration owing to the maximum value of the net signal ΔF. Due to that the concentration of RP could significantly affect the amplification efficiency, the RP concentration was further investigated. The RP concentration was varied from 150 nM to 375 nM. As shown in Fig. S3, the value of ΔF gradually increased with the increasing concentration of RP from 150 nM to 300 nM and reached a plateau beyond 300 nM. Thus, 300 nM of RP was employed in the sensing system. The fluorescence signal of the sensing system depended on the amount of NMM bound to G4 structures, so the concentration of NMM was further optimized. The NMM concentration was varied from 1.00 µM to 10.0 µM. As depicted in Fig. S4, the value of ΔF reached its maximum with 4.00 µM and then decreased at higher concentration. Therefore, 4.00 µM of NMM was used in the subsequent experiment. 3.4 Analytical performance of the developed strategy To study the analytical performance of the developed strategy, a series of samples with various concentrations of UDG were measured under the optimal conditions. Fig. 2A showed the fluorescence emission spectra of the sensing system. As can be seen, the fluorescence intensity gradually increased as the UDG concentration varied from 0 to 1.0 U/mL, which indicated that more and more G4 structures were formed. As shown in Fig. 2B, the corresponding calibration curve between the net signal ΔF and UDG concentration was obtained. Notably, the inset of Fig. 2B clearly showed that the net signal ΔF exhibited a good linear relationship with UDG concentration in the range from 0.00020 U/mL to 0.0080 U/mL. This linear relationship was described as ΔF = 30.13 +
9.936 × 104 C with a correlation coefficient of R2 = 0.9993, where C represented the concentration of UDG. And the detection limit for UDG activity was calculated to be 2.7 × 10-5 U/mL in terms of the 3δ rule. As shown in Table S2, the detection limit of this strategy was lower than that of the previously reported UDG activity assay (Hu et al., 2011; Zhang et al., 2012; Leung et al., 2013; Lee et al., 2015; Wu et al., 2015). In this strategy, two strand displacement reactions were involved. The first was the TSDR, the second was the SDA reaction, namely, the polymerization and nicking amplification reaction. Due to the AP sites generated from the removal of only two uracil bases could inhibit the TSDR, the highly efficient signal transduction was achieved. Moreover, due to the high signal amplification capability of SDA, the detection sensitivity was further improved. Therefore, this strategy offered a promising tool for sensitive quantification of UDG activity in UDG-related function study and disease prognosis.
Fig. 2 is here
The precision and reproducibility of the developed strategy were investigated due to they were vital parameters to evaluate the performance of a sensing system. To investigate the precision, three repetitive experiments for the samples were performed on the same day. According to the experimental results, the relative standard deviations (RSD) were 4.1%, 3.7%, 3.1% at 0.001 U/mL, 0.004 U/mL and 0.006 U/mL of UDG, respectively. In addition, to investigate the repeatability, three repetitive experiments for the samples were performed on three different days. The RSD for samples containing the
above concentrations were 5.8%, 5.9% and 5.5%, respectively. The results displayed that the precision and reproducibility of the developed strategy were acceptable. Since the selectivity was an important factor to assess the utility of the sensing system in biological samples, the selectivity of the proposed strategy for UDG activity was further investigated. The nucleases including hOGG1, hAAG and DNase I were selected as interference enzymes to assess the selectivity of this proposed strategy. And all enzymes had the same concentration of 1.0 U/mL. The results were presented in Fig. 3. In the presence of UDG, a high relative fluorescence response was obtained. In contrast, in the presence of hOGG1, hAAG or DNase I, the relative fluorescence response was very low and comparable to that in the blank solution. Moreover, when UDG was spiked in the mixed sample consisting of UDG, hOGG1, hAAG and DNase I, the relative fluorescence response was as high as that in the system containing UDG only. The results suggested that the strategy we proposed here had high selectivity toward UDG and could distinguish UDG in the mixed sample containing other interference enzymes. The high selectivity of our strategy was mainly due to the specific site recognition of UDG toward its substrate.
Fig. 3 is here
In order to investigate the practical applicability of the developed strategy, this strategy was further used for the detection of UDG activity in HeLa cells lysate. As shown in Fig. 4, when the lysis buffer instead of UDG was added to the sensing system,
the fluorescence intensity was very low. In contrast, when 1.0 µL of HeLa cells lysate instead of UDG was added into the sensing system with a final concentration of 1.0 × 104 cells/µL, an obvious increase in the fluorescence intensity was observed. Furthermore, when UGI, an inhibitor of UDG, was added to the sensing system containing HeLa cells lysate, the fluorescence intensity was very low. These results indicated that the fluorescence enhancement was derived from UDG activity rather than any other component in the lysate. Taken together, the developed strategy could be tolerant to the cellular components and held a great potential for detecting UDG activity in complex biological samples.
