Simple Detection of DNA Methyltransferase with an Integrated Padlock Probe

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.

Automated summary

DNA methyltransferases can be used as biomarkers for genetic diseases. A padlock probe was designed to detect the activity of DNA methyltransferases without the need for purification or other probe assistance. This method has high sensitivity and a wide linear range, and can be applied in complex biological environments, as well as for screening drugs or inhibitors for methyltransferases.