Autonomous Exonuclease III-Assisted Isothermal Cycling Signal

Sep 7, 2014 - Caili Fu , Hao Yu , Lingshan Su , Chang Liu , Yanling Song , Shaoyun Wang ... Juan Hu , Zi-yue Wang , Chen-chen Li , Chun-yang Zhang...
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Autonomous Exonuclease III-Assisted Isothermal Cycling Signal Amplification: A Facile and Highly Sensitive Fluorescence DNA Glycosylase Activity Assay Xiuzhong Wang,† Ting Hou,† Tingting Lu,‡ and Feng Li*,† †

College of Chemistry and Pharmaceutical Sciences, Qingdao Agricultural University, Qingdao 266109, People’s Republic of China College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, People’s Republic of China

‡

ABSTRACT: One common form of DNA damage is the oxidation of guanine to 8-oxo-7,8-dihydroguanine (8-oxoG), which can be carcinogenic. Human 8-oxoguanine DNA glycosylase (hOGG1) is a key base excision repair (BER) enzyme that repairs 8-oxoG, and the expression level of hOGG1 is closely related to many types of human cancers. Herein, a novel and highly sensitive fluorescence biosensing platform for hOGG1 activity detection has been constructed based on autonomous exonuclease III (Exo III)-assisted signal amplification. Two hairpin probes (HP1 and HP2) are ingeniously designed. In the presence of hOGG1, HP1 is cleaved at the 8-oxoG site, and the stem is subsequently digested by Exo III, releasing the trigger DNA fragment (tDNA1). Successively, tDNA1 partially hybridizes with HP2 to initiate the Exo III-assisted cycling cleavage to release another trigger DNA fragment (tDNA2), which in turn triggers the cycling cleavage of DNA fluorescence probe (FP). Therefore, large amount of fluorophore fragments are released, leading to a significantly amplified fluorescence signal toward hOGG1 activity detection. A directly measured detection limit down to 0.001 U/mL is obtained, which is much lower than that of the approaches reported in literature. In addition to high sensitivity and good selectivity, the as-proposed strategy also exhibits the advantages of isothermal experimental condition, simplicity, and convenience. Furthermore, the Exo III-assisted autonomous cycling cleavage approach we proposed here is a universal sensing strategy and has great potential in assays of many other biological analytes.

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hOGG1 is closely related to many types of human cancers, including lung cancer, gastric cancer, gallbladder cancer and bladder cancer.14−18 Traditional methods for hOGG1 assay, including polymerase chain reaction-restriction fragment length polymorphism (PCR-RELP) assay, enzyme-linked immunosorbent assay (ELISA), radioactive labeling and HPLC are usually adopted.14−19 These methods, however, suffer from the intrinsic drawbacks of potential radioactive hazards, sophisticated instrumentation, and complicated and time-consuming procedures. To overcome these limitations, a few sensitive and facile hOGG1 activity assays based on colorimetric strategies have been developed.20,21 Liu et al. designed a colorimetric assay for hOGG1 activity based on the self-assembly of the active HRPmimicking DNAzyme coupled with lambda exonuclease cleavage.20 Wu et al. developed a novel activity-based probe, and realized the colorimetric assay of hOGG1 activity.21 Although the aforementioned colorimetric methods have achieved great advances toward the hOGG1 activity assay,

aintaining the genome integrity is crucial for all living organisms. However, DNA is vulnerable to damage from a variety of sources, both extrinsic and intrinsic to cells, and DNA damage can threaten the genetic integrity by increasing the frequency of mutations and exacerbating replication errors unless the damage is repaired.1 One of the most common forms of DNA damage is the oxidation of guanine to 8-oxo-7,8dihydroguanine (8-oxoG) by reactive oxygen species, or even as a byproduct of normal cellular metabolism.2,3 The formation of 8-oxoG leads to a permanent alteration of the DNA base sequence, and during replication it will eventually cause the G:C to T:A transversion mutation, which is one common somatic mutation in human cancers.4−8 Human cells have the ability to repair damaged DNA, and the base excision repair (BER) pathway is one of the most important cellular protection mechanisms that responds to oxidative DNA damage.9,10 Human 8-oxoG DNA glycosylase 1 (hOGG1) is a key BER enzyme that repairs 8-oxoG by specifically catalyzing the removal of 8-oxoG bases from 8-oxoG:C base pairs within DNA.2,11,12 Subsequently, the original G:C pair is restored via the trimming of the sugar fragment, nucleotide insertion and ligation.13 hOGG1 acts both as a DNA N-glycosylase and an apurinic/apyrimidinic (AP)-lyase. The expression level of © 2014 American Chemical Society

