Homogeneously Sensitive Detection of Multiple DNA Glycosylases

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Homogeneously Sensitive Detection of Multiple DNA Glycosylases with Intrinsically Fluorescent Nucleotides Yan Zhang, Chen-chen Li, Bo Tang, and Chun-yang Zhang Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 16 Jun 2017 Downloaded from http://pubs.acs.org on June 16, 2017

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Analytical Chemistry

Homogeneously

Sensitive

Detection

of

Multiple

DNA

Glycosylases with Intrinsically Fluorescent Nucleotides Yan Zhang,† Chen-chen Li,† Bo Tang* and Chun-yang Zhang* College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals, Shandong Normal University, Jinan 250014, China. * Corresponding author. Tel.: +86 0531-86186033; Fax: +86 0531-82615258. E-mail: [email protected].

Tel.:

+86

0531-86180010;

Fax: +86

[email protected].

1

ACS Paragon Plus Environment

0531-86180017;


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ABSTRACT: DNA glycosylases are responsible for recognition and excision of the damaged bases in the base excision repair pathway, and all mammals express multiple DNA glycosylases to maintain genome stability. However, simultaneous detection of multiple DNA glycosylase still remains a great challenge. Here, we develop a rapid and sensitive fluorescent method for simultaneous detection of uracil DNA glycolase (UDG) and human 8-oxoG DNA glycosylase 1 (hOGG1) using exonuclease-assisted recycling signal amplification in combination with fluorescent bases 2-aminopurine (2-AP) and pyrrolo-dC (P-dC) as the fluorophores. We design a bifunctional DNA probe modified with one 8-oxoG and five uracil bases, which can hybridize with the trigger probes to form a sandwiched DNA substrate for hOGG1 and UDG. In addition, we design 2-AP and P-dC signal probes as the hairpin structures with 2-AP and P-dC in the stems. The presence of hOGG1 and UDG may initiate the signal amplification process by the recycling lambda exonuclease digestion and generates distinct fluorescence signals, with 2-AP indicating the presence of hOGG1 and P-dC indicating the presence of UDG. This method can simultaneously detect multiple DNA glycosylases with the detection limits of 0.0035U/mL for hOGG1 and 0.0025U/mL for UDG, and it can even measure DNA glycosylases at the single-cell level. Moreover, this method can be applied for the measurement of enzyme kinetic parameters and the screening of DNA glycosylase inhibitors, holding great potential for further applications in biomedical research and clinical diagnosis.

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INTRODUCTION Endogenous DNA damage is largely a consequence of various endogenous and environmental agents, and it may initiate carcinogenesis as a result of mutations and replication errors.1,2 Base excision repair (BER) is the primary DNA repair pathway that can deal with endogenous DNA base damage resulting from oxidative, alkylation and deamination.2 The BER pathway is initiated by DNA glycosylases that catalyze the cleavage of damaged / mismatched bases and generate an apurinic/apyrimidinic site for the downstream BER repair processes.3 Aberrant expression of glycosylases has profound implication in a variety of human diseases.4-11 For example, abnormal uracil DNA glycolase (UDG) activity is closely associated with cancer, aging and neurodegenerative diseases.5-7 Dysregulation of human 8-oxoG DNA glycosylase 1 (hOGG1) is associated with the risk of many types of human cancers including lung, gallbladder, gastric and bladder cancers.8-11 The DNA glycosylases have become important biomarkers and potential therapeutic targets for related diseases.12 Therefore, accurate detection of DNA glycosylase is very important for understanding the DNA damage repairing process and clinical diagnosis. Classic strategies for DNA glycosylases assay include radioactive labeling,13, electrophoresis,15 chromatography11,

16

14

gel

and streptavidin paramagnetic bead capturing

techniques.17 But they are usually labor-consuming with low sensitivity15 and restrained by the requirement of costly labeling reagents,13,14 sophisticated instrumentation11,16 and complicated procedures.17 To overcome these issues, fluorescent methods are introduced due to their distinct advantages of safety, simplicity and high sensitivity.18-21 Nevertheless, fluorescent methods rely on external labeling with a fluorophore and a quencher for homogeneous assay, 3



