DNAzyme and rGO based fluorescence assay for Fpg activity analysis, drug screening, and bacterial imaging

摘要

Due to the significant role of formamidopyrimidine DNA glycosylase (Fpg) in physiological processes and DNA oxidative damage-related diseases, it is essential to establish sensitive methods for monitoring the Fpg activity in vitro and in vivo so as to illustrate its concrete role in these events. In this work, a sensitive, simple and reliable fluorescence assay was developed by taking the advantages of DNAzyme assisted cascade signal amplification and ultra-high fluorescence quenching efficiency of reduced graphene oxide (rGO). This detection system consisted of DNAzyme, rGO and fluorescence probe allows the activity of Fpg to be detected in a linear range from 0 to 80 U/mL with a detection limit of 0.66 U/mL. With the help of this method, 11 natural compounds were screened, and 7 compounds were identified as activators of Fpg. More importantly, the developed assay was used to monitor the activity of Fpg through fluorescence imaging in living Escherichia coli for the first time. The imaging results visually demonstrated the dynamic activation effect of natural compound Ginsenoside Re on the Fpg of Escherichia coli. In summary, these results indicated that this DNAzyme and rGO based fluorescence assay provides a potent strategy for Fpg quantitative assay in vitro and real-time monitoring in living bacteria, which holds great potential for applying on biological study and Fpg-targeted drug screening.