Construction Of A Chemiluminescent Sensor For Sensitive Detective Of DNA Methyltransferase

Genomic DNA methylation is a predominant epigenetic modification in genomic DNA,playing critical roles in the regulation of gene transcription,chromatin structure,embryonic development,and cellular senescence.Recent research demonstrated that the DNA methylation patterns depend upon the DNA methyltransferase(DNA MTase)activity.DNA MTase may specifically recognize the short palindromic sequences and transfer a methyl group from S-adenosyl-L-methionine to target cytosine/adenine.The aberrant DNA MTase activity may destroy the normal DNA methylation patterns,leading to the occurrence of a variety of human diseases.Currently,most of the reported DNA MTase assays are based on a single type of DNA methyltransferas as the model,and few human methyltransferases have been identified,and few methods enable the detection of both bacteria methyltransferases and human methyltransferases.Herein,we construct a universal and label-free chemiluminescent sensor for accurate quantifification of both bacteria methyltransferases(e.g.,M.Sss I methyltransferase(M.Sss I MTase))and human methyltransferases(e.g.,DNA(cytosine-5)-methyltransferase 1,(Dnmt1))by integrating a dumbbell probe with Bss HII endonuclease-mediated rolling circle amplifification(RCA).We ingeniously design a structure-switchable dumbbell probe which integrates target-recognition,Bss HII endonuclease-cleavage,RCA and signal transduction in one probe for the detection of both M.Sss I MTase and Dnmt1.Dumbbell probe can be recognized by M.Sss I MTase and Dnmt1 for methylation,thus blocking the specific recognition cleavage of Bss HII endonuclease.The closed dumbbell probe can effectively prevent Exo I/Exo III-mediated digestion.Subsequently,the dumbbell probe may function as the circular template to induce RCA in the presence of d NTPs,primer and DNA polymerase,producing a long single stranded DNA(ss DNA)fragment with abundant repeated G-rich sequences which are complementary to the C-rich part of dumbbell template.The resultant G-rich products can fold into the G-quadruplex secondary structures for the formation of hemin-G-quadruplex DNAzymes with the assistance of cofactor hemin.These DNAzymes can catalyze the H₂O₂decomposition,and the decomposition product of H₂O₂can oxidize luminol to produce a distinct chemiluminescence signal.This chemiluminescent sensor can accurately quantify M.Sss I MTase in both 10%serum and various cell lysis buffffers,and even sensitively detect Dnmt1 activity in MCF-7 cells.Furthermore,this chemiluminescent sensor can be used to screen the inhibitors of Dnmt1 and M.Sss I MTase,with promising applications in disease diagnosis and drug discovery.