Received: June 9, 2014 Accepted: September 5, 2014 Published: September 7, 2014 9626

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Table 1. Sequences of the Oligonucleotides Used in the Experimentsa

a

In HP1, Goxo indicates the guanine modified with 8-oxoG, and the boldface letters represent the sequence of tDNA1. Unmodified-HP1 has the same sequence as HP1 except that the guanine marked in italics was not modified with 8-oxoG. The singly underlined letters in HP1 (UnmodifiedHP1) and HP2, and the doubly underlined letters in HP2 and FP represent the sequences complementary to each other, respectively.

the best of our knowledge, it is the first time to realize highly sensitive fluorescence turn-on assay of hOGG1 activity based on Exo III-assisted signal amplification. Furthermore, as a universal sensing strategy, the Exo III-assisted autonomous cycling cleavage method we proposed here has great potential in the assays of many other biological analytes.

further improvement of the analytical performances, particularly sensitivity, is still in high demand. Fluorescence is a highly sensitive analytical tool, and recently fluorescence turnon assays have been developed for the detection of different proteins and enzyme activities.22−24 For example, Zhu and coworkers developed a novel binding-induced fluorescence turnon assay for protein detection based on affinity binding-induced DNA hybridization and fluorescence enhancement of silver nanoclusters.22 Yu’s group demonstrated a novel strategy for fluorescence turn-on assay of acetylcholinesterase activity based on the in situ formation of metal coordination polymers.23 Although fluorescence turn-on assays exhibit high sensitivity and excellent selectivity, so far there is no fluorescence assay being reported for the detection of hOGG1 activity. Exonuclease III (Exo III) is a nuclease that selectively catalyzes the stepwise hydrolysis of mononucleotides from the blunt or the recessed 3′-hydroxyl termini of duplex DNA.25 Since no specific recognition sequences are required, Exo IIIassisted signal amplification strategy is very suitable for constructing universal biosensing platforms, and has recently been applied in the detection of nucleic acids, DNA-binding proteins, enzyme activity, and metal ions.26−33 For example, Qu and co-workers developed a label-free fluorescence turn-on assay for DNA detection based on Exo III-assisted amplification and aggregation-induced quenching.27 Zhang et al. reported an ultrasensitive fluorescence polarization DNA assay based on target assisted Exo III catalyzed signal amplification strategy.28 Willner and co-workers developed an Exo III-based aptasensor for the amplified detection of thrombin.29 Hsing’s group demonstrated an ultrasensitive electrochemical method for Hg2+ detection utilizing the conformation-dependent activity of Exo III.30 Herein, we developed a facile and isothermal Exo III-assisted signal amplification strategy for highly sensitive fluorescence turn-on detection of hOGG1 activity. Upon the scission of a hairpin probe (HP1) at the 8-oxoG site by hOGG1 and the successive digestion by Exo III, a trigger DNA fragment (tDNA1) originally caged in HP1 is released and it initiates the Exo III-assisted cycling cleavage to release another trigger DNA fragment (tDNA2) caged in another hairpin probe (HP2), which in turn triggers the cycling cleavage of the DNA fluorescence probe (FP). Eventually, large amount of the fluorophore fragments are released from FP, resulting in a significantly amplified fluorescence signal toward hOGG1 activity detection. By taking full advantage of the unique features of Exo III and the high sensitivity of fluorescence strategy, the as-proposed method can provide a facile and convenient biosensing platform for fluorescence assay of hOGG1 activity with high sensitivity and good selectivity. To