Fig. 4 is here
3.5 Evaluation of UDG activity inhibition Since the inhibition of UDG activity could enhance the therapeutic efficacy of DNA-damaging chemotherapeutic agents (Bulgar et al., 2012; Moeller et al., 2010; Priet et al., 2005), it was attractive to evaluation of the inhibition of UDG activity. Here, the capability of this strategy for detecting the inhibition of UDG activity was also evaluated. UGI, which was selected as a model inhibitor, could form a specific complex with UDG at a 1:1 stoichiometry (Kaushal et al., 2008). As presented in Fig. 5, the relative fluorescence intensity gradually decreased with the increasing concentration of UGI. The result demonstrated that the proposed strategy could be used to detect the inhibition of UDG activity and had a great potential to be used as a platform to screen UDG inhibitors.
Fig. 5 is here
4. Conclusions In summary, the TSDR-dependent fluorescent strategy was developed for sensitive detection of UDG activity. This strategy contained two strand displacement reactions: the first was the TSDR, and the second was the SDA reaction, namely, the polymerization and nicking amplification reaction. Upon the removal of uracil bases by UDG, the generated AP sites, which served as mismatched bases, could inhibit the first strand displacement reaction TSDR, leaving the trigger sequence in the ssDNA probe still free. Then, the free trigger sequence could initiate the second strand displacement reaction SDA. Due to the AP sites generated from the removal of only two uracil bases could inhibit the TSDR, the highly efficient signal transduction was achieved. And due to the high signal amplification capability of SDA, the detection sensitivity was further improved. The detection limit of this strategy was as low as 2.7 × 10-5 U/mL, which was better than that of the previously reported UDG activity assay (Table S2). Additionally, the strategy could distinguish UDG well from other interference enzymes. Furthermore, the strategy was also applied for detecting UDG activity in HeLa cells lysate with low effect of cellular components. These results indicated that the proposed strategy offered a promising tool for sensitive quantification of UDG activity in UDG-related function study and disease prognosis.
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Figure Captions
Scheme 1. Schematic presentation of the TSDR-dependent fluorescent strategy for detecting UDG activity.
Fig. 1. Fluorescence emission spectra for the sensing system containing: (a) UP, UDG, TP, RP, Vent (exo-) polymerase and Nt.BstNBI nicking enzyme; (b) UP, TP, RP, Vent (exo-) polymerase and Nt.BstNBI nicking enzyme; (c) UDG, TP, RP, Vent (exo-) polymerase and Nt.BstNBI nicking enzyme; (d) UP, UDG, TP, Vent (exo-) polymerase and Nt.BstNBI nicking enzyme.
Fig. 2. (A) Fluorescence emission spectra of the sensing system with various concentrations of UDG. (B) Calibration curve between the net signal ΔF and UDG
concentration. Inset: the linear relationship between the net signal ΔF and UDG concentration ranging from 0.00020 U/mL to 0.0080 U/mL. Error bars show the standard deviations of the results from three independent experiments.
Fig. 3. Relative fluorescence responses of the sensing system towards UDG against other nucleases: (1) blank; (2) UDG; (3) mixed sample consisting of UDG, hOGG1, hAAG and DNase I; (4) hOGG1; (5) hAAG; (6) DNase I. Error bars show the standard deviations of the results from three independent experiments.
Fig. 4. Fluorescence emission spectra in the absence and presence of HeLa cells lysate, and the inhibitory effect of 2.0 U/mL UGI on the UDG activity in the HeLa cells lysate.
Fig. 5. Relative fluorescence intensity of the sensing system in the presence of UGI at increasing concentration (0 U/mL, 0.020 U/mL, 0.10 U/mL, 0.20 U/mL, 0.60 U/mL, 1.0 U/mL, 2.0 U/mL). The concentration of UDG is 1.0 U/mL. Error bars show the standard deviations of the results from three independent experiments.
A fluorescence strategy for sensitive detection of UDG activity was proposed.
The strategy depended on the toehold-mediated strand displacement reaction (TSDR).
AP sites generated from the removal of two uracil bases by UDG inhibit the TSDR.
The highly efficient signal transduction was achieved.
The strategy could detect UDG activity as low as 2.7 × 10-5 U/mL.
Scheme 1
Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5