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which is very expensive with the involvement of complicated design. In addition, the reported fluorescent methods enable the detection of only a single type of DNA glycosylases.18-21 In fact, all mammals express multiple DNA glycosylases to maintain genome stability, each one with unique substrate specificity.2 DNA glycosylases fall into two classes: monofunctional DNA glycosylases and bifunctional DNA glycosylases.22 Monofunctional DNA glycosylases catalyze one-step removal of the lesion base to generate an abasic site.22 Bifunctional DNA glycosylases not only excise the substrate base but also possess an associated AP-lyase activity that hydrolyzes the 3′-phosphodiester bond of the abasic site.22 BER facilitates the repair of the damaged DNA via monofunctional and bifunctional DNA glycosylases to maintain genome stability necessary for all mammals.2 Among them, human 8-oxoG DNA glycosylase 1 (hOGG1) is a type of bifunctional DNA glycosylases that is able to remove the damaged 8-hydroxyguanine (8-oxoG) bases from 8-oxoG/C base pairs within the DNA substrate and hydrolyze the 3′-phosphodiester bond of the abasic site,22, 23,24 while uracil DNA glycolase (UDG) is a type of monofunctional DNA glycosylases that is able to remove uracil from DNA through catalyzing the hydrolysis of the N-glycosidic bond between the deoxyribose and uracil base for the generation of an abasic site.25 Both hOGG1 and UDG take part in the BER pathway and deal with endogenous DNA base damage,2 while aberrant expression of hOGG1 and UDG has profound implication in many types of human cancers.5,8-11 Therefore, simultaneous detection of multiple DNA glycosylase may bring new opportunities for understanding the DNA damage repairing process and improving the accuracy of early clinical diagnosis. In this research, we use hOGG1 and UDG as the models to demonstrate the development 4


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of a rapid and sensitive fluorescent method for simultaneous detection of multiple DNA glycosylases using exonuclease-assisted recycling signal amplification in combination with fluorescent bases 2-aminopurine (2-AP) and pyrrolo-dC (P-dC) as the fluorophores. With DNA molecules as the intrinsic quenchers, some fluorescent nucleotide analogs such as 2-AP and P-dC exhibit weak fluorescence when being incorporated into the double-stranded DNA (dsDNA) owing to an efficient stacking interaction among the bases, but an enhanced fluorescence when being free in the solution.26-29 In contrast to the conventional molecular beacons, the quenching of 2-AP and P-dC fluorescence results from a more efficient stacking interaction with the neighbor bases, avoiding the use of a relatively large organic quencher externally attached to the oligonucleotide strand.28 Moreover, the fluorophores of 2-AP and P-dC can be attached at any position of the signal probe,28 with no significant spectral overlap and cross-talk between the emission spectrum of 2-AP (365 nm) and that of P-dC (450 nm) (see Supporting Information, Figure S1). We design a bifunctional DNA probe modified with one 8-oxoG and five uracil bases, which can hybridize with the trigger probes to form a sandwiched DNA substrate for hOGG1 and UDG, enabling the discrimination of hOGG1 and UDG from other DNA glycosylases. The presence of hOGG1 and UDG may initiate the signal amplification process by the recycling lambda exonuclease digestion and generates distinct fluorescence signals, with 2-AP indicating the presence of hOGG1 and P-dC indicating the presence of UDG. The proposed method can detect multiple DNA glycosylases simultaneously with high sensitivity and good selectivity, and it can even measure DNA glycosylases at the single-cell level.

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EXPERIMENTAL SECTION Materials. All oligonucleotides (Table 1) were synthesized by Takara Biotechnology Co. Ltd. (Dalian, China). Human 8-oxoguanine-DNA glycosylase 1 (hOGG1), 10× NEB buffer 2 (500 mM sodium chloride (NaCl), 100 mM trizma hydrochloride, 100 mM magnesium chloride (MgCl2), 10 mM DL-Dithiothreitol (DTT), pH 7.9), 10 mg/mL bovine serum albumin (BSA), uracil DNA glycosylase (UDG), 10× UDG reaction buffer (200 mM Tris-HCl, 10 mM EDTA, 10 mM DTT, pH 8.0), the nicking enzymes of Nb.BtsI, 10× CutSmart buffer, lambda exonuclease, and 10× lambda buffer (670 mM Glycine-KOH, 25 mM MgCl2, 50 µg/mL BSA, pH 9.4) were obtained from New England BioLabs (Beverly, MA, USA). Thymine DNA Glycosylase (TDG) was bought from R&D System (Minneapolis, MN, USA). Magnesium chloride (MgCl2), ethylenediaminetetraacetic acid (EDTA), trizma hydrochloride (Tris-HCl, pH 8.0), sodium chloride (NaCl) and 5-fluorouracil (5-FU) were obtained from Sigma-Aldrich Company (St. Louis, MO, USA). SYBR Gold was purchased from Invitrogen Corporation (California, CA, USA). Nuclear extract kit was brought from Active Motif (Carlsbad, CA, USA). All other reagents were of analytical grade and used without further purification. Ultrapure water was prepared by a Millipore filtration system (Millipore, Milford, MA, USA).

