Subscriber access provided by UNIVERSITY OF TOLEDO LIBRARIES
Article
Terminal deoxynucleotidyl transferase and T7 exonuclease-aided amplification strategy for ultrasensitive detection of uracil-DNA glycosylase Yi-Chen Du, Yun-Xi Cui, Xiao-Yu Li, Guo-Ying Sun, YuPeng Zhang, An-Na Tang, Kwangil Kim, and Deming Kong Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01928 • Publication Date (Web): 18 Jun 2018 Downloaded from http://pubs.acs.org on June 19, 2018
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 7 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Analytical Chemistry
Terminal deoxynucleotidyl transferase and T7 exonucleaseaided amplification strategy for ultrasensitive detection of uracil-DNA glycosylase Yi-Chen Du†,‡, Yun-Xi Cui†, Xiao-Yu Li†, Guo-Ying Sun†, Yu-Peng Zhang†, An-Na Tang†, Kwangil Kim‡,§, De-Ming Kong†,‡,* †
State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Biosensing and Molecular Recognition, Research Centre for Analytical Sciences, College of Chemistry, Nankai University, Tianjin, 300071, P R China ‡ Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin, 300071, P R China. § Institute of Analysis, Kim Chaek University of Technology, Pyongyang, 999093, Democratic People’s Republic of Korea. ABSTRACT: As one of the key initiators of base excision repair process, uracil-DNA glycosylase (UDG) plays an important role in maintaining genomic integrity. It has been found that aberrant expression of UDG is associated with a variety of diseases. Thus, accurate and sensitive detection of UDG activity is of critical significance for biomedical research and early clinical diagnosis. Here, we developed a novel fluorescent sensing platform for UDG activity detection based on terminal deoxynucleotidyl transferase (TdT) and T7 exonuclease (T7 Exo)-aided recycling amplification strategy. In this strategy, only two DNA oligonucleotides (DNA substrate containing one uracil base and Poly-dT probe labelled with a fluorophore/quencher pair) are used. UDG catalyzes the removal of uracil base from the enclosed dumbbell-shape DNA substrate to give an apyrimidinic site, at which the substrate oligonucleotide is cleaved by endonuclease IV. The released 3′-end can be elongated by TdT to form a long adenine-rich (Poly-dA) tail, which may be used as a recyclable template to initiate T7 Exo-mediated hybridization-digestion cycles of the Poly-dT probe, giving a significantly enhanced fluorescence output. The proposed UDG-sensing strategy showed excellent selectivity and high sensitivity with a detection limit of 1.5×10-4 U/mL. The sensing platform was also demonstrated to work well for UDG inhibitor screening and inhibitory activity evaluation, thus holding great potential in UDG-related disease diagnosis and drug discovery. The proposed strategy can be easily used for the detection of other DNA repair-related enzymes by simply changing the recognition site in DNA substrate, and might also be extended to the analysis of some DNA/RNA-processing enzymes, including restriction endonuclease, DNA methyltransferase, polynucleotide kinase and so on.
