Signal-on electrochemical detection of DNA methylation based on the target-induced conformational change of a DNA probe and exonuclease III-assisted target recycling
Introduction
DNA methylation is an essential component of epigenetic modification associated with many biological processes, such as eukaryote development, cellular differentiation, and gene expression (Jones and Takai, 2001; Jones and Baylin, 2007; Schübeler, 2015; Berney and McGouran, 2018). Aberrant DNA methylation in mammals is associated with various genetic diseases and tumors, including bladder carcinoma, breast cancer, colon tumor, and prostate cancer (Ushijima, 2005). Consequently, accurate and sensitive recognition of DNA methylation is vital for understanding the mechanism of epigenetic regulation of genetic information and molecular pathology for early cancer diagnosis (Wang et al., 2010; Heyn and Esteller, 2012). Restriction enzyme-based detection and bisulfite conversion analysis are two important recognition methods. Restriction enzymes catalyze the scission reaction of unmethylated DNA, while they are limited to analyzing specific base sequences (Zhou et al., 2016). In bisulfite conversion analysis, cytosine is converted to uracil, whereas methylated cytosine remains unchanged in any DNA sequence (Feng et al., 2008). Therefore, bisulfite conversion analysis can expand the scope of the detection target for DNA methylation analysis (Kato et al., 2011).
Electrochemical detection methods are simple, fast, selective, and suitable for real-time in situ assays, and have thus attracted substantial attention for analyzing DNA methylation (Kato et al., 2008; Tanaka et al., 2007; Feng et al., 2019; Liu et al., 2011; Li et al., 2012; Tsutsui et al., 2011; Wang et al., 2013, 2015; Gao et al., 2018). For example, Muren and Barton developed a multiplexed chip platform for a robust, nonradioactive assay of bacterial and human methyltransferase activity (Muren and Barton, 2013). Moreover, an innovative secondary electrode array sensing system was designed for analyzing colorectal tumor tissue based on low-density DNA monolayer patterning and DNA charge transport (Furst et al., 2014). Interestingly, Kurita et al. reported the on-chip evaluation of cytosine methylation level in genomic DNA using an excellent nanocarbon film electrode with nanocrystalline structure (Kurita et al., 2017). We used bisulfite conversion to develop a label-free electrochemical biosensor for profiling the methylation information of the p53 gene according to the current signal of a [Ru(NH3)6]3+ indicator (Wang et al., 2012). Recently, the methylation status of a tumor suppressor gene was assessed rapidly in human serum and tissue samples with elegant results using immunomagnetic beads (Povedano et al., 2018). Although these approaches obtained DNA methylation information, the improvement of sensitivity is highly required because DNA samples are tiny in many diagnostic scenarios.
An electrochemical DNA (E-DNA) sensor based on the hybridization-induced conformational change of a redox-tagged stem-loop DNA probe has been developed for bioassays (Xiao et al., 2007a, 2007b). Compared with cumbersome traditional sensors, the E-DNA sensor provided a convenient, reagentless, and single-step DNA detection method (Xiao et al., 2005, 2006), which was suitable for real-world oligonucleotide detection. Fan et al. pioneered a signal-off E-DNA sensor for the sequence-specific detection of DNA, in which target binding reduced the redox current (Fan et al., 2003). Ricci et al. confirmed that the E-DNA signal arose from the hybridization-linked conformational change, which altered the electron transfer efficiency between the redox moiety and the electrode surface (Ricci et al., 2007). However, these methods suffered from limited signal gain; no more than 100% of the original current could be obtained. Moreover, surrounding contaminants also produced false positives, which were difficult to distinguish from the signal change arising from the intended target (Xiao et al., 2005; Cash et al., 2009). In consideration of the above problems from signal-off E-DNA system, it is necessary to design a signal-on E-DNA sensor, which can increase the signal response after target recognition.
To achieve a highly sensitive electrochemical biosensing system, various amplification strategies have been explored, such as polymerase chain reaction (PCR) (Clark et al., 2006; Burke et al., 2009), hybridization chain reaction (HCR) (Chen et al., 2019), DNA structure-based assembly (Li et al., 2013; Zhou et al., 2017), and rolling circle amplification (RCA) (Bi et al., 2013; Gao et al., 2017). These recycling amplification systems are mainly used for recycling probes, signals, and targets. Target regeneration is a promising recycling amplification strategy, in which a single target could produce multiple binding events. For example, exonuclease III (Exo III)-assisted target recycling amplification allowed the recognition of the blunt 3′-terminus of a molecular beacon in a double-stranded DNA probe and the selective digestion of a molecular beacon (Zuo et al., 2010; Xiong et al., 2015). Consequently, the autonomous generation of target DNA allowed continuous hybridization, resulting in higher sensitivity and a lower detection limit.