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Table 1. Sequences of the Oligonucleotidesα note

sequence (5′-3′)

bifunctional DNA probe

T*T*A TGC AAC GGA CAA CAA GTT TTT TTT ATG CCU UAC UUA GCU TC

trigger 1

A*T*C TTG TTG TCC GTT GCA GTG AGT T

trigger 2

T*G*G AAG CTA AGT AAG GCA GTG TAT A

2-AP signal probe

A*A*A ACT↓CAC TGC AAC GGA CAA CAATCC GTT GCA G

P-dC signal probe

A*A*T ATA↓CAC TGC CTT ACT TAG CTT AGT AAG GCA G

α

The asterisks indicate the phosphorothioate modifications. The bold G and U bases in the

bifunctional

DNA

probe

indicate

the

damaged

guanine

(8-oxoG)

and

uracil

deoxyribonucleotide modification, respectively. The underlined region and the underlined italic region of bifunctional DNA probe indicate the binding sequence of trigger 1 and trigger 2, respectively. The underlined bold region of trigger 1 and the underlined italic bold region of trigger 2 indicate the binding sequence of bifunctional DNA probe. The arrows in 2-AP signal probes and P-dC signal probes indicate the nicking positions of Nb.BtsI. The bold regions of 2-AP signal probes and P-dC signal probes represent the complementary regions within the hairpin structure probes.

Preparation of DNA Stock Solutions. The 10 µM bifunctional DNA probes, 10 µM trigger probe 1 and 10 µM trigger probe 2 were incubated in a buffer containing 50 mM NaCl , 10 7



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mM Tris-HCl (pH 8.0) and 1 mM EDTA at 95 °C for 5 min, followed by slowly cooling to the room temperature to form the sandwiched DNA substrates. The 10 µM 2-AP signal probes and 10 µM P-dC signal probes were incubated in a buffer containing 1.5 mM MgCl2 and 10 mM Tris-HCl (pH 8.0), respectively, at 95 °C for 5 min and then slowly cooled to the room temperature to make 2-AP signal probes and P-dC signal probes fold into hairpin structures. The obtained DNA stock solutions were stored at -20 °C for further use. Fluorescence Measurement of hOGG1 and UDG. The detection of hOGG1 and UDG involves two consecutive steps. Firstly, 0.4 µL of DNA substrates (10 µM) was added into the excision reaction system (20 µL) containing different-concentration hOGG1 and UDG, 2 µL of 10× NEB buffer 2, 100 µg/mL BSA, 2 µL of 10× UDG reaction buffer and incubated at 37 °C for 60 min. Secondly, 0.8 µL of 2-AP signal probes and P-dC signal probes (10 µM), 3 U Nb.BtsI, 2 U lambda exonuclease, 3 µL of 10× lambda buffer were added into the excision reaction system with a total volume of 30 µL and then incubated at 37 °C in the dark for 60 min. The 30 µL of reaction products were diluted to a final volume of 60 µL with ultrapure water. All fluorescence spectra were measured using a quartz cuvette on a Hitachi F-7000 fluorescence spectrophotometer (Tokyo, Japan). The spectra of 2-AP and P-dC were recorded at the excitation wavelengths of 310 nm and 350 nm, respectively. The fluorescence intensity at the emission wavelengths of 365 nm and 450 nm was used for quantitative analysis of hOGG1 and UDG, respectively. Inhibition

Assay.