Genome, which is constituted of pairing DNA, contains important genetic information of organism’s life. Integrity of genome is a prerequisite property in gene transcription. DNA damage caused by various endogenous and exogenous agents may lead to mutations or breaks in DNA strands during replication1–3, thus resulting in genome instability or even carcinogenesis4,5. Base excision repair (BER) is a significant mechanism which helps to maintain the genomic integrity through removing of the damaged DNA bases. Uracil-DNA glycosylase (UDG) is a well-known initiator in BER system, it can catalyze the hydrolysis of the N-glycosylic bond between uracil and sugar, leaving an apyrimidinic (AP) site in uracilcontaining DNA. This process has been demonstrated involving in various of DNA-repair pathways6–8. Abnormal activity of UDG has been reported to disturb the uracilincluded DNA-repair process, and resulting in different diseases such as cancer, Bloom syndrome, lymphoma and neurodegenerative diseases2,4,9–11. Therefore, development of highly sensitive and specific sensing platform for UDG activity analysis has attracted intense interest of scientists. Conventional method of UDG activity detection applies gel electrophoresis coupled with radioactive labeling12. However,
this method is limited by several intrinsic drawbacks include hazardous radiation, low sensitivity, time-consuming and complicated manipulation13. In recent years, a variety of new technologies have been developed for the measurement of UDG activity, such as colorimetry14–16, electrochemistry17, chemiluminescence18 and fluorescence19–22. For the regular methods, DNA substrates containing multiple uracil bases are usually applied as the identification sites of UDG. The presence of UDG enables the denaturing of DNA substrates, which may release the primers or signal probes. Nevertheless, in the case of extremely low UDG concentration, especially for biosamples which are usually under the cellular or serous environment, the activity of UDG is not enough. In such situation, targeted uracil bases keep their positions in the DNA substrates. So the DNA strands will maintain their original conformation, thus reducing the efficiency of signal transduction22. Although incorporated with signal amplification strategy, many of those methods still cannot achieve the detection of UDG with low activity, thus limiting their applications. Terminal deoxynucleotidyl transferase (TdT) is a kind of DNA polymerase that catalyzes the addition of
ACS Paragon Plus Environment
Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
deoxyribonucleotides to the 3′-end of a DNA strand, and has been exploited as powerful tools for bioanalysis23, DNA hydrogel preparation24 and cell apoptosis bioimaging25. Different from general DNA polymerases, it is a templateindependent enzyme. Thus, it may work using any DNA substrates as primers26,27. The sequence of the extension product is highly dependent on the components of the deoxyribonucleotides (dNTP) pool applied in the polymerization reaction. For example, if only dATP is added in, the extended sequence would contain repeated deoxyadenine (dA) only. Here, we proposed a simple but high sensitive fluorescent method for detection of UDG activity using an enclosed dumbbell-shape DNA substrate containing one uracil base. In presence of UDG, the uracil base will be removed, initiating the subsequent amplification cycles based on the cooperated activity of TdT and T7 exonuclease (T7 Exo), which can catalyze the removal of mononucleotides from the blunt or recessed 5′-termini of duplex DNA28–30. Thus, the fluorophore labeled on a Poly-dT probe will be released and the quenched fluorescence signal is recovered. Taking advantage of the unique polymerization property of TdT and DNA digestion function of T7 exonuclease, the proposed amplified strategy showed extremely high selectivity and sensitivity for UDG activity detection. What's more, we also demonstrated that our novel sensing platform could be applied to screen UDG inhibitors and to detect cellular UDG activity. EXPERIMENTAL SECTION Materials and Reagents. The oligonucleotides used in this work (Table 1) were synthesized and purified by Sangon Biotech. Co. Ltd. (Shanghai, China). Uracil-DNA glycosylase (UDG), 10 × UDG buffer (200 mM Tris-HCl, 10 mM EDTA, 10 mM DTT, pH 8.0), terminal deoxynucleotidyl transferase (TdT), 10 × TdT buffer (500 mM KAc, 200 mM Tris-Ac, 100 mM Mg(Ac)2, 10 mM DTT, pH 7.9), CoCl2 (2.5 mM), T7 exonuclease (T7 Exo), 10 × NEBuffer 4 (500 mM KAc, 200 mM Tris-Ac, 100 mM Mg(Ac)2, 10 mM DTT, pH 7.9), endonuclease IV (Endo.IV), UDG inhibitor (UGI), T4 DNA ligase, exonuclease I (Exo I), exonuclease III (Exo III), Bst DNA polymerase, T4 polynucleotide kinase (PNKP), restriction endonuclease EcoRI, human alkyl adenine DNA glycosylase (hAAG), 10 × T4 DNA ligase buffer (500 mM Tris-HCl, 100 mM MgCl2, 100 mM dithiothreitol, 10 mM ATP, pH 7.5) and deoxyadenosine triphosphate (dATP) were obtained from New England Biolabs (Beijing, China). Table 1. The oligonucleotides used in this work Oligonucleotide DS-U Poly-dT
Sequence (5′→3′) P -TAT UCT GGA TAC GTC TAA CTC CAG AAT ATG TAT CGT AGC TAA GTC CCT ACG ATA CA FAM-TTT TTT TTT TTT-BHQ1
Italic regions in DS-U are complementary and underlined regions are complementary, respectively. ‘P’ indicates phosphate group. ‘FAM’ indicates 6-carboxyfluorescein. ‘BHQ1’ indicates black hole quencher 1 modification.
