A Controlled T7 Transcription-driven Symmetric Amplification Cascade Machinery For Single-molecule Detection Of Multiple Repair Glycosylases

Genomic oxidation and alkylation are two most important forms of cytotoxic damages that may induce mutagenesis,carcinogenicity and teratogenicity.Human 8-oxoguanine（h OGG1）and alkyladenine DNA glycosylases（h AAG）are responsible for two major oxidative and alkylative damages repair,and their aberrant activities may cause repair deficiencies that are associated with a variety of human diseases including cancers.Due to the complicated catalytic pathways and hydrolysis mechanisms,simultaneous and accurate detection of multiple repair glycosylases has remained a great challenge.Herein,by taking advantage of unique features of T7-based transcription and intrinsic superiorities of single-molecule imaging technique,we demonstrate for the first time the development of a controlled T7 transcription-driven symmetric amplification cascade machinery for single-molecule detection of h OGG1 and h AAG.The presence of h OGG1 and h AAG can remove damaged 8-oxo G and deoxyinosine from the dumbbell substrate,respectively,resulting in the break of dumbbell substrate,the unfolding of two loops,and the exposure of two T7 promoters simultaneously.The T7promoters can activate symmetric transcription amplifications with unfolded loops as templates,inducing efficient transcription to produce numerous two different single-stranded RNA transcripts（i.e.,reporter probes 1 and 2）.Reporter probes 1 and 2 hybridize with signal probes1 and 2,respectively,to initiate duplex-specific nuclease-directed cyclic digestion of signal probes,liberating large amounts of Cy3 and Cy5 fluorescent molecules.The released Cy3 and Cy5 molecules can be simply measured by total internal reflection fluorescence（TIRF）-based single-molecule detection,with Cy3 signal indicating the presence of h OGG1 and Cy5 signal indicating the presence of h AAG.This method exhibits good specificity and high sensitivity with a detection of limit of 3.52×10^-8 UμL^-1for h OGG1 and 3.55×10^-7 UμL^-1 for h AAG,and it can even quantify repair glycosylases at the single-cell level.Moreover,it can be applied for the measurement of kinetic parameters,the screening of potential inhibitors,and the detection of repair glycosylases in human serum,providing a new paradigm for repair enzymes-related biomedical researches,drug discovery and clinical diagnosis.