For

DNA glycosylase

inhibition

assay,

different-concentration

5-fluorouracil (5-FU) was incubated with the DNA substrates at 25 °C for 15 min, followed 8


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by the addition of hOGG1 and UDG into the solution and incubation at 37 °C for 60 min. The subsequent reactions followed the above steps, and fluorescence intensities of 2-AP and P-dC were measured as described above. The relative activity of DNA glycosylases (RA) was measured according to RA =

Fi − F 0 × 100% , where F0 is the fluorescence intensity in the Ft − F 0

absence of hOGG1 or UDG, and Ft is the fluorescence intensity in the presence of 32 U/mL hOGG1 or 50 U/mL UDG, and Fi is the fluorescence intensity in the presence of both hOGG1 and 5-FU or in the presence of both UDG and 5-FU. The IC50 value was calculated from the curve of RA versus the 5-FU concentration. Cell Culture and Preparation of Cell Extracts. Human cervical carcinoma cell line (HeLa) was cultured in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, USA) supplemented with 10% fetal bovine serum (FBS, Invitrogen, USA) and 1% penicillin-streptomycin. The cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2. The number of cells was measured by Countstar cell counter. The nuclear extracts were prepared using the nuclear extract kit (ActiveMotif, Carlsbad, CA, USA) according to the manufacturer’s protocol.

RESULTS AND DISCUSSION Principle of Fluorescence Monitoring of DNA Glycosylases. With hOGG1 and UDG as the model analytes, we demonstrated for the first time the simultaneous detection of multiple DNA glycosylases on the basis of the specific recognition and removal of damaged bases by DNA glycosylases and the subsequent lambda exonuclease-assisted fluorescence signal amplification. The principle of DNA glycosylase assay is illustrated in Scheme 1. We 9



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designed a bifunctional DNA probe modified with one 8-oxoG and five uracil bases, which can hybridize with the trigger probe 1 and the trigger probe 2 to form a sandwiched DNA substrate for hOGG1 and UDG. Both the trigger probe 1 and the trigger probe 2 have 3′-protruding termini that contain a recognition site for Nb.BtsI nicking enzyme (Scheme 1, purple color). Especially, the 5′ end of the probes is modified with phosphorothioate to prevent the nonspecific digestion of lambda exonuclease. We designed the 2-AP signal probe and the P-dC signal probe as the hairpin structure which consists of a loop and a stem with a 5′-protruding terminus, with 2-AP fluorophore and P-dC fluorophore being incorporated into the stem, respectively. Both the 2-AP signal probe and the P-dC signal probe exhibit weak fluorescence due to an efficient stacking interaction among the bases, but an enhanced fluorescence when being free in the solution. 28,29 The 5′-protruding termini of both the 2-AP signal probe and the P-dC signal probe contain a recognition site for Nb.BtsI nicking enzyme (Scheme 1, purple color), which may be recognized by Nb.BtsI nicking enzyme upon the formation of a double-stranded DNA (dsDNA). The proposed assay involves two steps: (1) hOGG1-induced 8-oxoG excision repair and UDG-induced uracil excision repair, (2) lambda exonuclease-mediated cyclic cleavage of signal probes and the liberation of fluorescence signal. The presence of hOGG1 may promote the removal of the damaged 8-oxoG base from 8-oxoG/C base pairs in the DNA substrate and subsequently hydrolyze the 3′-phosphodiester bond of the abasic site, resulting in the dissociation of bifunctional probe-trigger probe 1 hybrid and the release of trigger probe 1. The released trigger probe 1 may hybridize with the 2-AP signal probe to form a new stable dsDNA with the recognition sites for Nb.BtsI nicking enzyme. The subsequent cleavage of 10


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2-AP signal probes by Nb.BtsI nicking enzyme may yield the site of phosphate group (PO4) for the lambda exonuclease digestion. The digestion of the DNA duplex by lambda exonuclease releases the free 2-AP molecules and trigger probe 1 which can further hybridize with new signal probes to initiate new cycles of nicking-digestion-hybridization, generating abundant free 2-AP molecules for significant fluorescence enhancement at the emission wavelength of 365 nm. Similarly, the presence of UDG may promote the removal of five uracil bases in the DNA substrate, resulting in the dissociation of the bifunctional probe-trigger probe 2 hybrids and the release of trigger probe 2. The released trigger probe 2 can