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EXPERIMENTAL SECTION Reagents. Dithiothreitol (DTT) was purchased from Shanghai Generay Biotech Co., Ltd. (Shanghai, China). NaCl, MgCl2, KCl, and [tris(hydroxymethyl)aminomethane] (Tris) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Exonuclease III (Exo III) was purchased from Thermo Fisher Scientific (China) Co., Ltd. Human 8oxoguanine DNA glycosylase (hOGG1) and bovine serum albumin (BSA) were bought from New England Biolabs, Ltd. (Ipswich, MA, USA). Escherichia coli uracil DNA glycosylase (UDG) and 10× UDG reaction buffer (200 mM Tris-HCl, 10 mM EDTA, 100 mM NaCl, pH 8.2) were purchased from Fermentas China Co., Ltd. All oligonucleotides used in this work were synthesized and purified by Shanghai Sangon Biotechnology Co., Ltd. (Shanghai, China), and the sequences of the oligonucleotides are listed in Table 1. Other reagents were all of analytical grade and used without further purification. Doubly distilled water (DDW) was used through the experiments. Instrument. Fluorescence measurements were performed using a Hitachi F-4600 spectrofluorimeter (Tokyo, Japan) with a scan rate of 1200 nm/min. The excitation wavelength was set to 490 nm, and the 24 photomultiplier tube voltage was set to 700 V. The slits for excitation and emission were set at 5 nm/5 nm. Preparation of DNA Stock Solutions. All oligonucleotides were used as provided and diluted in 10 mM Tris-HCl buffer (50 mM NaCl, 10 mM MgCl2, pH 7.9) to give the stock solutions. The concentrations of HP1 and Unmodified-HP1 stock solutions were 2 μM, the concentration of HP2 stock solution was 4 μM, and the concentration of FP stock solution was 8 μM. Each oligonucleotide was pretreated with a procedure of heat incubation at 90 °C for 2 min and then slowly cooled down to room temperature. The obtained DNA stock solutions were stored at 4 °C for further use. Exonuclease III-Assisted Fluorescent hOGG1 Activity Assay. In this assay, 5 μL of HP1, various amounts of hOGG1, 6 μL of exonuclease III (30 U), 5 μL of HP2 and 5 μL of FP were added into the reaction buffer (10 mM Tris-HCl, 50 μM NaCl, 10 mM MgCl2, 1 mM DTT, pH 7.9) to give a total volume of 100 μL, and the final concentrations of HP1, HP2, and FP were 0.1, 0.2, and 0.4 μM, respectively. The above 9627

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Scheme 1. Schematic Illustration of hOGG1 Activity Assay Based on Exo III-Assisted Signal Amplification

solutions were incubated at 37 °C for 2 h before fluorescence measurements. The control experiments were carried out under the same condition without adding hOGG1. All experiments were repeated three times.

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released to achieve the significant fluorescence signal amplification. In contrast, in the absence of hOGG1, the stem of HP1 modified with 8-oxoG cannot be selectively cleaved. So the protruding 3′-termini of HP1 remained unchanged, and the subsequent Exo III-assisted cleavage processes could not proceed, resulting in weak fluorescence background signal. It is noteworthy that we have made full use of Exo III in this strategy by applying this single tool enzyme in three different cleavage processes, namely the cleavage of HP1, and the subsequent cycling cleavage of HP2 and FP, to result in a significantly amplified fluorescence signal. Since only one tool enzyme was adopted and its reaction condition was similar to that of hOGG1, the reaction could be carried out in one-pot fashion under isothermal condition, making the as-proposed assay very simple and very convenient to implement. Therefore, this facile biosensing platform is a good candidate for highly sensitive fluorescence determination of hOGG1 activity. Feasibility Study of hOGG1 Activity Assay. The proofof-concept experiments were carried out to test the feasibility of the proposed fluorescence strategy for hOGG1 activity detection. Figure 1 depicts the fluorescence emission spectra of the biosensing platform in response to hOGG1 and in the control experiments, respectively. In FP, the fluorescence of FAM at the 5′ end was quenched by BHQ1 at the 3′ end through the fluorescence resonance energy transfer (FRET) effect, so in the presence of FP, HP1, and HP2, a very low fluorescence intensity was observed (curve a), and the spectrum was almost the same as that when only FP, or both FP and HP2 were present (data not shown here). When hOGG1 was added into the solution containing FP, HP1 and HP2, no appreciable increase of the fluorescence intensity was observed (curve b). But, after Exo III was added into the solution containing HP2 and FP, the fluorescence intensity exhibited a slight enhancement (curve c), which may be resulted from the Exo III digestion of small amount of duplex DNA formed between HP2 and FP. This explanation could also be applied to the result of the control experiment in which only hOGG1 was absent, and slightly higher fluorescence intensity than in curve c was observed (curve d). However, in the presence of hOGG1, HP1, HP2, FP, and Exo III, significant fluorescence enhancement was observed (curve f), indicating that HP1 was specifically recognized and selectively cleaved at the 8-oxoG site by hOGG1 to initiate the digestion of the stem of HP1 by

