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  • In the present study we implemented the dendritic RCA induce

    2019-08-08

    In the present study, we implemented the dendritic RCA-induced formation of amplifying DNAzyme catalytic labels strategy for sensitive and accurate assay of MTase activity and the detection of DNA methylation. In comparison with the traditional RCA process, the proposed strategy achieved high amplification. Moreover, incorporating the Mg2+-dependent DNAzyme as the recognition event to readout of the sensing process, this method provides a versatile tool for screening of suitable inhibitors for Dam MTase.
    Experimental
    Results and discussion
    Conclusion In conclusion, a RCA-stimulated dendritic synthesis of catalytic nucleic DMP 777 in (DNAzymes) system has been developed for methylation detection and MTase activity assay. In comparison with the traditional methods, this approach utilized the functional hairpin and the autonomous formation of DNAzymes to achieve the isothermal and exponential amplification of D-RCA for signal enhancement, which averts complicated PCR amplification and expensive instruments. As a result, this method exhibits excellent specificity and high sensitivity with a detection limit of 0.36 U mL−1. In addition, we also demonstrated the application of this assay for evaluation and screening of the inhibitors of MTase, which may be helpful for the discovery of anticancer drugs. Therefore, the developed method has a great potential application for DNA methylation related clinical diagnosis and drug screening.
    Acknowledgments This work was supported by the National Natural Science Foundation of China (21275003, 21505082), a project of Shandong Province Higher Educational Science and Technology Program (Grant J16LC10)and the Open Funds of the Shandong Province Key Laboratory of Detection Technology for Tumor Markers (KLDTTM2015-5).
    Introduction DNA methylation, a common gene protection approach, plays an important role in both prokaryotes and eukaryotes. It is extremely important to lots of normal cellular processes including development, transposon silencing, gene regulation, and X chromosome inactivation, among others [1], [2]. Alterations of methyltransferases (MTases) activity may lead to aberrant DNA methylation patterns, which are associated with several genetic diseases and various types of cancer [3], [4]. DNA MTases have become potential therapeutic targets and predictive biomarkers in a variety types of cancer of lung [5], gastric [6], colon [7], prostate [8] and so on. Moreover, abnormalities in MTase activity usually occur far before other signs of malignancy and could thus be used for early cancer diagnosis [9], [10]. Thus, sensitive activity assay and inhibitor (anti-methylation drugs) screening for MTases represent a valuable strategy to both clinical diagnostics and therapeutics. In addition, it’s also crucial in understanding how MTase activity contributes to cancer initiation and progression. The conventional methods for DNA MTase activity determination include radioactive labeling [11], [12] liquid chromatography/mass spectrometry [13], high performance capillary electrophoresis (HPCE) [14], fluorescence [15], [16], [17], [18], colorimetric [19], [20], electrochemical [21], [22], [23], electrochemiluminescence [24], [25] and chemiluminescence [26]. In recent years, various advanced techniques have been proposed. For example, Cai et al., reported an approach by coupling the fluorescence quenching of graphene oxide with site-specific cleavage of a restriction endonuclease [17]. Gao and coworkers described an electrochemical assay employing a threading intercalator, N,N′-bis(3-propylimidazole)-1,4,5,8-naphthalene diimide (PIND) functionalized with electrocatalytic redox Os(bpy)2Cl+ moieties (PIND-Os), for signal amplification [27]. Nevertheless, these methods still carry obvious drawbacks, such as radioisotope labeled substrate, expensive antibodies, fluorescently labeled substrates, bulky detection equipment, time-consuming sample preparation, or inflexible detection schemes. Therefore, further efforts are needed in the development of simple and sensitive MTase activity assays.