Preparation of enclosed dumbbell-shape UDG substrate. 50 µL reaction mixture containing 5 µL of DS-U solution (10 µM) and 5 µL 10 × T4 DNA ligase buffer was incubated at 95 °C for 5 min, and then cooled down to 25 °C to ensure the
Page 2 of 7
formation of the dumbbell-shape structure of DS-U. Next, 20 U of T4 DNA ligase was added into the mixture and the ligation reaction was conducted at 16 °C for 12 h. After that, 1 µL of Exo I (20 U/µL) and 1 µL of Exo III (100 U/µL) were added in, and the mixture was incubated at 37 °C for 1 h for complete digestion of the unligated substrates. The enzymes were then inactivated by heating the mixture at 80 °C for 20 min. The obtained solution containing ligated DS-U substrate was stored at -20 °C for further use. Detection of UDG activity utilizing TdT/T7 Exo-aided recycling amplification strategy. First, 2 µL of ligated DS-U substrate (1 µM) was added into the excision reaction system (20 µL) containing different concentrations of UDG, 0.5 U of Endo.IV and 2 µL of 10 × UDG reaction buffer. The mixture was incubated at 37 °C for 30 min. Second, 2 µL of dATP (100 mM), 3 µL of 10 × TdT buffer, 3 µL of CoCl2 (2.5 mM), and 15 U of TdT were added into the excision reaction system with a total volume of 30 µL and then incubated at 37 °C for 60 min. After that, TdT polymerase was inactivated by heating at 80 °C for 10 min. Next, 7 µL of 10 × NEBuffer 4, 2 µL of Poly-dT probe (10 µM), 5 U of T7 Exo were added in. The mixture with a total volume of 100 µL was incubated for 30 min at 25 °C. All fluorescence spectra were measured by Shimadzu RF-5301 fluorescence spectrometer (Shimadzu Ltd., Japan) exciting at 492 nm and measuring emission spectra between 500 nm and 580 nm. The fluorescence intensity at 517 nm was collected for quantitative assay of UDG activity. For UDG inhibition assay, different amounts of UGI were incubated with the ligated DS-U substrate before addition of 0.01 U/mL UDG. Polyacrylamide gel electrophoresis (PAGE) analysis. The reaction products were analyzed by 15% polyacrylamide gel electrophoresis (PAGE) in 1 × TBE buffer (89 mM Tris-boric acid, 2.0 mM EDTA, pH 8.3) at a 110 V constant voltage for 50 min. The gel was stained with 2 × Gel Red and photographed using a Gel Documentation system (Huifuxingye, Beijing, China). Cell culture and sample preparation. HeLa (human cervical carcinoma) and HepG2 (liver hepatocellular carcinoma) cells were grown in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with penicillin in a concentration of 100 U/mL, streptomycin (100 µg/mL) and 10% fetal bovine serum and incubated at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. Approximately 1 × 106 cells were dispensed in a 1.5 mL centrifuge tube, washed twice with PBS buffer and centrifuged at 2000 rpm for 3 min followed by discarding the supernatant. A volume of 100 µL lysis buffer (20 mM Tris with a pH value of 7.5, 150 mM NaCl, 1% Triton X-100, sodium pyrophosphate, βglycerophosphate, EDTA, Na3VO4 and leupeptin) was added into the cell residues. The mixture was incubated on ice for 30 min, with vortex for 15 s every 5 min. The resulting mixture was centrifuged at 12000 rpm for 20 min at 4 °C. The supernatant could be used immediately for UDG activity assay or frozen at -80 °C for long-term storage. Total amounts of protein in cell lysates were determined by a BCA protein assay kit. Cell lysates containing 2 µg protein were added into the sensing system with a total volume of 100 µL. Blank control was prepared by inhibiting UDG activity in cell lysates by UGI.
