Ligation-dependent Cycling Signal Amplification For Sensitive Detection 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.The accurate quantification of DNA methylation is not only of high importance to early diagnosis,prognosis,and therapy of cancers,but also of great significance to biochemical research and drug development.DNA methyltransferase?DNA MTase?is responsible for the genomic DNA methylation modification,and the aberrant DNA MTase activity may destroy the normal DNA methylation patterns,inducing a variety of cancers including lung,breast and liver cancers.Currently,conventional methods for DNA MTase assay are usually cumbersome and laborious with poor sensitivity.Alternatively,some signal amplification strategies are employed to improve the sensitivity,but they suffer from poor specificity and consequently limited sensitivity due to the nonspecific amplification.Therefore,the development of accurate and sensitive methods for DNA MTase assay is essential to the understanding of the carcinogenesis mechanism and the discovery of anticancer drugs.Herein,we develop for the first time a new fluorescence method to specifically and sensitively detect DNA MTase activity on the basis of single-ribonucleotide repair-mediated ligation-dependent cycling signal amplification.Ribonuclease HII?RNase HII?can excise the single ribonucleotide,resulting in the cyclic cleavage of signal probes and the generation of an enhanced fluorescence signal.Taking advantage of the high specificity of RNase HII-catalyzed single-ribonucleotide excision and the high amplification efficiency of cyclic ligation-dependent SDA,this assay exhibits the highest sensitivity reported so far with a detection limit of 4.8×10^-6 U/mL and a large dynamic range of 5 orders of magnitude.Moreover,this method can be used for the discrimination of Dam MTase from other DNA MTases,the accurate quantification of Dam MTase activity in E.coli cells,and the screening of Dam MTase inhibitors.The method holds great promise in the study of carcinogenesis mechanism and the discovery of anticancer drugs.