Ultra-sensitive detection of DNA N6-adenine methyltransferase based on a 3D tetrahedral fluorescence scaffold assisted by symmetrical double-ring dumbbells

Highlights

• An ultra-sensitive fluorescent system based on DTFSs assisted by SDRDs is proposed.

• DTFSs were constructed as target carriers for the detection of Dam MTase.

• The fluorescent system greatly enhanced Dam MTase detection.

• The developed biosensor enables early clinical detection of Dam MTase.

• Inhibitor and serum experiments support DTFS use in cancer diagnosis and treatment.

Abstract

DNA methylation is an epigenetic modification that plays a vital role in X chromosome inactivation, genome imprinting, and gene expression. DNA methyltransferase establishes and maintains a stable methylation state in genomic DNA. Efficient and specific DNA methyltransferase testing is essential for the early diagnosis and treatment of cancer. In this study, we designed an ultra-sensitive fluorescent biosensor, based on a 3D tetrahedral fluorescent scaffold assisted by symmetrical double-ring dumbbells, for the detection of DNA-[N 6-adenine]-methyltransferase (Dam MTase). Double-stranded DNA was methylated by Dam MTase and then digested by DpnI to form two identical dumbbell rings. The 3D tetrahedral fluorescent scaffold was synthesized from four oligonucleotide chains containing hairpins. When the sheared dumbbells reacted with the 3D tetrahedral fluorescent scaffold, the hairpins opened and a fluorescence signal could be detected. The strategy was successful over a wide detection range, from 0.002 to 100 U mL⁻¹ Dam MTase, and the lowest detection limit was 0.00036 U mL⁻¹. Control experiments with M.SssI methyltransferase and HpaII methylation restriction endonuclease confirmed the specificity of the method. Experiments with spiked human serum and the 5-fluorouracil inhibitor proved the suitability of the method for early cancer diagnosis.

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.