ACS Paragon Plus Environment
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Analytical Chemistry RESULTS AND DISCUSSION Principle of the UDG detection. The working mechanism of TdT/T7 Exo-aided recycling amplification strategy for UDG activity assay is illustrated in Scheme 1. Enclosed dumbbell-shape UDG substrate was prepared by self-template ligation of a 5'-phosphorylated DNA (called DS-U, Table 1) which contained one uracil base. In the presence of UDG, the uracil was removed from ligated DS-U, thereby generating an AP site that can be subsequently cleaved by Endo.IV. The released 3′-end could then be elongated by TdT in the dATP pool, forming long deoxyadenine-rich (Poly-dA) tail. When the dual-labelled reporter probe with deoxythymine-rich (Poly-dT) sequence was added, it would hybridize with the elongated Poly-dA sequence, forming a double-stranded DNA region with a calculated melting temperature of 36.2 oC,. T7 Exo specifically catalyzes the removal of 5′-mononucleotides from the Poly-dT probe, resulting in the separation of the fluorophore (FAM) and the quencher (BHQ1) labelled at the two ends of the probe, thus generating recovered fluorescence signal that was previously quenched in random-coiled structure of Poly-dT probe. Since the released Poly-dA sequence will be re-used for hybridization with another PolydT probe and thus initiate new cycles for fluorescence amplification, greatly amplified detection signal could be provided for UDG activity detection.
incubating with T4 DNA ligase. Highly efficient formation of enclosed dumbbell-shape UDG substrate was verified by polyacrylamide gel electrophoresis (PAGE) analysis (Figure S1).
(a)
500
5
400
Fluorescence
Page 3 of 7
300
1 Without UDG 2 Without Endo.IV 3 Without TdT 4 Without T7 Exo 5 With 0.01 U/mL UDG
200 100 0 500
1,2,3 4 525
550
575
Wavelength (nm)
Figure 1 (a) Fluorescence and (b) electrophoretic assay for different sensing systems. In (b), Lane M is the DNA ladder marker. Lane 1: DS-U; Lane 2: Ligated DS-U; Lane 3: Ligated DS-U + UDG + Endo.IV; Lane 4: Ligated DS-U + UDG + Endo.IV + TdT; Lane 5: Ligated DS-U + UDG + Endo.IV + TdT + Poly-dT; Lane 6: Ligated DS-U + UDG + Endo.IV + TdT + Poly-dT + T7 Exo.
Scheme 1. Illustration of TdT/T7 Exo-aided recycling amplification strategy for UDG activity assay. Since TdT can catalyze the addition of deoxyribonucleotides to the 3′-end(s) of both single- and double-stranded DNAs31, the UDG substrate used in this work must have no 3′-OH end before UDG treatment. Although some 3′-end modification techniques can be adopted, undesirable background fluorescence might also be given due to the destruction of the modification by enzymes or other reagents in the sensing system. Therefore, the most efficient way is to deprive the substrate DNA strand of 3′-end by ligating its two ends together to form an enclosed circle. Therefore, a 5'-phosphorylated dumbbell-shape DNA strand DS-U was designed, its two ends could be easily ligated by
Fluorescence analysis was then conducted to verify the feasibility of the proposed sensing platform for UDG activity detection. As expected, significantly enhanced fluorescence response was given by the sensing system towards UDG. On the contrary, none of the negative controls lacking UDG, Endo.IV, TdT or T7 Exo exhibited observable fluorescence signal changes (Figure. 1a). To further validate the fluorescence signal enhancement was really caused by the proposed TdT/T7 Exo-aided recycling amplification mechanism, non-denatured PAGE was carried out to analyze the products of each step (Figure 1b). 5'-phosphorylated DS-U could be ligated by T4 DNA ligase to form enclosed dumbbell-shaped UDG substrate (Figure 1b, Lane 2), which cannot be digested by Exo I and Exo III any more (Figure S1). In the presence of UDG and Endo.IV, uracil in the ligated DSU probe was removed and the enclosed dumbbell-shape structure was destroyed at the resulting AP site, making the migration rate of the DNA band slightly slower (Lane 3). Then, a long Poly-dA sequence could be extended from the released 3’-end by TdT, giving a new band located between 100 bp and 200 bp (Lane 4). That is, more than 100-nt Poly-dA sequence was extended, thus making simultaneous hybridization with several Poly-dT probes possible. After addition of Poly-dT probe, the new band appeared a little brighter, thus confirming
ACS Paragon Plus Environment
Analytical Chemistry the hybridization of the Poly-dT probe on the extended PolydA sequence. Further incubation with T7 Exo resulted in the appearance of a new band with very short length, which corresponds to the residual Poly-dT probe after digestion by T7 Exo. When the residual part is too short to form stable duplex with Poly-dA sequence, it would be released into the solution, thus making next round of probe hybridization/digestion possible. These results clearly demonstrate that the proposed TdT/T7 exonuclease-aided amplification recycles could really be initiated by UDG, thus can be applied for the detection of UDG activity.