hybridize

with

the

P-dC

signal

probe

and

initiate

the

cycle

of

nicking-digestion-hybridization, inducing significant fluorescence enhancement at the emission wavelength of 450 nm. When both hOGG1 and UDG are present, one 8-oxoG and five uracil bases may be removed from the DNA substrate by hOGG1 and UDG, respectively, leading to the release of trigger probe 1 and trigger probe 2. The released trigger probe 1 and trigger probe 2 may hybridize with 2-AP signal probe and P-dC signal probe, respectively, initiating the signal amplification process and generating significant fluorescence enhancement at the emission wavelengths of 365 nm and 450 nm simultaneously. While in the absence of hOGG1 and UDG, neither 8-oxoG base nor uracil bases can be removed, and neither trigger probe 1 nor trigger probe 2 is released. As a result, neither nicking nor digestion reaction may occur, and no significant fluorescence enhancement is observed for 2-AP and P-dC.

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Scheme

1.

Schematic

illustration

of

multiple

DNA

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glycosylases

assay

using

exonuclease-assisted recycling signal amplification in combination with fluorescent bases 2-AP and P-dC as the fluorophores

To demonstrate the feasibility of the proposed method for DNA glycosylases assay, we performed fluorescence measurement. In the presence of hOGG1, a distinct 2-AP fluorescence signal with the characteristic emission peak of 365 nm is observed (Figure 1A, green line), but no P-dC fluorescence signal at the emission wavelength of 450 nm is observed. On the contrary, no significant 2-AP fluorescence signal is observed in the absence of hOGG1 (Figure 1A, black line). These results indicate that hOGG1 can remove the damaged 8-oxoG base from 8-oxoG/C base pairs specifically and releases the trigger probe 1 12


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from the bifunctional probe-trigger probe 1 hybrid, initiating the signal amplification process and generating an enhanced 2-AP fluorescence signal. While in the presence of UDG, only a distinct P-dC fluorescence signal with the characteristic emission peak of 450 nm is observed (Figure 1B, red line), but no significant 2-AP fluorescence signal is observed. On the contrary, no significant P-dC signal is observed in the absence of UDG (Figure 1B, blue line). These results indicate that UDG can specifically excise the uracil from A/U base pairs and release the trigger probe 2 form the bifunctional probe-trigger probe 2 hybrid, initiating the signal amplification process and generating an enhanced P-dC fluorescence signal. Notably, only the co-existence of hOGG1 and UDG may induce the enhancement of both 2-AP and P-dC fluorescence signals simultaneously (Figure 1C). These results clearly demonstrate that the proposed method can be used for simultaneous detection of multiple DNA glycosylases.

Figure 1. (A) Normalized fluorescence emission spectra of 2-AP in the presence (green line) and in the absence (black line) of hOGG1, respectively. (B) Normalized fluorescence emission spectra of P-dC in the presence (red line) and in the absence (blue line) of UDG, respectively. (C) Normalized fluorescence emission spectra of 2-AP and P-dC in the presence and in the absence of hOGG1 and UDG, respectively. The hOGG1 concentration is 32 U/mL, and the UDG concentration is 50 U/mL.

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Optimization of Experimental Conditions. To obtain the optimal reaction condition, we investigated the performance of the designed probes in CutSmart buffer, lambda buffer and the mixture buffer, respectively. As shown in Figure 2A, the value of (F-F0)/F0 in response to lambda buffer is much higher than those in response to CutSmart buffer and mixture buffer, where F and F0 are the fluorescence intensity in the presence and in the absence of DNA glycosylases, respectively. Thus, the lambda buffer is used in the subsequent research. In this assay, the signal amplification relies on the cooperation of Nb.BtsI nicking enzyme and lambda exonuclease, and thus the amounts of Nb.BtsI and lambda exonuclease should be carefully optimized. We investigated the influence of Nb.BtsI upon the fluorescence signal with a fixed amount of lambda exonuclease (5U).30 As shown in Figure 2B, the value of (F-F0)/F0 increases with the increasing amount of Nb.BtsI from 1 to 3 U, and the highest value of (F-F0)/F0 is obtained at 3U Nb.BtsI (F and F0 are the fluorescence intensity in the presence and in the absence of DNA glycosylases, respectively). We further investigated the influence of lambda exonuclease upon the fluorescence signal with a fixed amount of Nb.BtsI (3U). As shown in Figure 2C, the maximum value of (F-F0)/F0 is obtained at 2 U lambda exonuclease (F and F0 are the fluorescence intensity in the presence and in the absence of DNA glycosylases, respectively). Therefore, 3 U Nb.BtsI and 2 U lambda exonuclease are used in the subsequent research. We investigated the influence of reaction time upon the fluorescence signal as well. As shown in Figure 2D, the value of (F-F0)/F0 increases with the reaction time from 30 to 60 min and reaches a plateau at 60 min (F and F0 are the fluorescence intensity in the presence and in the absence of DNA glycosylases, respectively). Thus, the reaction time of 60 min is used in 14


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the subsequent research.

