Ultra-sensitive detection of DNA N6-adenine methyltransferase based on a 3D tetrahedral fluorescence scaffold assisted by symmetrical double-ring dumbbells
Graphical abstract
Introduction
DNA methylation is a form of epigenetic modification on genomic DNA. It is catalyzed by DNA methyltransferase (DNA MTase), which adds a methyl group from S-adenosyl-l-methionine (SAM) to the 5′ end carbon atom of CpG dinucleotide cytosine [1,2]. The methylated forms in eukaryotes and prokaryotes mainly include N6-methyladenine (6 mA) [3,4], 5-methylcytosine (5 mC) [5], and N4-methylcytosine (4 mC) [6]. The methylated forms of 5-mC in eukaryotes and 6 mA in prokaryotes are essential for genetic regulation [7,8]. DNA methyltransferase significantly promotes genomic DNA methylation and plays a vital role in the regulation of X chromosome inactivation, genomic imprinting, and gene expression changes. DNA methylation is a common covalent modification in eukaryotes and a major epigenetic marker in mammals. However, in the pathogenic bacteria such as Escherichia coli, the DNA-[N 6-adenine]-methyltransferase (Dam MTase) encoded by the Dam gene is used to catalyze the adenine of 5′-GATC-3′ sites and makes it methylated [9], Dam MTase exerts key methylation effects on DNA. Bacterial epigenetics also mainly exists in the form of DNA methylation [10,11], however, it has received lesser attention than eukaryotic methylation.
DNA MTase establishes and maintains a stable methylation state of the genome [12], and it has been closely associated with the occurrence of various genetic diseases and cancers, making it an important clinical biomarker [[13], [14], [15], [16]]. Studies have shown that Dam MTase is closely involved in the occurrence of a variety of genetic diseases and cancers as well as the development of anticancer and antibacterial drugs [14,15,17,18]. Traditional DNA MTase detection methods include high-performance liquid chromatography (HPLC) [12,19,20], methylation-specific PCR [21,22], and radiation labeling analysis [23,24], which generally involve complex experimental protocols, elevated toxicity, and expensive equipment. More rapid and simpler alternatives, such as colorimetric detection [25,26], electrochemical detection [[27], [28], [29]], electrochemiluminescence [30,31], and chemiluminescence [32,33], can greatly improve the efficiency of DNA MTase detection. However, instability of the detection probes and interference from surrounding environmental factors affect experimental accuracy. Recently, stable and sensitive detection of Dam MTase was achieved through the use of a fluorescent biosensor [34]. The latter was developed based on different signal amplification strategies in a uniform reaction environment, such as the hyperbranching rolling ring [35], G-quadruplet-assisted rolling ring [36], and entropy-driven hairpin signal replacement [37].
The strategy based on a 3D tetrahedral fluorescent scaffold (DTFS) assisted by a symmetric dumbbell ring enabled the ultra-sensitive detection of Dam MTase. A single-stranded chain containing 5′-GATC-3′ sites, which could form a stable symmetrical dumbbell ring, was uniquely designed. The 5′-GATC-3′ sites of the double-stranded dumbbell stem were methylated following the recognition and catalytic activities of Dam MTase, followed by a specific cleavage reaction of Dpn I. Dpn I can specifically recognize methylated 5′-GATC-3′ sites in the DNA sequence [18,19]. The long single-stranded chains of DTFS modified with groups were designed for the first time. Each single-stranded chain of the DTFS was first treated at 95 °C to form a hairpin. The DTFS containing hairpins was then synthesized [38,39] to bring the fluorescent groups and quenching groups closer to each other, following which the fluorescence was quenched. In presence of the Dam MTase, the entire experimental reaction proceeded smoothly; the dumbbell was sheared and hybridized with the hairpins at the end of the DTFS to generate a strong fluorescent signal. The stable 3D tetrahedral structure could notably reduce the interference background signal; the fluorescence signal increased with an increase in the Dam MTase concentration, thus enabling detection in a wide linear range. Furthermore, experiments involving human serum and 5-fluorouracil inhibitors confirmed the superiority of this method. This approach would be valuable in the early diagnosis and treatment of cancer.
Section snippets
Materials and reagents
Dam MTase, 10 × Dam MTase buffer, SAM, DpnI methylation restriction endonuclease, HpaII methylation restriction endonuclease, 10 × CutSmart buffer, T4 DNA ligase, 10 × T4 DNA ligase reaction buffer, exonuclease I, exonuclease III, CpG MTase (M.SssI), and 1 × NEBuffer 2 were purchased from NEB Co., Ltd (Beijing, China); 5-fluorouracil was purchased from Solarbio Biotech Co., Ltd (Beijing, China).
TE buffer, Tris powder, MgCl2·6H2O, ammonium persulfate, 30% acrylamide/methylene bisacrylamide
Construction of the fluorescent biosensor
As shown in Scheme 1, the fluorescence signal amplification system based on DTFSs assisted by SDRDs consisted of two parts (part A and part B). As shown in part A, the dumbbell-shaped probe designed in the experiment was composed of a single-stranded chain of 40 bases, which was modified with a phosphate group at the 5′ end. The single-stranded chain was divided into two parts comprising 20 bases each. After the dumbbell rings were formed, the 5′-phosphate and 3′-OH could be linked by a T4 DNA
Conclusion
The fluorescent Dam MTase detection strategy described in this study is based on a DTFS coupled to symmetrical double-ring dumbbells. The structure of the dumbbell was stable, and the two symmetrical single-stranded chains generated upon selective digestion of the methylated dumbbells quickly reacted with the tetrahedral scaffolds. The four corners of the stable DNA tetrahedrons combined with the sheared dumbbells, greatly increasing the fluorescence signal corresponding to captured Dam MTase.
CRediT authorship contribution statement
Yuqi Huang: Conceptualization, Methodology. Wenxiu Zhang: Validation, Formal analysis. Shuhui Zhao: Investigation, Resources, Qiuyue Duan, Data curation, Visualization. Zuowei Xie: Writing – original draft. Siyi Chen: Writing – review & editing. Gang Yi: Supervision, Project administration, Funding acquisition.
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 supported by the Key Project of Science and Technology Research Program of Chongqing Education Commission (grant No. KJZD-K202000404); and the Science and Technology Research Program of Chongqing Yuzhong District Science Technology Commission (grant No. 20180127).
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