(a)
500
0.1 U/mL
300
0
200 100 0 500
525
550
575
Wavelength (nm)
(b)
500 400
concentration range of 5×10-4 ~ 1×10-2 U/mL, the fluorescence intensity at 517 nm showed a good linear correlation with the logarithm of UDG concentration. The linear regression equation is F = 1168.49 + 351.44 lgC with a correlation coefficient (R2) of 0.9768, where F is the fluorescence intensity and C (U/mL) is the concentration of UDG. Based on 3 times of the standard deviation over the blank response (3σ/S), the detection limit was estimated to be 1.5×10-4 U/mL, which is better than or comparable to the reported methods (Table S1).15,19,20,32-35 The high sensitivity may be attributed to the excellent amplification efficiency of TdT-/T7 Exo-aided amplification strategy and low background signal due to the high exactitude of TdT36 and T7 exonuclease and the use of ligated UDG substrate. Selectivity of UDG assay. To evaluate the specificity of the proposed method, a series of contrast experiments were carried out using T4 DNA ligase, Bst DNA polymerase, T4 polynucleotide kinase (PNKP), restriction endonuclease EcoRI and human alkyl adenine DNA glycosylase (hAAG) as negative controls. hAAG is also a DNA glycosylase. But different from UDG that recognizes the uracil RNA base(s) in uracil-containing DNA, hAAG is responsible for recognizing alkylated and deaminated purines. The results showed that none of the tested negative-control enzymes introduced observable fluorescence enhancement compared to the background (Figure 3), thus indicating the high selectivity of our proposed method because of the highly specific uracil identification of UDG.
500
500
400
Fluorescence
300
1: UDG 2: T4 ligase 3: Bst polymerase 4: T4 PNKP 5: EcoRI 6: hAAG 7: Blank
300
200
400
200 100
100
0 -3.5
0
-3.0
-2.5
-2.0
lg UDG 0.00
0.01
0.02
0.03
0.04
0.05
UDG Concentration (U/mL) Figure 2 (a) Fluorescence spectra of the sensing systems containing different concentrations of UDG. The concentrations of UDG are (arrow direction): 0, 1×10-4, 5×10-4, 1×10-3, 2.5×10-3, 5×10-3, 1×10-2, 5×10-2 and 0.1 U/mL. (b) UDG concentration-dependent change in the fluorescence intensity at 517 nm. The insert shows the linear relationship between the fluorescence intensity and the logarithm of UDG concentration in the range of 5×10-4~1×10-2 U/mL.
