Aberrant DNA methylation, which is caused by the abnormal level of DNA methyltransferase (MTase), has been considered associated with a growing number of human diseases. Although there are various methods paying close attention to DNA methyltransferase (MTase) detection, most of them are generally complex and expensive. Here, a simple electrochemical strategy for sensitive detection of DNA methyltransferase (MTase) and inhibitor screening based on DpnI digestion triggering the formation of G-quadruplex DNAzymes has been developed. In this paper, a probe richness of guanine (G) was first self-assembled on the surface of the electrode through Au–S bond and then hybridized with the complementary DNA. Without DNA methylation, G-quadruplex DNAzymes cannot be formed due to the double helix structure and a weak electrochemical response can be observed. On the contrary, an obvious enhancement of the electrochemical response can be achieved after the cleavage of the methylated double-strand DNA by DpnI since G-quadruplex DNAzymes can be obtained, which catalyze the oxidation of hydroquinone by H 2 O 2 with the assistance of the cofactor hemin. This method is under a detection limit of 0.96 U/mL and can monitor the change of DNA methylation level selectively. Moreover, RG108 was selected as a representative inhibitor for studying the inhibition activity of DNA MTase.
Xue Li,Zhipeng Xie,Wei Wang,Yunlei Zhou,Huanshun Yin,Zhiqing Yang,Shiyun Ai
The DNA isothermal amplification cycle is an effective expansion strategy, which can overcome the inherent limitation of the target to signal ratio of 1 : 1. In this work, we fabricated an electrochemical biosensor for the rapid detection of Dam methyltransferase activity based on the exonuclease III (Exo III) assisted isothermal amplification cycle. First, double stranded DNA (ds-DNA) containing the symmetric sequence of 5′-GATC-3′ was immobilized on the magnetic bead surface. After the ds DNA was methylated by Dam MTase, it could be further cleaved by DpnI and formed a blunt end, which could lead to the digestion of a DNA strand by Exo III and release of a target single stranded DNA (ts-DNA). Then, the ts-DNA can further hybridize with DNA probes (DNA3) immobilized on the AuNPs/Au electrode surface and form a blunt end DNA–DNA duplex. Subsequently, the DNA3 in the DNA–DNA duplex can be digested by Exo III and release ts-DNA. The released ts-DNA can further hybridize with the unhybridized single stranded DNA (ss-DNA) and an isothermal amplification cycle is achieved. The method showed high sensitivity with a low detection limit of 0.031 unit per mL. Moreover, inhibition investigation indicates that Dam MTase activity can be inhibited by using 5-fluorouracil with an IC50 value of 131.22 μM.
Fengzhe Li,Yingying Chen,Jinhua Shang,Qing Wang,Shizhen He,Xiwen Xing,Fuan Wang
Abnormal DNA methylation contributes to the annoying tumorigenesis and the elevated expression of methylation-related methyltransferase (MTase) is associated with many diseases. Hence DNA MTase could serve as a promising biomarker for cancer-specific diagnosis as well as a potential therapeutic target. Herein, we developed an isothermal autocatalytic hybridization reaction (AHR) circuit for the sensitive detection of MTase and its inhibitors by integrating the catalytic hairpin assembly (CHA) converter with the hybridization chain reaction (HCR) amplifier. The initiator-mediated HCR amplifier could generate amplified fluorescent readout, as well as numerous newly activated triggers for motivating the CHA converter. The CHA converter is designed to expose the identical sequence of HCR initiators that reversely powered the HCR amplifier. Thus, the trace amount of target could produce exponentially amplified fluorescent readout by the autocatalytic feedback cycle between HCR and CHA systems. Then an auxiliary hairpin was introduced to mediate the assay of Dam MTase via the well-established AHR circuit. The Dam MTase-catalyzed methylation of auxiliary hairpin leads to its subsequent efficient cleavage by DpnI endonuclease, thus resulting in the release of HCR initiators to initiate the AHR circuit. The programmable nature of the auxiliary hairpin allows its easy adaption into other MTase assay by simply changing the recognition site. This proposed AHR circuit permits a sensitive, robust, and versatile analysis of MTase with the limit of detection (LOD) of 0.011 U/mL. Lastly, the AHR circuit could be utilized for MTase analysis in real complex samples and for evaluating the cell-cycle-dependent expression of MTase. This developed MTase-sensing strategy holds promising potential for biomedical analysis and clinical diagnosis.
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