A time-dependent fluorescent biosensor for uracil-DNA glycosylase detection based on the uracil inhibition effect towards archaebacterial DNA polymerases

Abstract

Uracil-DNA glycosylase (UDG) plays important role in DNA lesion repair. It can remove the uracil lesion in DNA and is involved in multiple biological processes. UDG activity detection methods based on DNA probe are developed fast recently due to its high sensitivity and simplicity. They typically use two strategies: the conformational change or the local environmental change of the DNA probe after UDG cleavage. Here we proposed a new strategy for UDG activity detection, which utilized uracil-containing oligo to inhibit archaebacterial DNA polymerases, and after UDG treatment the archaebacterial DNA polymerases could be activated to generate linear or exponential amplification fluorescent signal. The exponential methods showed a time-dependent signal output and achieved limit of detection of 0.0025 U/mL. More importantly, the feature of time-dependence allowed us to easily modulate the resolution or the dynamic range of our method to meet different demands in various occasions. The strategy was also demonstrated to be highly selective toward DNase and exonucleases and had been applied to detect the UDG activity in cell lysate.