Sensitive Detection Of DNA Methyltransferase Based On Isothermal Amplification

DNA methylation participates in the crucial epigenetic processes and gets involved in many essential biological processes including cell proliferation,genomic imprinting,and gene transcription in all eukaryotic and prokaryotic cells.Aberrant methylation is demonstrated to closely relate to many human diseases,including systemic lupus erythematosus,Hodgkin's lymphoma,leukemia,and even lethal cancers.Abnormal MTase expression has thus been recognized as an emerging cancerous indicator,and the associated methyltransferase has been considered as a new pharmacological object in antitumor treatments.Traditional single-stage amplification means often achieve linear amplification that might not fulfill the increasing demands for detecting trace amount of target.It is desirable to construct multistage cascaded amplifiers that allow for enhanced signal amplifications.Taking advantage of signal amplification and fluorescence,we established two signal amplification strategies for detecting MTases based on F|rster resonance energy transfer FRETU mechanism.These are listed as follow in detail:Firstly,we have successfully constructed a nonenzymatic cascaded CHA-HCR method for amplified MTase assay and inhibitor screening by rational integration of CHA and HCR circuits.An auxiliary hairpin H_Mwas engineered and utilized as Hpa II substrate and thus could release a fragmented product to trigger the CHA-HCR-mediated FRET signal generation.Yet the M.Sss I-methylated substrate could not be cleaved by Hpa II enzyme,thus prohibiting the subsequent CHA-HCR process.As an isothermal homogeneous sensing strategy,this CHA-HCR amplifier enables the selective and ultrasensitive detection of M.Sss I with a low detection limit corresponding to 1.2×10^-4U/m L.The CHA-HCR circuit was able to screen MTase inhibitors,which were of great importance for future clinical diagnosis and new drug design.The CHA-HCR-based MTase assay strategy is a universal detecting system for probing more different enzyme-mediated biotransformations,and for analyzing other restriction enzymes,e.g.,Dam,by altering merely the specific sequence of the auxiliary hairpin substrate.The high specificity of the multiple-guaranteed recognitions of MTase/endonuclease,together with the synergistic amplification feature of concatenated CHA-HCR strategy,contributes to the ultrasensitive detection of MTase and its inhibitors in complex biological environments,including serum and E.coli cells.Our CHA-HCR circuit is thus anticipated to improve the signal gain of the system for accurately detecting other important biomarkers,and thus holds great promise for cancerous treatment and biomedical research.Secondly,we designed a multiple signal amplification strategy for sensitive DNA MTases analysis by coupling a target-initiated HCR with Mg²⁺-DNAzyme-feedback mediated target regeneration.In the absence of M.Sss I MTase,Hpa II endonuclease could specifically recognize the auxiliary hairpin substrate and then catalytically cleave the corresponding recognition site,releasing a DNA fragment that triggers the HCR-DNAzyme-mediated FRET transduction.Yet the M.Sss I-methylated hairpin substrate could not be cleaved by Hpa II enzyme,and thus prohibiting the HCR-DNAzyme-mediated FRET generation.This provides a substantial signal difference with that of MTase-absent system.The HCR-DNAzyme-based MTase assay strategy is a universal detecting system for probing more different enzymes,e.g.,Dam,by altering merely the specific sequence of the hairpin substrate.The method can be successfully implemented in screening MTases inhibitors and quantifying endogenous Dam in E.coli cells.Our system demonstrates massive potential of the proposed strategy in clinical diagnosis and cancerous treatment.