Figure 2. (A) The value of (F-F0)/F0 in response to CutSmart buffer, lambda buffer and mixture buffer, respectively. (B) The value of (F-F0)/F0 in response to different amount of Nb.BtsI nicking enzyme at a fixed amount of lambda exonuclease (5 U). (C) The value of (F-F0)/F0 in response to different amount of lambda exonuclease at a fixed amount of Nb.BtsI (3 U). (D) Variance of (F-F0)/F0 value with the reaction time. F and F0 are the fluorescence signals in the presence and in the absence of 32 U/mL hOGG1 and 50 U/mL UDG, respectively. Error bars show the standard deviation of three experiments.

Sensitivity and Selectivity of DNA glycosylase Assay. To assess the sensitivity of the proposed method, we measured hOGG1 and UDG at various concentrations under the optimally experimental conditions. Figure 3A shows the variance of fluorescence emission 15



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spectra with the concentration of hOGG1. The normalized fluorescence intensity at the emission wavelength of 365 nm improves with the increasing hOGG1 concentration from 0 to 40 U/mL. Moreover, the normalized fluorescence intensity shows a linear correlation with the logarithm of hOGG1 concentration in the range from 0.005 U/mL to 1 U/mL (inset of Figure 3B). The regression equation is F = 0.5012 + 0.1552 log10 C (R2 = 0.9909), where F is the normalized fluorescence intensity and C is the concentration of hOGG1, respectively. The detection limit is calculated to be 0.0035 U/mL by evaluating the average signal of blank plus 3 times standard deviation. Notably, the sensitivity of the proposed method has improved by 2 orders of magnitude compared with that of AuNP-based colorimetric assay (0.7 U/mL),31 and nearly 1 order of magnitude compared with that of DNAzyme-based colorimetric assay (0.01 U/mL),32 and it is comparable to that of Exo III-assisted isothermal amplification-based fluorescent assay (0.001 U/mL).20 Figure 3C shows the variance of fluorescence emission spectra with the concentration of UDG. The fluorescence intensity at the emission wavelength of 450 nm enhances with the increasing UDG concentration from 0 to 50 U/mL. Moreover, the normalized fluorescence intensity exhibits a linear correlation with the logarithm of UDG concentration in the range from 0.005 U/mL to 5 U/mL (inset of Figure 3D). The regression equation is F = 0.602 + 0.1377 log10 C (R2 = 0.9902), where F is the normalized fluorescence intensity and C is the concentration of UDG, respectively. The detection limit is calculated to be 0.0025 U/mL, which has improved by 1 order of magnitude compared with those of G-quadruplex-based luminescent assay (0.02 U/mL)33 and the label-free colorimetric assay (0.02 U/mL),34 and it is comparable to that of RCA-based assay (0.002 U/mL).19 The improved sensitivity might be ascribed to (1) the specific hOGG1-induced 8-oxoG-excision 16


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repair and UDG-induced uracil-excision repair, (2) the low background signal resulting from an efficient stacking interaction among the bases of signal probes,28 and (3) the efficient signal amplification induced by lambda exonuclease-catalyzed multiple turnover reactions.

Figure 3. (A) Fluorescence spectra in response to different concentrations of hOGG1. (B) Linear relationship between the normalized fluorescence intensity at 365 nm and the logarithm of hOGG1 concentration in the range from 0.005 U/mL to 1 U/mL. (C) Fluorescence spectra in response to different concentrations of UDG. (D) Linear relationship between the normalized fluorescence intensity at 450 nm and the logarithm of UDG concentration in the range from 0.005 U/mL to 5 U/mL. Error bars show the standard deviation of three experiments.

