Label-free detection of exonuclease III activity and its inhibition based on DNA hairpin probe

In this paper, we have developed a label-free and rapid fluorescence assay for the detection of exonuclease III (exo III) activity via thioflavin T (ThT) as the G-quadruplex inducer. In this assay, a hairpin probe (HP) with a 5′-guanine-rich (G-rich) sequence is employed as the substrate for exo III. In the presence of exo III, HP can be digested at 3′-OH termini releasing 5′-G-rich sequence. Then, the 5′-G-rich sequence folds into a G-quadruplex, which can be recognized quickly by the ThT dye resulting in an increase in fluorescence emission. This strategy can detect exo III activity as low as 0.5 U/mL. This assay is simple and of low cost without the requirement of labeling with a fluorophore-quencher pair.