Sensitivity of UDG assay. Above experiments demonstrated that the proposed method could be used for UDG activity assay. To achieve the best assay performance, the experimental conditions, including amounts of Endo.IV, TdT, T7 Exo and Poly-dT probe, were optimized (Figure S2 ~ Figure S5). Under the optimal conditions (0.5 U of Endo.IV, 15 U of TdT, 5 U of T7 Exo and 200 nM of Poly-dT probe), the sensitivity of the sensing platform was evaluated by recording the fluorescence signal change as a function of UDG concentration (Figure 2). As expected, with an increase of UDG concentration, more and more ligated DS-U strands were converted into TdT substrates, thus providing more PolydA products for subsequent Poly-dT probe digestion by T7 Exo, which is reflected by the continuous increase in the fluorescence intensity of the sensing system. In the UDG
Fluorescence
Fluorescence
400
Fluorescence
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 4 of 7
300 200 100 0 1
2
3
4
5
6
7
Figure 3 Fluorescence responses of the UDG-sensing system to 0.01 U/mL UDG, 0.1 U/mL T4 DNA ligase, 0.1 U/mL Bst DNA polymerase, 0.1 U/mL T4 polynucleotide kinase (PNKP), 0.1 U/mL restriction endonuclease EcoRI and 0.1 U/mL human alkyl adenine DNA glycosylase (hAAG). UDG inhibition assay. Recent research reported that the activity of UDG was associated with base repairing process and gene mutation. Inhibition of UDG activity could enhance the therapeutic efficacy of DNA-damaging chemotherapeutic agents.37,38 Thus the screening of inhibitor for UDG has attracted intense attention. Our UDG-sensing platform might also be used for inhibitory ability evaluation of UDG inhibitor and thus for screening of inhibitor. To demonstrate this, we selected the uracil-DNA glycosylase inhibitor (UGI) as the model inhibitor. UGI can bind with UDG at a stoichiometric ratio of 1:1, forming a stable protein complex. The result presented in Figure 4a showed that the fluorescence signal was completely disappeared in the presence of one equivalent UGI. However, when UGI was added after treatment of ligated DSU with UDG, similar fluorescence signal output to that
ACS Paragon Plus Environment
Page 5 of 7
without UGI addition was given, thus indicating that UGI has no obvious effect on the activities of Endo.IV, TdT and T7 exonuclease, and above-observed fluorescence decrease is really related to the repression of UDG activity by UGI. According to the dose-dependent relationship between fluorescence signal output and UGI concentration, a IC50 value (the UGI concentration resulting in 50% inhibition of UDG activity) of 0.004 U/mL was obtained, which is consistent with that reported in the literature20. These results demonstrate that the sensing platform could be applied to screen UDG inhibitors and evaluate their inhibitory activities, thus holding great potential in UDG-related disease diagnosis and drug discovery.
fluorescence intensity obtained with and without UGI were brought into the linear correlation function constructed in Figure 2, the UDG activities were calculated to be 6.2×10-3 U/mL and 7.6×10-3 U/mL in HeLa and HepG2 reaction systems with a total volume of 100 µL. And then, the UDG activities in HeLa and HepG2 cell lysates were calculated to be 0.31 U and 0.38 U per 1 mg total protein, respectively, which are consistent with those reported in other literatures39,40. These results confirmed that the UDG sensing platform can be used for measurement of UDG activity in complex biological samples. 500
(a) Fluorescence
400
Fluorescence
Cell lysate + UGI Cell lysate
400
500
300 200
300 200 100
100
0
0
A
B
C
D
(b)
HeLa
HepG2
Figure 5 Fluorescence responses of the sensing platform towards HeLa and HepG2 cell lysates (2 µg protein) in the absence or presence of UGI.
100
Relative activity (%)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Analytical Chemistry
75
IC50=0.004 U/mL
50 25 0 0.000
0.005
0.010
0.015
UGI Concentration (U/mL)
0.020
