Elsevier

Analytica Chimica Acta

Volume 1223, 29 August 2022, 340220
Analytica Chimica Acta

Sensitive detection of abasic sites in double-stranded DNA based on the selective reaction of enzymes

https://doi.org/10.1016/j.aca.2022.340220Get rights and content

Highlights

  • A sensitive and clinical available system was developed for detecting AP site.

  • Two compatible assays methods were developed for AP site in long and short dsDNA.

  • APE1 triggered the secondary signal amplification with TdT.

  • Substrate preference of TdT was discovered to simplify the reaction procedure.

  • AP site detecting system was applied to λ DNA, plasmid and human genomic DNA.

Abstract

The apurinic/apyrimidinic (AP) site is one of the most common DNA lesions and a critical intermediate during the base excision repair pathway. Therefore, AP sites are essential in clinical diagnosis, treatment and detection. However, the existing detection methods are complicated in design and synthesis and have high instrument requirements, limiting their wide application. Therefore, there is an urgent need for a sensitive and straightforward detection method without time-consuming and heterogeneous reactions. Herein, we developed two compatible detection methods for AP sites in long and short dsDNA. For long and short dsDNA, the background signal was successfully suppressed by the affinity difference of Terminal deoxynucleotidyl transferase (TdT) and 3’ -end blocking, respectively, thus achieving high detectability and specificity. The detection limit was 13 pM in 20 μL, meaning that the LOD was 0.26 fmol for AP site amount and 0.05% for AP site abundance. The method has been successfully applied to detect AP sites in various biological samples quickly. Therefore, it has broad clinical application prospects, catering for the need for a point of care.

Introduction

Maintaining the integrity of the genome is of great significance for all organisms to complete the relevant physiological processes [1]. However, due to the existence of physical effects (such as UV) and chemical agents (such as oxidative agents and reactive oxygen species) [2], genomic DNA may undergo a variety of potential damage [[3], [4], [5]], including strand breaks, debasing, base modification and so on. One of the most common oxidative damage is the apurinic/apyrimidinic (AP) site [6]. Oxidative attack by physical effects and chemical reagents will break the N-glycosidic bond connecting the base and the deoxyribonucleic skeleton in DNA [7,8], thus releasing free bases and generating exogenous AP sites. At the same time, the spontaneous hydrolysis of the N-glycosidic bond of DNA will produce endogenous AP sites. Under physiological conditions, an average of 12000 purines will be lost in each lactation cell within 20 h of growth due to spontaneous hydrolysis [9].

Furthermore, the above spontaneous physiological processes will rapidly accelerate under heating or low pH conditions to produce AP sites [7,10]. In addition to the above pathway, AP sites are the central intermediates in oxidative damage's base-excision repair (BER) [11,12]. Usually, these AP site intermediates are repaired in subsequent processes. However, AP sites may “escape” from the BER process and cause DNA strand breaks, leading to DNA replication and transcription interference [7,12]. The escaped AP sites can eventually lead to gene mutation, cell death, cancer, or other genetic diseases [7,[13], [14], [15]]. Because of the importance of AP sites in molecular biology, the quantitation of AP sites plays a vital role in clinical diagnosis, treatment monitoring, screening and evaluation of the potential genotoxicity of environmental pollutants.

Many methods have been developed for quantitatively detecting AP sites in DNA, such as enzyme-linked immunosorbent assay (ELISA), covalent chemical probe assay and noncovalent chemical probe assay. ELISA was easy to perform, and the limit of detection (LOD) was one abasic site per 104–105 nucleotides [16]. However, the specificity of the method was not satisfactory because of the cross-reaction of antibodies. The covalent chemical probe method can be divided into O-(pyridin-3-methyl) hydroxylamine (PMOA) probes and aldehyde reaction probes (ARP) [17]. The former was often combined with the LC-MS/MS method [18], and its detection results were accurate and reliable. The LOD was 2.2 lesions per 108 nucleotides (0.9 fmol), but its application was limited because it required demanding heterogeneous reaction pretreatment of samples and professionals to operate expensive instruments. The latter was the most commonly used AP site detection method because it did not need to derive or digest DNA and had high sensitivity and specificity advantages [19]. ARP is often coupled with capillary electrophoresis with laser-induced fluorescence (CE-LIF) [20] and electrochemiluminescence [21]. The mechanism is that hydroxylamine reacts with the ring-opening aldehyde structure of the AP site and bounds covalently [19,22]. The biotin-labelled ARP probe was most commonly used. The LOD was one lesion in 512 nucleotides (8.5 fmol) [21]. However, it required complex multistep cleaning and separation operations because it was in a heterogeneous system. To avoid the deficiency of the traditional heterogeneous system with biotin-labelled ARP probes, the researchers designed a fluorescent ARP probe (FARP) to be detected with CE-LIF [20], and its LOD was 1.2 AP sites per 106 nucleotides (20 amol). However, this method requires time-consuming precipitation, centrifugation and incubation pretreatment and expensive instruments. It is worth mentioning that the ARP probe will cross-react with other reactive aldehydes [22], such as formylated bases (5-fC, 5-fU). The interference of these bases on the determination of AP sites cannot be ignored. In addition, because hydroxylamine reactions require nucleophilic catalysis under acidic conditions [22], acidic environments will produce additional AP sites [18]. Because of the advantages of avoiding covalent labelling reactions and cleaning and separating markers, noncovalent probes were also used to detect AP sites [24]. The noncovalent probe could produce stable and reversible current signals or fluorescence by infiltrating small molecules with complementary shapes and binding forces into the cavity of AP sites [25]. However, the binding ability of the noncovalent probe was generally lower than that of a covalent reaction probe, and its detectability was poor (1 AP per 74 bases) [24]. In summary, these methods are inevitably subject to one or more limitations, such as poor detectability, complex design, expensive instruments and time-consuming pretreatment, which limit their application in clinical and environmental genotoxicity monitoring.