RESULTS AND DISCUSSION

Principle of Fluorescent hOGG1 Activity Assay. Our strategy for highly sensitive hOGG1 activity detection was based on the specific recognition and removal of 8-oxoG by hOGG1 and the subsequent Exo III-assisted fluorescence signal amplification. As shown in Scheme 1, an ingeniously designed hairpin probe (HP1) with protruding 3′-terminus was modified with one 8-oxoG in the stem region. In the presence of hOGG1, HP1 was selectively cleaved by hOGG1 at the 8-oxoG site, resulting in a new hairpin DNA with recessed 3′-terminus. The stem of the resultant hairpin DNA was then specifically hydrolyzed by Exo III in the direction from 3′ to 5′, releasing the trigger DNA (tDNA1, in red) originally caged in HP1. tDNA1 was complementary with the green region of another hairpin DNA probe (HP2), and it could open HP2 through the toehold-mediated strand displacement reaction (SDR) to form a new duplex DNA with blunt 3′-terminus, which was again digested by Exo III in the direction from 3′ to 5′, thus releasing both tDNA1 and the second trigger DNA (tDNA2, in blue) originally caged in HP2. During this process, tDNA1 maintained its complete sequence because its 3′-termini remained in the protruding condition to prevent the digestion by Exo III. Then the released tDNA1 participated in another SDR and the cycling cleavage process (cycle I), while the released tDNA2 could partially hybridize with the DNA fluorescence probe (FP), in which the fluorescence of the fluorophore (FAM) at one end was quenched by the quencher (BHQ1) at the other end due to the fluorescence resonance energy transfer (FRET) effect. In the resultant duplex DNA, FP with the recessed 3′-termini was selectively digested by Exo III, but tDNA2 with the protruding 3′-termini remained intact. As a consequence, the fluorophore in FP was separated from the quencher to restore its fluorescence signal, and tDNA2 was released to hybridize with another FP to initiate the subsequent cycling cleavage process (cycle II). On the basis of the autonomous cleavage in cycles I and II, large amount of fluorophore fragments originally contained in FP could be 9628

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Figure 1. Fluorescence emission spectra for the biosensing platform containing: (a) HP1, HP2, and FP; (b) HP1, HP2, FP, and hOGG1; (c) HP2, FP, and Exo III; (d) HP1, HP2, FP, and Exo III; (e) unmodified-HP1, HP2, FP, hOGG1, and Exo III; (f) HP1, HP2, FP, hOGG1, and Exo III. The concentrations of hOGG1, Exo III, HP1 (unmodified-HP1), HP2, and FP were 1 U/mL, 30 U, 0.1 μM, 0.2 μM and 0.4 μM, respectively.

Exo III and the subsequent autonomous Exo III-assisted cleavage processes. To further demonstrate the specific recognition of the 8-oxoG site by hOGG1, a comparative experiment was conducted by substituting unmodified-HP1 (without 8-oxoG in the sequence) for HP1. The corresponding fluorescence intensity (curve e) was comparable with that with HP1, HP2, FP, and Exo III present in curve d, which indirectly verified the Exo III-assisted signal amplification effect of the proposed biosensing strategy. The aforementioned results clearly demonstrated the feasibility of the proposed fluorescence biosensing platform for hOGG1 activity detection. Optimization of Experimental Conditions. As illustrated in Scheme 1, in the presence of hOGG1, tDNA1 was released to trigger cycle I and then the subsequent cycle II. To ensure high sensitivity for the biosensing system, sufficient amount of HP2 and FP needed to be added into the reaction system to carry out enough rounds of the cycles. Also, as shown in Figure 1d, in the absence of hOGG1, an obvious background signal was observed, which could be resulted from the Exo IIIcatalyzed digestion of small amount of duplex DNA formed between different DNA probes. Therefore, to obtain the best performance of the biosensing system, the amount of Exo III and the reaction time for hOGG1 induced scission and Exo III digestion were optimized in the presence of 1 U/mL hOGG1, respectively. As shown in Figure 2A, at a reaction time of 2 h, the background signal increased as the Exo III amount increased, but the fluorescence signal initially increased and then fluctuated at higher amount of Exo III. As a result, the signal to background ratio (S/N) reached the maximum value at the Exo III amount of 30 U. As shown in Figure 2B, in the presence of 30 U of Exo III, both the fluorescence signal and the background signal increased as the reaction time increased, but the signal to background ratio (S/N) reached maximum at the reaction time of 2 h. Thus, 30 U of Exo III and 2 h of reaction time were chosen as the optimal experimental conditions, and used in the subsequent experiments. Fluorescence Detection of hOGG1 Activity. Under the optimal experimental conditions, the analytical performance of the proposed fluorescence biosensing platform was investigated

Figure 2. (A) Fluorescence intensity versus the Exo III amount in the absence (a) or presence (b) of 1 U/mL hOGG1, respectively, and the signal-to-noise ratio (S/N) versus the Exo III amount. (B) The fluorescence intensity versus the reaction time in the absence (a) or presence (b) of 1 U/mL hOGG1, respectively, and the signal-to-noise ratio (S/N) versus the reaction time.