Figure 4 (a) Fluorescence intensities of the UDG-sensing system in the absence or presence of UGI. The concentrations of UGI and UDG were both 0.01 U/mL. A: without UDG and UGI; B: treatment of UDG with UGI before adding into the system; C only UDG; D: UGI was added after UDG-actuated uracil-excision process. (b) Relative activity of UDG in the presence of increasing concentrations of UGI. Cellular UDG assay. To further investigate the potential application of our proposed UDG-sensing platform in real and complex biological samples, we detected UDG activity in HeLa and HepG2 cell lysates. As shown in Figure 5, addition of HeLa and HepG2 lysates induced significant fluorescence enhancement. To verify that the observed fluorescence enhancement was really related with active UDG activity in the cell lysates and to deduct the effect of enhanced background caused by degradation of dumbbell-shape DS-U substrate by other components in the cell lysates (Figure S6), the UDG inhibitor UGI was added into the cell lysates. As expected, inhibition of UDG activity by UGI resulted in greatly reduced fluorescence output. When the values of the
CONCLUSION In summary, we have developed a novel fluorescent sensing platform for the detection of UDG activity by utilizing a TdT/T7 Exo-aided recycling amplification strategy. This strategy shows the following advantages: (1) The use of ligated dumbbell-shape UDG substrate without 3’-end, combing with the high exactitude of TdT and T7 Exo, confer the sensing platform with low background and thus high signal-to-noise ratio; (2) Since the ligated UDG substrate can be prepared in large quantity or commercially available, the UDG activity detection operation can be finished in a short time (~2 h); (3) Isothermal signal amplification strategy eliminates the needs for any expensive instruments. (4) Long Poly-dA extension sequence that can simultaneously hybridize with several Poly-dT probes, combing with high efficiency of T7 Exo-aided signal amplification strategy, confer the sensing platform with high detection sensitivity. As low low as 1.5×104 U/mL UDG can be detected with high specificity. Cellular UDG activity can also be accurately detected; (5) The sensing system is relatively simple, and only two DNA oligonucleotides are used. Using the same Poly-dT probe, the proposed method can be easily extended to the activity assay of other DNA-repair related enzymes by simple changing the enzyme recognition site in the substrate oligonucleotide; (6) This method might also be extended to the analysis of some DNA/RNA-processing enzymes, including restriction endonuclease, DNA methyltransferase, polynucleotide kinase and so on.
ASSOCIATED CONTENT Supporting Information
ACS Paragon Plus Environment
Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
The Supporting Information is available free of charge on the ACS Publications website.
AUTHOR INFORMATION Corresponding Author
*
[email protected]; Fax: +86-22-23502458 Notes The authors declare no competing financial interest.
ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (Nos. 21728801, 21322507, 21275081) and the National Natural Science Foundation of Tianjin (No. 16JCYBJC19900).
REFERENCES (1) Kunkel, T. A.; Erie, D. A. Annu. Rev. Biochem. 2005, 74, 681710. (2) Seal, G.; Brech, K.; Karp, S. J.; Cool, B. L.; Sirover, M. A. Proc. Natl. Acad. Sci. USA 1988, 85, 2339-2343. (3) Zharkov, D. O.; Mechetin, G. V.; Nevinsky, G. A. Mutat. Res. 2010, 685, 11-22. (4) Hoeijmakers, J. H. Nature 2001, 411, 366-374. (5) Wood, R. D. J. Biol. Chem. 1997, 272, 23465-23468. (6) Cole, H. A.; Tabor-Godwin, J. M.; Hayes, J. J. J. Biol. Chem. 2010, 285, 2876-2885. (7) Savva, R.; McAuley-Hecht, K.; Brown, T.; Pearl, L. Nature 1995, 373, 487-493. (8) Krusong, K.; Carpenter, E. P.; Bellamy, S. R.; Savva, R.; Baldwin, G. S. J. Biol. Chem. 2006, 281, 4983-4992. (9) Imai, K.; Slupphaug, G.; Lee, W.; Revy, P.; Nonoyama, S.; Catalan, N.; Yel, L.; Forveille, M.; Kavli, B.; Krokan, H. E.; Ochs, H. D.; Fischer, A.; Durandy, A. Nat. Immunol. 2003, 4, 1023-1028. (10) Sousa, M. M. L.; Krokan, H. E.; Slupphaug, G. Mol. Aspects Med. 2007, 28, 276-306. (11) Lindahl, T. Nature 1993, 362, 709-715. (12) Vaughan, P.; Mccarthy, T. V. Nucleic Acids Res. 1998, 26, 810815. (13) Ischenko, A. A.; Saparbaev, M. K. Nature 2002, 415, 183-187. (14) Liu, X.; Chen, M.; Hou, T.; Wang, X.; Liu, S.; Li, F. Biosens. Bioelectron. 2014, 54, 598-602. (15) Nie, H.; Wang, W.; Li, W.; Nie, Z.; Yao, S. Analyst 2015, 140, 2771-2777.