To evaluate the selectivity of the proposed method, we used thymine DNA glycosylase 17



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(TDG) as an interference enzyme. TDG is a monofunctional enzyme, and it can selectively remove T from G/T mismatches through the DNA BER pathway.35 As shown in Figure 4, in the presence of hOGG1 and UDG, both 2-AP and P-dC fluorescence signals can be simultaneously observed. In the presence of hOGG1, only a distinct 2-AP fluorescence signal is observed. While in the presence of UDG, only a distinct P-dC fluorescence signal is observed. In contrast, neither 2-AP nor P-dC fluorescence signal can be observed in the presence of TDG in spite of its higher concentration (100 U/mL) than hOGG1 (32 U/mL) and UDG (50 U/mL). These results clearly demonstrate the capability of the proposed method to discriminate hOGG1 and UDG against other interference DNA glycosylases.

Figure 4. Selectivity of the proposed method for the detection of hOGG1 and UDG. The hOGG1 concentration is 32 U/mL, and the UDG concentration is 50 U/mL, and the TDG concentration is 100 U/mL. F and F0 are the fluorescence signals in the presence and in the absence of DNA glycosylases, respectively. Error bars show the standard deviation of three experiments.

Kinetic Analysis. We further applied this method to evaluate the enzyme kinetic parameters 18


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of hOGG1 and UDG. We measured the initial velocity in the presence of 5 U/mL hOGG1 and 10 U/mL UDG, respectively, and different-concentration DNA substrates in 10-min reaction at 37 ℃. As shown in Figure 5, the initial velocities of both hOGG1 (Figure 5A) and UDG (Figure 5B) increase with the increasing concentration of DNA substrates. The experimental data are fitted to the Michaelis-Menten equation V = Vmax [S] / (Km + [S]), where Vmax is the maximum initial velocity, and [S] is the concentration of DNA substrate, and Km is the Michaelis-Menten constant. The Vmax of hOGG1 is evaluated to be 0.6184 s-1 and Km is calculated to be 0.01472 µM, consistent with that obtained by the single quantum dot (QD)-based sensor (10.7 nM).36 The Vmax of UDG is evaluated to be 0.8981s-1 and Km is calculated to be 0.1641 µM, consistent with that obtained by the fluorescent assay (0.12 µM).37

Figure 5. Variance of initial velocity with the concentration of DNA substrates in response to hOGG1 (A) and UDG (B). Error bars show the standard deviation of three experiments.

Inhibition Assay. To demonstrate the feasibility of the proposed method for DNA glycosylase inhibition assay, we selected 5-fluorouracil (5-FU) as a model inhibitor. Some chemical drugs such as 5-FU and gentamicin may interact with DNA glycosylases and affect 19



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their activity.38 Among them, 5-FU is the most widely used chemotherapy drug for the treatment of various tumors.39 Because the proposed method involves Nb.BtsI and lambda exonuclease, it is necessary to investigate the effect of 5-FU on these two enzymes. Our research indicates that 5-FU shows no obvious effect upon the activities of Nb.BtsI and lambda exonuclease (see Supporting Information, Figure S2). We used the IC50 value to evaluate the inhibition effect of 5-FU upon hOGG1 and UDG. The IC50 value is the inhibitor concentration required to reduce enzyme activity by 50%.38 Figure 6A shows that the relative activity of hOGG1 decreases with the increasing concentration of 5-FU. On the basis of the plot of relative activity of hOGG1 versus 5-FU concentration, the IC50 value is calculated to be 4.9667 mM. Figure 6B shows that the relative activity of UDG decreases monotonically with the increasing concentration of 5-FU in a dose-dependent manner. The calculated IC50 value is 4.6304 mM, consistent with that obtained by quadruplex-based functional molecular beacon assay (5 mM).38 These results clearly demonstrate the feasibility of the proposed method as a simple and rapid platform for the screening of hOGG1 and UDG inhibitors.

Figure 6. (A) Variance of

the relative

activity of hOGG1

in response

to

different-concentration 5-FU. (B) Variance of the relative activity of UDG in response to different-concentration 5-FU. The hOGG1 concentration is 32 U/mL, and the UDG 20


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concentration is 50 U/mL. Error bars show the standard deviations of three experiments.

