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  • br Experimental section br Results and discussion


    Experimental section
    Results and discussion
    Conclusions In conclusion, we have presented an autocatalytic recycling amplification electrochemical biosensing platform for simple, rapid and ultrasensitive detection of MTase activity by combining methylation-sensitive restriction endonuclease cleavage and Exo III-assisted dual-signal amplification. This unique strategy offers an impressive detection limit as low as 4.8×10units/mL, which is superior to the traditional methods. This new assay could discriminate Dam from other MTases with high selectivity, due to the specific site recognition of MTases and showed excellent performance for screening of the MTase inhibitors, which is helpful in anticancer drugs discovery. Taking advantage of the simple principle and easy operation of recent various electrochemical assays, this general approach is valuable in the design of very sensitive dual-signal electrochemical sensors for practical use.
    Conflict of interest
    Acknowledgments We gratefully appreciate the support from the National Natural Science Foundation of China (21475056 and 21365015).
    Introduction DNA methylation, a well-known epigenetic modification in human genomes, plays crucial roles in regulation of gene expression, eukaryotes development, cellular differentiation as well as the pathogenesis of various human diseases [1], [2], [3]. Generally, the DNA methylation is catalyzed by DNA methyltransferases(MTases), occurring at the C-5/N-4 positions of GDC-0349 receptor and at the N-6 position of adenine, with a process of DNA MTases transferring a methyl group from S-adenosylmethionine (SAM) to the C5-position of cytosine in CpG islands. In cancer cells, if promoter CpG islands are methylated, tumor suppressor genes would be consistently silenced. Thus, an analysis of DNA methylation and MTase activities is of great importance on the early clinical diagnosis of cancer. Currently, the existing methods for analyzing DNA methylation and MTase activities include various PCR-based techniques [4], HPLC [5], [6], gel electrophoresis [7], and radioactive labeling [8]. And as is known, a high-precision thermocycler always necessary for these methods, and most of them also require radioactive materials. More recently, some new approaches have been developed to avoid the shortcomings of traditional methods, such as the hairpin DNA probe-based fluorescence method [8], [9], gold nanoparticle-based enzyme-linkage reaction assay [10], graphene oxide-based electrochemical method [11], QCM-based technique [12], surface enhanced Raman spectroscopy [13], and chemiluminescent method [14]. However, the development of highly sensitive, versatile, and low-cost methods for the detection of DNA methylation and DNA MTase assay still remains a great challenge. Previously, a linear rolling circle amplification (RCA) has been employed to create DNA nanochains or DNAzyme chains for DNA [15], miRNA [16], protein [17] detection. In RCA-based assays, target quantification is achieved through the quantification of the RCA products. In order to improve the sensitivity of this method, several novel strategies were developed by introducing a second primer [18], dumbbell-shaped DNA probe [16], [19], DNAzyme [20], [21] and Q-STAR reporters [22]. In contrast with the conventional linear rolling circle amplification, recently the an exponential mode dendritic RCA was reported [23] with a process of a cascade reaction of linear rolling circle amplification and hairpin-based hyper-branched RCA. Catalytic nucleic acids (DNAzyme) have been found increasing interest as amplifying labels for sensing event. The DNAzyme structures are flexible mastered by encoding recognition functions into DNAzyme sequences, which could reduce nonspecific absorption of other nucleic acids. Thus, the DNAzyme are the ideal candidates for the development of bioanalytical platforms. For example, the metal-dependent DNAzymes and the hemin/G-quadruplex were extensively used as catalytic labels for the amplified signal detection of target [24], [25], [26], [27]. The flexibility to encode in the circular template and the synthesis of DNAzyme chains, together with the replication and generation of the catalytic nucleic acids, provide high amplification efficiency of the recognition events in bioanalytical applications [28], [29].