Page 6 of 7
(16) Du, Y.-C.; Jiang, H.-X.; Huo, Y.-F.; Han, G.-M.; Kong, D.-M. Biosens. Bioelectron. 2016, 77, 971-977. (17) McWilliams, M. A.; Anka, F. H.; Balkus. K. J.; Slinker, J. D. Biosens. Bioelectron. 2014, 54, 541-546. (18) Dong, J.; Lian, J.; Jin, Y.; Li, B. Anal. Methods 2017, 9, 276-281. (19) Zhang, Y.; Li, C. C.; Tang, B.; Zhang, C. Y. Anal. Chem. 2017, 89, 7684-7692. (20) Wang, L.; Ren, M.; Zhang, Q.; Tang, B.; Zhang, C. Anal. Chem. 2017, 89, 4488-4494. (21) Wu, Y.; Wang, L.; Jiang, W. Biosens. Bioelectron. 2017, 89, 984-988. (22) Du, Y.-C.; Zhu, L.-N.; Kong, D.-M. Biosens. Bioelectron. 2016, 86, 811-817. (23) Liu, Z.; Li, W.; Nie, Z.; Peng, F.; Huang, Y.; Yao, S. Chem. Commun. 2014, 50, 6875-6878. (24) Xiang, B.; He, K.; Zhu, R.; Liu, Z.; Zeng, S.; Huang, Y.; Nie, Z.; Yao, S. ACS Appl. Mater. Interfaces 2016, 8, 22801-22807. (25) Liu, Z.; Luo, X.; Li, Z.; Huang, Y.; Nie, Z.; Wang, H.-H.; Yao, S. Anal. Chem. 2017, 89, 1892-1899 (26) Faber, J.; Kantarjian, H.; Roberts, M. W.; Keating, M.; Freireich, E.; Albitar, M. Arch. Pathol. Lab. Med. 2000, 124, 92-97. (27) Yuan, Y.; Li, W.; Liu, Z.; Nie, Z.; Huang, Y.; Yao, S. Biosens. Bioelectron. 2014, 61, 321-327. (28) Wang, H. B.; Wu, S.; Chu, X.; Yu, R.-Q. Chem. Commun. 2012, 48, 5916-5918. (29) Kerr, C.; Sadowski, P. D. J. Biol. Chem. 1972, 247, 305-310. (30) Liu, X.; Li, W.; Hou, T.; Dong, S.; Yu, G.; Li, F. Anal. Chem. 2015, 87, 4030-4036. (31) Du, Y.-C.; Zhu, Y.-J.; Li, X.-Y.; Kong, D.-M. Chem. Commun. 2018, 54, 682-685. (32) Leung, K. H.; He, H. Z.; Ma, V. P.; Zhong, H. J.; Chan, D. S.; Zhou, J.; Mergny, J. L.; Leung, C. H.; Ma, D. L. Chem. Commun. 2013, 49, 5630-5632. (33) Du, W.; Li, J.; Xiao, F.; Yu, R.; Jiang, J. Anal. Chim. Acta 2017, 991, 127-132. (34) Xiang, Y.; Lu, Y. Anal. Chem. 2012, 84, 9981-9987. (35) Zhang, L.; Zhao, J.; Jiang, J.; Yu, R. Chem. Commun. 2012, 48, 8820-8822. (36) Zhang, Y.; Wang, X.-Y.; Zhang, Q.; Zhang, C.-Y. Anal. Chem. 2017, 89, 12408-12415. (37) Bulgar, A. D.; Weeks, L. D.; Miao, Y.; Yang, S.; Xu, Y.; Guo, C.; Markowitz, S.; Oleinick, N.; Gerson, S. L.; Liu, L. Cell Death Dis. 2012, 3, e252. (38) Priet, S.; Gros, N.; Navarro, J. M.; Boretto, J.; Canard, B.; Que´rat, G.; Sire, J. Mol. Cell 2005, 17, 479-490. (39) Tao, J.; Song, P. S.; Sato, Y.; Nishizawa, S.; Teramae, N.; Tong, A. J; Xiang, Y. Chem. Commun. 2015, 51, 929–932. (40) Wu, Y.; Yan, P.; Xu, X.; Jiang, W. Analyst 2016, 141, 17891795.
ACS Paragon Plus Environment
Page 7 of 7 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Analytical Chemistry
For TOC only
ACS Paragon Plus Environment
7
