Abstract
DNA methylation is a significant epigenetic mechanism involving processes of transferring a methyl group onto cytosine or adenine. Such DNA modification catalyzed by methyltransferase (MTase) plays important roles in the modulation of gene expression and other cellular activities. Herein, we develop a simple and sensitive biosensing platform for the detection of DNA MTase activity by using only two oligonucleotides. The fluorophore labeled molecular beacon (MB) can be methylated by MTase and subsequently cleaved by endonuclease DpnI at the stem, giving a shortened MB. The shortened MB can then hybridize with a primer DNA, initiating a cycle of strand displacement amplification (SDA) reactions. The obtained SDA products can unfold new MB and initiate another cycle of SDA reaction. Therefore, continuous enlargement of SDA and exponential amplification of the fluorescence signal are achieved. Because the triple functions of substrate, template and probe are elegantly integrated in one oligonucleotide, only two oligonucleotides are necessary for multiple amplification cycles, which not only reduces the complexity of the system, but also overcomes the laborious and cumbersome operation that is always a challenge in conventional methods. This platform exhibits an extremely low limit of detection of 3.3 × 10-6 U mL-1, which is the lowest to our knowledge. The proposed MTase-sensing platform was also demonstrated to perform well in a real-time monitoring mode, which can achieve a further simplified and high-throughput detection. The sensing strategy might be extended to the activity detection of other enzymes, thus showing great application potential in bioanalysis and clinical diagnosis.
Highlights
Genomic DNA methylation is a crucial epigenetic DNA modi cation which commonly occurs in nature, and plays important roles in the regulation of gene expression.[1,2] The DNA methylation process is catalyzed by a series of methyltransferases (MTase) that transfer a methyl group from S-adenyl methionine (SAM) to the C-5/N-4 positions of cytosine (C) or the N-6 position of adenine (A).[3]
Studies on cancer pathology have revealed that the abnormal pattern of DNA methylation induced by aberrant MTase activities is involved in various types of cancer, including breast, prostate, lung, liver and colon aState Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Biosensing and Molecular Recognition, Research Centre for Analytical Sciences, College of Chemistry, Nankai University, Tianjin 300071, P
We investigated the capability of the proposed biosensor in the application of a Dam MTase assay in a human serum sample
Summary
Genomic DNA methylation is a crucial epigenetic DNA modi cation which commonly occurs in nature, and plays important roles in the regulation of gene expression.[1,2] The DNA methylation process is catalyzed by a series of methyltransferases (MTase) that transfer a methyl group from S-adenyl methionine (SAM) to the C-5/N-4 positions of cytosine (C) or the N-6 position of adenine (A).[3]. Conventional analytical methods for the detection of MTase activity include methylation-target polymerase chain reaction (PCR), radioactive labeling-based gel electrophoresis, and highperformance liquid chromatography (HPLC).[13,14,15,16] most of these methods are time-consuming and harmful to biological systems because of their radioactive substances.[17] Recently, various alternative approaches have been developed for an MTase assay, such as uorescence, chemiluminescence, colorimetric and electrochemical methods.[18,19,20,21,22,23,24,25,26,27,28] The emerging methods have attracted intense attention due to their cheap instrumentation and low toxicity. DNA-based biosensors, in particular, can provide high sensitivity and selectivity because
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