Label-free fluorescence strategy for sensitive detection of exonuclease activity using SYBR Green I as probe

A label-free and sensitive fluorescence assay for exonuclease activity is developed using commercially available SYBR Green I (SG) dye as signal probe. A proof-of-concept of this assay has been demonstrated by using exonuclease III (Exo III) as a model enzyme. In this assay, double-stranded DNA (dsDNA) can bind SG, resulting in a strong fluorescence signal of SG. Upon the addition of Exo III, dsDNA would be digested, and SG emits very weak fluorescence. Thus, Exo III activity can be facilely measured with a simple fluorescence reader. This method has a linear detection range from 1 U/mL to 200 U/mL with a detection limit of 0.7 U/mL. This label-free approach is selective, simple, convenient and cost-efficient without any complex DNA sequence design or fluorescence dye label. The method not only provides a platform for monitoring activity and inhibition of exonuclease but also shows great potential in biological process researches, drug discovery, and clinic diagnostics.