Construction Of Self-directed Replication System For Glycosylase Assay

Genomic DNA,constituted of Watson–Crick-pairing bases,is selected over the course of evolution as the ideal carrier for preserving and transmitting the genetic information.However,the genomic DNAs are frequently damaged by various exogenous and endogenous factors.Persistent DNA damage leads to genomic instability and induces cancer.Cells have evolved multiple protection mechanisms to specially repair a wide range of DNA lesions.Among them,the base-excision repair?BER?pathway is the most important repair mechanism,and DNA glycosylase represents one of the most important repair enzymes.In the BER pathway,DNA glycosylase initiates a critical first step by cleaving the N-glycosidic bond between the damaged base and the DNA carbon backbone.The dysregulation of DNA glycosylases is closely linked to multiple human diseases.The development of new methods for accurate and sensitive detection of DNA glycosylation enzymes is of great significance for the study of carcinogenesis and the development of anti-cancer drugs.Inspired by the natural repair mechanism in vivo,we demonstrate for the first time the construction of self-directed replication system for label-free and real-time sensing of repair glycosylases with zero background.The presence of DNA glycosylase can catalyze the excision repair of damaged base,successively autostarting the self-directed replication through recycling polymerization extension and strand-displacement DNA synthesis for the generation of exponentially amplified dsDNAs.The resultant dsDNA products can be label-free and real-time monitored with SYBR Green I as the fluorescent indicator.Owing to the high efficiency of self-directed exponential replication and the absolute zero background resulting from the efficient inhibition of nonspecific amplification induced by multiple primer-dependent amplification,this strategy exhibits high sensitivity with a detection limit of 1×10^-8 U/?L in vitro and 1 cell in vivo,and it can be further applied to screen inhibitors,quantify DNA glycosylase from diverse cancer cells,and even monitor various repair enzymes by simply changing the specific damaged base in DNA template.Importantly,this assay can be performed in a label-free,real-time and isothermal manner with the involvement of only a single type of polymerase,providing a simple,robust and universal platform for repair enzymes-related biomedical research and clinical diagnosis.