Simple Detection of DNA Methyltransferase with an Integrated Padlock Probe

Abstract

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 SYBRTM 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.

A Short Review on Miniaturized Biosensors for the Detection of Nucleic Acid Biomarkers

“…Wang et al [ 114 ] designed a padlock probe (PP) to sense nucleic acid MTases, which syndicates target identification with the rolling circle amplification (RCA) technique without refinement or using other probes. By introducing MTase to the PP, which served as the enzyme’s substrate, the PP was methylated and protected against the cleavage reactions of HpaII, lambda exonuclease, and ExI, as well as absorption, and the PP started RCA.…”

“… ( A ) Schematic representation of the components, including digitally barcoded nanoparticles, microfluidic cartridge, and an integrated instrument for the detection of molecular biomarkers [ 112 ] ( B ) Working principle of the nucleic acid-based nanotweezer sensor [ 113 ] ( C ) MTase activity uncovering by padlock probe with RCA technique [ 114 ] ( D ) Multi-view of the DMF device and accumulation [ 115 ]. …”

Fluorescence turn-off strategy for sensitive detection of DNA methyltransferase activity based on DNA-templated gold nanoclusters

“…In recent years, the detection of methyltransferases has been roughly divided into two categories. The first method depends on the amplification processes of rolling circle amplification (RCA) [ 14 , 15 ], hybridization chain reaction (HCR) [ 16 , 17 ], and strand displacement amplification (SDA) [ [18] , [19] , [20] , [21] ], which aim to amplify the signal, improve the detection efficiency, and achieve lower detection limits. The second method is based on novel materials, such as nanoparticles [ 22 , 23 ] and quantum dots [ 24 , 25 ], which are used as signal outputs for molecular detection.…”