Integration Of A Dumbbell Probe With Dual Signal Amplification For Ultrasensitive Detection Of Multiple DNA Methyltransferases

DNA methyltransferase(DNA MTase)can catalyze the DNA methylation reaction.It catalyzes the methylation of adenine/cytosine residues in specific regions of the genome and is involved in the establishment of epigenetic patterns.The aberration of DNA MTase activity is associated with a variety of cancers such as lung cancer,breast cancer,and colon cancer.Therefore,the ultrasensitive detection of DNA MTase and the screening of its inhibitors are of great significance for basic biomedical research and early clinical diagnosis.The conventional methods usually involve expensive equipment or complicated experimental procedures,and cannot be used to simultaneously detect multiple DNA MTases.We have demonstrated for the first time the integration of a dumbbell probe with dual signal amplification for the simultaneous detection of multiple DNA MTases.The stem of the dumbbell probe contains two specific recognition sequences for DNA MTases.After being catalyzed by DNA MTase,dumbbell probe can be specifically recognized and cleaved by endonuclease to release the primer.The primer specifically hybridizes with the circular template and triggers rolling circle amplification(RCA).The product of RCA can specifically hybridize with the signal probe to initiate the Endonuclease?(Endo IV)-assisted signal amplification reaction,and generate a strong fluorescent signal.This method has the following advantages:(1)the designed dumbbell probe integrates target-recognition and signal amplification into one multifunctional probe,greatly simplifying the probe design and reducing the experimental cost;(2)this assay may be performed in a homogeneous format without the involvement of any washing and separation steps;(3)the integration of dumbbell probe and RCA can eliminate the high background signal induced by non-specific amplification;(4)this method exhibits good specificity and high sensitivity with a limit of detection of 2.2×10^-5U/m L for DNA adenine methyltransferase(Dam MTase)and 3.2×10^-6 U/m L for CpG Methyltransferase(M.SssIMTase).In addition,this method can be used to screen inhibitors of enzymes and simultaneously detect Dam MTase and M.SssI MTase in complex biological samples.Importantly,this method can be easily extended to the simultaneous detection of multiple endonucleases/enzymes and the screening of the corresponding inhibitors by simply changing the two recognition sequences,holding great potential in biomedical research,disease diagnosis,and drug discovery.