Therefore, developing a rapid, simple and high-detectability detection system is imperative. Because the AP site has no base, it cannot be amplified by DNA polymerase, so polymerase chain reaction (PCR) technology is limited. Additionally, the randomness of the location of the AP site and the unknown upstream and downstream sequences limits the application of hybridization-based DNA probes. To solve these problems, we introduced apurinic/apyrimidinic endonuclease I (APE1) to identify AP sites, avoiding false positives and complex heterogeneous cleaning and separation steps. Terminal deoxynucleotidyl transferase (TdT) is a template-independent DNA polymerase that can add nucleotides to 3′ ends [26,27]. APE1 is a multifunctional DNA nuclease having AP-endonuclease, 3′ phosphodiesterase, 3′ to 5′ exonuclease and even RNA cleavage activities [28,29]. The last decade has witnessed rapid exploration of TdT and APE1 for a wide range of applications and across many fields due to their unique properties [[26], [30], [31], [32], [33], [34], [35], [36], [37], [38], [39], [40], [41], [42], [44]]. TdT is introduced into the APE1 detection system (Scheme 1) to elongate the resulting 3’ –OH to form a poly A tail, thus transforming the unamplified AP site into an amplified long poly A sequence for further amplification. Finally, multiple poly T probes (FL1) will hybridize with the poly A tail. The fluorophore (carboxyfluorescein, FAM) and the black hole quencher-1 (BHQ-1) are labelled on two sides of the AP site in FL1, and BHQ initially quenched the fluorescence signal of FAM. As the APE1 catalyzes the cleavage of DNA phosphodiester backbone at AP sites in dsDNA, FL1 could be digested after hybridizing with the poly A tail. The FAM and BHQ-1 groups will depart from each other after the digestion, and the fluorescent signals will be generated. Thus, the number of AP sites will be reflected by the fluorescence signal of FAM. To ensure high specificity of the detection system, the blocking strategy is introduced; that is, before detection, the 3’ –OH of the DNA substrate is blocked to avoid interference from the substrate itself. These lifting designs that aim at the deficiencies in the current field make our method simple and cost-effective to detect AP sites quickly and sensitively. The method has been successfully applied to detect AP sites in various biological samples and has broad clinical application prospects.

Section snippets

Materials and reagents

All synthesized and purified oligonucleotides were obtained from Sangon Biotech Co., Ltd. (Shanghai, China). TdT, rSAP, APE1, TdT buffer, CutSmart buffer, and CoCl2 solution were obtained from New England Biolabs Inc. (Ipswich, MA, USA). Endo IV was purchased from Thermo Fisher (MA, USA). Dideoxy ribonucleotide triphosphates (ddNTPs) were purchased from TriLink BioTech. Deoxynucleotide adenine triphosphate (dATP), λ-DNA (0.2 mg/ml), pUC19 plasmid, pBC322 plasmid, 1 × Tris-EDTA buffer (1 mmol/L

The principle of the 3’ terminal-blocking-based AP biosensor

Scheme.1 illustrates the principle of the novel abasic site biosensor with a 3′ terminal-blocking strategy for short DNA. We used APE1 to recognize the AP site in DNA instead of the commonly used aldehyde reaction probe to avoid complex organic synthesis processes and harsh reaction conditions. APE1 cleaves the AP site and produces a 3’ –OH end for subsequent signal amplification. However, the endogenous 3’ –OH ends of DNA can also be extended by TdT, resulting in leakage of the background

Conclusion

We have developed a highly sensitive and specific AP site detection system based on APE1 recognition and TdT elongation. We used a ddNTP-blocking amplification strategy for short-chain DNA, and the AP site concentration and abundance LOD reached 13 pM and 0.05%, respectively. We utilized an unblocking-amplification strategy for long-chain DNA to detect AP sites rapidly without complicated preprocessing, even with dozens of dsDNA interferences. However, our approach has several limitations, such

CRediT authorship contribution statement

Zhe Hu: Methodology, Investigation, Writing - original draft. Weicong Ye: Investigation, Writing - original draft. Zhen Zhang: Investigation. Tianci Xie: Investigation. Wenqian Yuan: Investigation. Tongbo Wu: Supervision, Conceptualization, Funding acquisition, Writing - review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was financially supported by the National Natural Science Foundation of China (No. 82172372 and 21904045), and Training Program of Innovation and Entrepreneurship for Undergraduates of Hubei Province (No. S202110487381).

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  • Cited by (2)

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    These authors contributed equally to this work.

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