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标题：Simple Detection of DNA Methyltransferase with an Integrated Padlock Probe
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作者：Yuehua Wang ; Yingli Han ; Fangyu Zhou 等
期刊名称：Biosensors
电子版ISSN：2079-6374
出版年度：2022
卷号：12
期号：8
DOI：10.3390/bios12080569
语种：English
出版社：MDPI Publishing
摘要：DNA methyltransferases (MTases) can be regarded as biomarkers, as demonstrated by many studies on genetic diseases. Many researchers have developed biosensors to detect the activity of DNA MTases, and nucleic acid amplification, which need other probe assistance, is often used to improve the sensitivity of DNA MTases. However, there is no integrated probe that incorporates substrates and template and primer for detecting DNA MTases activity. Herein, we first designed a padlock probe (PP) to detect DNA MTases, which combines target detection with rolling circle amplification (RCA) without purification or other probe assistance. As the substrate of MTase, the PP was methylated and defended against HpaII, lambda exonuclease, and ExoI cleavage, as well as digestion, by adding MTase and the undestroyed PP started RCA. Thus, the fluorescent signal was capable of being rapidly detected after adding SYBR TM Gold to the RCA products. This method has a detection limit of approximately 0.0404 U/mL, and the linear range was 0.5–110 U/mL for M.SssI. Moreover, complex biological environment assays present prospects for possible application in intricacy environments. In addition, the designed detection system can also screen drugs or inhibitors for MTases.
关键词：DNA methyltransferases;padlock probe;rolling circle amplification;fluorescence