Research On Sensitive Detection Of DNA Methyltransferase By Hyperbranched Amplification

DNA methylation plays important role in cell proliferation,senescence,and gene transcription.Both excessive methylation?hypermethylation?and deficient methylation?hypomethylation?have been identified in a variety types of cancers.DNA methyltransferases?MTases?may specifically recognize the short palindromic sequences and transfer a methyl group from S-adenosyl-L-methionine to target cytosine/adenine.The aberrant DNA methylation is linked to the abnormal DNA MTase activity,and some DNA MTases have become promising targets of anticancer/antimicrobial drugs.However,the reported DNA MTase assays often involve laborious operation,expensive instruments,and radio-labeled substrates.Here,we develop a simple and label-free fluorescent method to sensitively detect DNA adenine methyltransferase?Dam?on the basis of terminal deoxynucleotidyl transferase?TdT?-activated Endonuclease IV?Endo IV?-assisted hyperbranched amplification.We design a hairpin probe with a palindromic sequence in the stem as the substrate and a NH₂-modified 3?end for the prevention of nonspecific amplification.The substrate may be methylated by Dam and subsequently cleaved by DpnI,producing three single stranded DNAs,two of which with 3?-OH termini may be amplified by hyperbranched amplification to generate a distinct fluorescence signal.The enhanced sensitivity can be ascribed to three factors:?1?highly efficient TdT-activated Endo IV-assisted hyperbranched amplification induces enhanced fluorescence signal;?2?the modification of 3?termini of hairpin probe and assistant probe by NH₂ efficiently prevents the TdT-activated nonspecific amplification;and?3?high exactitude of TdT enables the occurrence of amplification only in the presence of free 3?-OH termini and Endo IV can only hydrolyze the intact AP sites,resulting in zero background signal.This method exhibits excellent selectivity and high sensitivity with a limit of detection of 0.003U/mL for pure Dam and 9.61×10^-6 mg/mL for Dam in E.coli cells.Moreover,it can be used to screen the Dam inhibitors,holding great potentials in disease diagnosis and drug development.