A label free fluorescent assay for uracil-DNA glycosylase activity based on the signal amplification of exonuclease I

Abstract

We introduced a simple, fast and label free fluorescent assay for uracil-DNA glycosylase activity based on the signal amplification of exonuclease I. This method exhibits a high sensitivity for UDG activity assay with a low detection limit of 0.0070 U mL⁻¹.

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The base excision repair (BER) pathway that removes damaged DNA bases plays an important role in maintaining genomic integrity.¹ The uracil residue is a common damaged base in DNA, whether due to the misincorporation during DNA replication or deamination of cytosine.² If the cytosine is deaminated to a uracil residue, the transition mutation (G:C → A:T) occurs after the next round of replication. This transition mutation may disturb the protein–nucleic acid interactions and generate false code information.^2a As a highly conserved damage repair protein, uracil-DNA glycosylase (UDG) catalyses the cleavage of the N-glycosidic bond between uracil and the deoxyribose phosphate backbone of DNA, generating an abasic site (AP site).³ This process then initiates the BER pathway to repair the damaged uracil base. Thus, UDG plays a crucial role in the BER pathway, and has been demonstrated to be related with human diseases such as Bloom syndrome and human immunodeficiency.⁴ Detection of UDG activity therefore represents a critical step toward the understanding of DNA damage repairing and the development of tools for molecular diagnostics.

Traditional methods for UDG detection include gel electrophoresis and autoradiography.⁵ But these methods have some shortcomings of being time-consuming, labor-intensive and of insufficient sensitivity. Thus, great efforts have been made to develop more UDG activity assays with better performance, such as fluorescence resonance energy transfer based fluorescent methods,⁶ enzymatic amplification based electrochemical assay,⁷ gold nanoparticle or G-quadruplex based colorimetric methods.⁸ Although these methods have achieved great advances toward the UDG activity assay, there still are some limitations in each method. For examples, labeling of DNA probes with fluorescent dyes was required in the above mentioned fluorescent methods. Multi-step modifications or washings were commonly existed in electrochemical assay. In G-quadruplex based assays, DNA probes need specific design. These weaknesses make the above assays expensive, complicated and/or tedious. Obviously, considering the significance of UDG in BER-related biological events, there is a continuous demand for developing more convenient and sensitive analytical methods for UDG detection.

A label free detection strategy can provide a fast, convenient and costless assay, which has gained much interest. Due to the favorable photophysical properties, thermal stability and low-cost, SYBR green I (SG I) as an effective DNA interacting dye is most frequently used to construct label free detection methods for various biomolecules.⁹ In this work, we developed a convenient, costless and label free fluorescent method for highly sensitive UDG activity assay using SG I as a fluorescence indicator and coupled with the signal amplification of exonuclease I (Exo I). As we known, SG I dye shows stronger binding ability to double strand DNA (dsDNA) over single strand DNA (ssDNA). However, the nonspecific interaction between SG I dye and ssDNA through electrostatic interactions will still cause a relatively large signal, leading to a poor signal to background ratio and sensitivity.^9a,10 Exo I is a sequence-independent nuclease that catalyzes the stepwise removal of 3′ mononucleotides from the 3′ termini of ssDNA and does not hydrolyze the dsDNA.¹¹ Obviously, the Exo I-catalyzed digestion of ssDNA will reduce the undesired signal that originates from the nonspecific interaction between SG I and ssDNA. And the change of fluorescence intensity is therefore enhanced, improving the assay's sensitivity.

In our design (Scheme 1), a hairpin-structured probe (HP) with a blunt 3′-end in the duplex stem was used as the UDG substrate. Four uracil deoxyribonucleotides (U, as indicated in the scheme) were modified in the stem region. In the absence of UDG, the HP probe with a hairpin-structure was the predominant form. But it was not the favorite substrate for Exo I since Exo I is a ssDNA-specific exonuclease. Thus, no significant digestion of HP probe could be occurred. Accordingly, SG I dye would bind with the stem region by the both intercalation and minor groove binding, producing a strong fluorescence signal. After adding the UDG, uracil bases were removed from the deoxyribose phosphate backbone of HP. AP sites were generated simultaneously, which lowered the melting temperature of HP. Thereby, the HP probe was opened to form a single strand structure. The Exo I would then catalyze the digestion of new generated ssDNA into mononucleotides. Taking the advantage of the weaker affinity between SG I and mononucleotides, the fluorescence signal of final system was further remarkably reduced. As a result, the Exo I could enhance the change of fluorescence signal, providing an improvement in the sensitivity for UDG assay.