In this work, we developed a signal-on E-DNA biosensor sensitized with Exo III-assisted recycling strategy for analyzing DNA methylation. In this approach, one terminus of the stem-loop probe DNA was modified with a thiol group that bound chemically to the Au nanoparticles (nano-Au) modified electrode via an Au–S bond. The other terminus was covalently modified with a methylene blue (MB) tag. After the introduction of auxiliary DNA, the stem-loop structure of the probe DNA opened to form double-stranded probe DNA/auxiliary DNA with a rod-like structure, sequestering MB from the electrode surface, and exhibiting a lower current signal. The methylated cytosine in target DNA remained unchanged after the treatment of bisulfite. Hence, upon the addition of methylated target DNA, probe DNA/auxiliary DNA/methylated target DNA complex was formed through complementary hybridization. With the help of Exo III, the auxiliary DNA was digested from the 3′-terminus, and the released methylated target DNA would hybridize with another auxiliary DNA. The continuous hybridization of methylated target DNA constantly triggered digestion, resulting in large signal amplification. Consequently, the stem-loop structure of the probe DNA was regenerated, and the rate at which the MB tag hit the electrode surface was improved, increasing the electron transfer and Faradic current (“signal-on” status). Therefore, based on the target-induced conformational change of the MB tag-bound probe DNA, signal transduction for methylated target DNA analysis with a detectable increase in redox current was achieved.
Section snippets
Reagents and apparatus
Trihydrate chloroauric acid (HAuCl4·3H2O), tris(2-carboxyethyl) phosphine hydrochloride (TCEP), K3[Fe(CN)6], and K2[Fe(CN)6] were obtained from Sigma-Aldrich (St. Louis, MO, USA), and used without further purification. Exo III was purchased from Takara Biotechnology Co., Ltd (Dalian, China). Phosphate buffered solution (PBS; pH 7.4) was prepared by mixing stock solutions of 0.1 M KH2PO4, Na2HPO4, and NaCl. Unless otherwise stated, all reagents were of analytical grade, and all aqueous solutions
Construction of the signal-on E-DNA sensor
Scheme 1 shows schematics of the construction of the electrochemical sensing platform, the Exo III-assisted target recycling amplification, and the target-induced conformational change of the electrode-bound oligonucleotides. The stem-loop structural probe DNA modified with a thiol group at the 3′ end was immobilized on the nano-Au modified GCE via an Au–S bond. The probe had an MB tag at the 5′ end. Upon hybridization with complementary auxiliary DNA, the stem-loop structure of the probe DNA
Conclusions
We proposed a signal-on electrochemical biosensing platform for sensitive recognition and detection of DNA methylation. In light of bisulfite conversion, the DNA methylation information was translated into nucleic acid sequence information, which provided a promising strategy for the hybridization detection of target DNA methylation. With the assistance of auxiliary DNA, the Exo III-based recycling amplification was triggered by target DNA hybridization, causing the conformational rearrangement
CRediT authorship contribution statement
Qiumei Feng: Conceptualization, Project administration, Writing - original draft. Li Qin: Data curation, Methodology. Mengying Wang: Formal analysis, Validation. Po Wang: Supervision, 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.
Acknowledgements
This work was financially supported by the National Natural Science Foundation of China (Nos. 21705062, 21205052, and 21675067), the Natural Science Foundation of Jiangsu Province (Nos. BE2019645 and BK20170228), and the Natural Science Foundation of Xuzhou City (No. KC19161).
References (55)
- et al.
Biosens. Bioelectron.
(2016) - et al.
Biosens. Bioelectron.
(2018) - et al.
Biosens. Bioelectron.
(2018) - et al.
Biosens. Bioelectron.
(2018) - et al.
Sens. Actuators, B
(2018) - et al.
Cell
(2007) - et al.
Anal. Chim. Acta
(2018) - et al.
Biosens. Bioelectron.
(2019) - et al.
Biosens. Bioelectron.
(2013) - et al.
Electrochem. Commun.
(2015)