Ultrasensitive Detection Of Intracellular Uracil DNA Glycosylase

The structural characteristics of DNA bases may be affected by many factors.If these mutagenesis are not repaired in time,it will lead to the instability of genome and induce cancer.In this regard,biological organisms have corresponding repair methods,such as the base-excision repair pathway(BER),which is a highly conserved DNA cutting activity that is initiated by DNA glycosylase.Uracil-DNA glycosylase(UDG)is an important base-excision repair enzyme responsible for the repair of uracil-induced DNA lesion and the maintenance of genomic integrity,while the aberrant expression of UDG is associated with a variety of cancers.Thus,the accurate detection of UDG activity is essential to biomedical research and clinical diagnosis.At present,the isothermal amplification assay of nucleic acids is a promising ultrasensitive method,which is a simple,ultrasensitive detection assay can be applied to various types of targets,such as proteins,cells,small molecules and ions.Here,we develop a fluorescent method for ultrasensitive detection of UDG activity using excision repair-initiated enzyme-assisted bicyclic cascade signal amplification.This assay involves(1)UDG-actuated uracil-excision repair,(2)excision repair-initiated nicking enzyme-mediated isothermal exponential amplification,(3)ribonuclease H(RNase H)-induced hydrolysis of signal probes for generating fluorescence signal.The presence of UDG enables the removal of uracil from U·A pairs and generates an apurinic/apyrimidinic(AP)site.Endonuclease IV(Endo IV)subsequently cleaves the AP site,resulting in the break of DNA substrate.The cleaved DNA substrate functions as both a primer and a template to initiate isothermal exponential amplification,producing a large number of triggers.The resultant trigger may selectively hybridize with the signal probe which is modified with FAM and BHQ1,forming a RNA-DNA heterogeneous duplex.The subsequent hydrolysis of RNA-DNA duplex by RNase H leads to the generation of fluorescence signal.This assay exhibits ultrahigh sensitivity with a detection limit of 0.0001 U/mL,and it can even measure UDG activity at the single-cell level.Moreover,this method can apply for the measurement of kinetic parameters and the screening of inhibitors,thereby providing a powerful tool for DNA repair enzyme-related biomedical research and clinical diagnosis.