Scheme 1 Schematic illustration of UDG assay based on the amplification of exonuclease I.

The fluorescence spectra in the presence and absence of Exo I were explored to confirm the feasibility of the present strategy. As shown in Fig. 1A, the HP-contained solution without UDG and Exo I exhibited a strong fluorescence (curve a). The sole addition of UDG induced a loss of fluorescence intensity (curve b) with a F₀/F value of ∼3.7 (F₀ is the fluorescence intensity at 528 nm in the absence of UDG, F is the fluorescence intensity in the presence of UDG). As expected, when the Exo I was introduced into the sensing system, a further reduction of fluorescence signal was observed (curve d). More importantly, the Exo I has no remarkable effect on the background fluorescence signal of HP probes (curve c). Accordingly, a larger F₀/F value (∼12.1) was achieved. These satisfactory results demonstrated that Exo I could indeed amplify the UDG-triggered fluorescence change, assuring a higher sensitivity. The agarose gel stained by SG I dye was also used to demonstrate the UDG-triggered digestion of HP by Exo I (Fig. 1A, inset). Lane a is the band of HP probe without UDG and Exo I. Treating HP probe with UDG still results in a faint band (lane b), probably due to the nonspecific binding of SG I with opened ssDNA-structured HP probe. Incubation of HP probe with Exo I does not obviously alter the brightness or position of HP band in gel image (lane c). However, after the treatment of HP by UDG and Exo I together, no bands were observed (lane d), implying the occurrence of the digestion of opened HP into mononucleotides. Moreover, a HP2 probe without uracil modification in the stem region was used as a control probe to further confirm that the fluorescence signal change was originated form the UDG-catalyzed removal of uracil bases. The results in Fig. 1B clearly showed that no obvious decrease of fluorescence signal was detected, indicating that the HP2 probe still was intact even in the presence of UDG and Exo I together. This result was also verified by gel images (Fig. 1B, inset). The above experiments imply that the present strategy may provide a sensitive, selective method for UDG assay.

Fig. 1 (A) Fluorescence spectra under different conditions. (a) HP; (b) HP + UDG; (c) HP + Exo I; (d) HP + UDG + Exo I. The concentrations of HP, UDG, Exo I were 50 nM, 15 U mL⁻¹ and 20 U, respectively. Inset is gel image of sensing system under different conditions. SG I dye was used to stain the gels. Lane (a): HP; lane (b): HP + UDG; lane (c): HP + Exo I; lane (d): HP + UDG + Exo I. (B) Fluorescence spectra of sensing solutions contained 50 nM HP2, 20 U Exo I with or without 10 U mL⁻¹ UDG. Inset is the corresponding gel images. Lane (1): in the absence of UDG; lane (2): in the presence of UDG.

In order to achieve the best assay performance, the amount of Exo I and enzymatic reaction time were optimized. The ratio of F₀/F increased with increasing the amounts of Exo I and then tended to level off after 15 U (Fig. S1, ESI†). Thus, to obtain an effective digestion, 20 U of Exo I was used in the following experiments. The effect of reaction time on the fluorescence response was illustrated in Fig. S2 (ESI†). The fluorescence decreased with the increasing reaction time. After 20 min, the fluorescence intensity tended to be constant, indicating the reaction process was complete. Consequently, 20 min was selected as the optimal enzymatic reaction time.

Under the optimal conditions, various concentrations of UDG were tested using the proposed method. The fluorescence intensity decreased upon increasing the UDG concentration (Fig. 2A). A linear relationship (R² = 0.9983) between peak fluorescence intensity and logarithmic UDG concentrations was obtained in the concentration range from 0.010 to 5.0 U mL⁻¹ (Fig. 2B, inset). The detection limit was estimated to be ∼0.0070 U mL⁻¹ (according to 3σ), which is lower or comparable to that of most previous assays (Table S1, ESI†). In addition, this method is fast. The assay time is much shorter than that of DNAzyme-amplified approach,^6c graphene oxide-based fluorescent assay,^6e nicking enzyme-amplified colorimetric method,^8b electrochemical detection,¹² and comparable to that of molecular beacon-based fluorescence method.^6b Furthermore, the relative standard deviations were 2.5%, 2.8% and 3.7% in three repetitive assays of 5.0, 0.50 and 0.050 U mL⁻¹ UDG. These results indicate that the developed label free fluorescent method can be used for sensitively detecting UDG activity with a good reproducibility.