Detection of Cellular DNA Glycosylases. To demonstrate the feasibility of the proposed method for cellular DNA glycosylase assay, we measured the hOGG1 and UDG activities in HeLa cells (Figure 7). In response to hOGG1, the normalized fluorescence intensity improves with the increasing number of HeLa cells, with a linear correlation being obtained between the normalized fluorescence intensity and the logarithm of the number of HeLa cells in the range from 5 to 10000 cells (Figure 7A). The regression equation is F = 0.3803 + 0.1473 log10 N with a correlation coefficient of 0.9855, where F is the normalized fluorescence intensity and N is the number of HeLa cells, respectively. The detection limit is calculated to be 4 cells based on the evaluation of the average response of the negative control plus 3 times standard deviation, which is comparable to that obtained by single quantum dot-based sensor (5 cells).36 In response to UDG, the normalized fluorescence intensity exhibits a linear correlation with the logarithm of the number of HeLa cells in the range from 5 to 10000 cells (Fig. 7B). The regression equation is F = 0.2760 + 0.1724 log10 N with a correlation coefficient of 0.9931, where F is the normalized fluorescence intensity and N is the number of HeLa cells, respectively. The detection limit is calculated to be 3 cells, which is comparable to that obtained by enzyme-assisted isothermal amplification-based fluorescent assay (3 cells).40 These results clearly demonstrate that this method may be applied for quantitative detection of multiple DNA glycosylases even at the single-cell level, holding great potential for further application in clinical diagnosis.

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Figure 7. (A) Linear relationship between the normalized fluorescence intensity at 365 nm and the logarithm of the number of HeLa cells. The cell number is 5, 10, 100, 1000, and 10000 cells, respectively. (B) Linear relationship between the normalized fluorescence intensity at 450 nm and the logarithm of the number of HeLa cells. The cell number is 5, 10, 100, 1000, and 10000 cells, respectively. Error bars show standard deviations of three experiments.

CONCLUSIONS In summary, we have developed a new fluorescent method for sensitive detection of hOGG1 and UDG simultaneously using exonuclease-assisted recycling signal amplification in combination with 2-AP and P-dC as the fluorophores. Especially, this assay is rapid and homogeneous without the involvement of either external quenchers or separation steps. In this research, we designed a bifunctional DNA probe modified with one 8-oxoG and five uracil bases, which can hybridize with both trigger probe 1 and trigger probe 2 to form a sandwiched DNA substrate for hOGG1 and UDG. This DNA substrate is capable of discriminating hOGG1 and UDG from other DNA glycosylases with high selectivity and sensitivity. In contrast to the conventional fluorescent probes for homogenous assay with the 22


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requirement of dyes and quenchers attached to the 5′ and 3′ ends,18-21 the 2-AP and P-dC signal probes used in this research are quenched through their stacking interaction with the adjacent bases without the involvement of any extra quenchers. Moreover, the 2-AP and P-dC may be attached at any positions of the signal probes.28 Due to the low background signal resulting from an efficient stacking interaction among the bases of signal probes28 and the signal amplification induced by lambda exonuclease-catalyzed multiple turnover reactions, the proposed method exhibits high sensitivity with the detection limits of 0.0035 U/mL for hOGG1 and 0.0025 U/mL for UDG, which are superior to the reported colorimetric assays 31, 32, 34

and luminescent assay.33 Furthermore, the proposed method may be applied for the

measurement of enzyme kinetic parameters and the simultaneous screening of hOGG1 and UDG inhibitors, and it can be used to detect the cellular DNA glycosylases even at the single-cell level. Importantly, the proposed method can be extended for multiplexed detection of other BER enzymes by rationally designing appropriate DNA probes, offering a powerful tool for further applications in biomedical research and clinical diagnosis.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge available free of charge on the ACS Publications website at DOI: Normalized absorption and emission spectra of 2-AP and P-dC;

Effect of 5-FU on Nb.BtsI nicking enzyme and lambda exonuclease (PDF). AUTHOR INFORMATION Corresponding Author 23



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*Tel.: +86 0531-86186033. Fax: +86 0531-82615258. E-mail: [email protected].. *Tel.: +86 0531-86180010. Fax: +86 0531-86180017. E-mail: [email protected]. Author Contributions †These authors contributed equally. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (Grant Nos. 21325523, 21527811 and 21605098), and the Award for Team Leader Program of Taishan Scholars of Shandong Province, China.

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For TOC only

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Homogeneously Sensitive Detection of Multiple DNA Glycosylases

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