by varying the concentration of hOGG1. As shown in Figure 3, the fluorescence intensity increased as the hOGG1 concentration increased from 0 to 1 U/mL, which demonstrated that the release of FAM fragments from FPs assisted by Exo III was highly dependent on the concentration of hOGG1. As shown in the inset of Figure 3, a good linear relationship between the fluorescence intensity change, F − F0 (in which F0 and F are the fluorescence intensity detected in the absence and presence of hOGG1, respectively) and the logarithm of hOGG1 concentration ranging from 0.001 to 1 U/mL was obtained, with a regression equation of (F − F0) = 1125.66 + 351.549·log C and a correlation coefficient of R2 = 0.9951. The directly measured limit of detection for hOGG1 activity was as low as 0.001 U/ mL, one or 2 orders of magnitude lower than that of the colorimetric methods (Table 2). The results indicated that the signal amplification effect of the proposed Exo III-assisted recycling strategy was indeed realized. In addition, the repeatability of the hOGG1 activity biosensor was investigated through 5 successive assays in the presence of 0.05 U/mL of hOGG1. The relative standard deviation (RSD) was determined to be 2.82%, indicating an acceptable repeatability of the as-proposed strategy. Selectivity of hOGG1 Activity Assay. The selectivity of the as-proposed method was further evaluated by adding a nonspecific protein bovine serum albumin (BSA), another BER 9629

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Figure 4. Comparison of the fluorescence intensity of the biosensing platform in the presence of BSA, UDG, and hOGG1, where F and F0 are the fluorescence intensities (at the emission frequency of 525 nm) of the biosensor in the presence and absence of proteins, respectively. The concentrations of BSA, UDG, and hOGG1 were 0.05 U/mL. The error bars represent the standard deviation of three measurements.

Figure 3. Fluorescence emission spectra of the biosensing system upon the addition of hOGG1 with different concentrations: (a) 0 (control), (b) 0.001, (c) 0.005, (d) 0.01, (e) 0.05, (f) 0.1, (g) 0.5, and (h) 1 U/mL. Inset: the linear relationship between the fluorescence intensity change (F − F0) and the logarithm of hOGG1 concentration ranging from 0.001 to 1 U/mL. The error bars represent the standard deviation of three measurements.

autonomous cycling cleavage method we proposed here can be used as a universal sensing strategy, and has great potential in the assays of many other biological analytes.

Table 2. Comparison of Analytical Performance for hOGG1 Activity Detection by Our Strategy and Those Reported in Literature method

detection limit (U/mL)

colorimetry

0.01

colorimetry

0.7

fluorescence

0.001

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AUTHOR INFORMATION

Corresponding Author strategy self-assembly of DNAzyme coupled with lambda exonuclease cleavage terminal protection of DNA−gold nanoparticle probes by covalent trapping of target enzymes Exo III-assisted signal amplification

*Tel: 86-532-86080855. Fax: 86-532-86080213. E-mail: [email protected].

ref 20

Author Contributions 21

X.Z.W and T.H. contributed equally to this work. Notes

The authors declare no competing financial interest.

This work

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Nos. 21375072 and 21175076), the Open Foundation of State Key Laboratory of Electroanalytical Chemistry (No. SKLEAC201402), and the Research Foundation for Distinguished Scholars of Qingdao Agricultural University (No. 631311).

enzyme uracil DNA glycosylase (UDG), and hOGG1 into the reaction system, respectively, and all proteins had the same concentration of 0.05 U/mL. The results are shown in Figure 4, where F and F0 are the fluorescence intensities of the biosensing platform in the presence and absence of proteins, respectively. High fluorescence signal was obtained only when the specific protein (hOGG1) was present, whereas in the presence of BSA or UDG the fluorescence signal was fairly small and comparable to that in the control experiment. Thus, the strategy we proposed here exhibited good performance for discriminating hOGG1 against other interfering proteins, and held great potential in clinical applications.

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CONCLUSIONS In summary, we have successfully developed a novel and facile fluorescent biosensing strategy for highly sensitive detection of hOGG1 activity based on autonomous Exo III-assisted signal amplification. This homogeneous sensing system exhibits good selectivity and very high sensitivity for hOGG1 activity assay, and a low detection limit of 0.001 U/mL is obtained, which is much lower than that of the colorimetric methods previously reported in literature. In addition to high sensitivity and good selectivity, the as-proposed strategy also exhibits other advantages such as isothermal experimental condition, simplicity and convenience. Furthermore, the Exo III-assisted 9630

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