Fig. 2 (A) Fluorescence spectra upon the addition of UDG with different concentrations from 0 to 20 U mL⁻¹. (B) The relationship between fluorescence intensity and UDG concentrations. Inset is the linearity of fluorescence intensity with respect to the logarithm of UDG concentrations. Error bars were estimated from three replicate measurements.

Some other enzymes or proteins, including M.SssI methyltransfer (M.SssI), Dam methyltransferase (Dam MTase), Nb.BbvCI restriction endonuclease (Nb.BbvCI) and bovine serum albumin (BSA), were tested to investigate the selectivity of the present method. The concentration of UDG used here was 10 U mL⁻¹ and it was 40 U mL⁻¹ for each other control enzyme. The BSA concentration was 200 μg mL⁻¹. The comparison result of fluorescence intensity in the presence of these enzymes or proteins was illustrated in Fig. S3 (ESI†). Only UDG could induce a remarkable fluorescence decrease. This result demonstrates that the proposed strategy has a good selectivity toward UDG.

The practicality of the proposed strategy was also tested by the detection of UDG in complex biological samples. Diluted crude cell extracts was used to simulate a complex biological environment. UDG with various concentrations was mixed with the diluted crude cell extracts (2%) and then analyzed. The dilution process may reduce the potential effect of coexisting interferents. As shown in Fig. S4 (ESI†), the fluorescence intensity decreased gradually with increasing UDG concentration. These results suggest that the proposed method may hold a potential application for real complex sample analysis.

We also explored the potential use of this method for evaluating the inhibition effect on UDG activity. The anticancer drugs 5-fluorouracil, which has been confirmed to be an inhibitor of UDG,^6b was introduced as a study model inhibitor. Since the Exo I was used in the sensing design, the effect of 5-fluorouracil on Exo I was investigated firstly. We treated HP with saturated UDG to obtain a ssDNA-structured probe. After that, Exo I was added into the above solution with or without 5-fluorouracil and the fluorescence intensity respectively recorded 20 min later. The results in Fig. S5 (ESI†) indicated that the 5-fluorouracil did not affect the Exo I activity in our assay conditions. Subsequently, the inhibition assay of UDG at different concentrations of 5-fluorouracil was carried out. As shown in Fig. 3, the activity of UDG was decreased with increasing 5-fluorouracil concentrations. The IC₅₀ value was estimated to be ∼7.0 mM, which is in the same order of magnitude as those of previous reports.^6b,13 Thus, the proposed strategy may provide a simple and rapid method for screening inhibitors of UDG.

Fig. 3 Influence of 5-fluorouracil on the activity of UDG. Error bars were estimated from three replicate measurements.

In summary, we have developed a simple, fast, and label free fluorescent strategy for detecting UDG activity based on the Exo I-assisted signal amplification. SG I, a dsDNA chelating dye, was used as the fluorescence signal indicator. The UDG-catalyzed uracil removal opened the hairpin substrate. Thus, the Exo I will digest the generated ssDNA-structured oligonucleotides, resulting in a remarkable enhancement in the change of fluorescence signal. The present label free fluorescent strategy possesses several important features. First, SG I-based label free design does not require costly labeling, tedious modification and/or separation, making the assay convenient in operation by one-step manner, inexpensive and fast. Second, the homogeneous assay format enables this method to be easily automated and high-throughput. Third, the efficient digestion ability of Exo I results in an improvement of sensitivity for UDG activity assay. A low detection limit of 0.0070 U mL⁻¹ was achieved, which is lower than that of most previously reported methods. Finally, the developed method exhibits an approving performance in complex biological samples, and can be used for evaluating inhibition effect of 5-fluorouracil on UDG activity. In view of these merits, the proposed method may hold potential applications in UDG-related clinical diagnosis and functional research.

Acknowledgements

Financial support from the National Natural Science Foundation of China (21305020, 21405025), Natural Science Foundation of Guangxi Province (2014GXNSFBA118047), Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources of Education Ministry (CMEMR2013-A12) and Guangxi Normal University (2013ZD002) is gratefully acknowledged.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra12958c

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