A label-free ratiometric fluorescence strategy for 3′–5′ exonuclease detection

A label-free ratiometric fluorescent platform has been developed for the detection of 3′–5′ exonuclease utilizing two individual DNA conformation-specific dyes. Initially, the DNA substrate maintains a stable HP (hairpin probe) structure, resulting in bright fluorescence at 455 nm for 4′,6-diamidino-2-phenylindole (DAPI), which is able to insert into A–T base pairs of the HP structure. Upon the addition of 3′–5′ exonuclease (exonuclease III), the HP structure was digested to single-stranded DNA (ssDNA), and the fluorescence intensity at 455 nm was decreased. However the emission of N-methyl mesoporphyrin IX (NMM) at 610 nm was increased significantly due to the formation of G-quadruplex. This label-free method is realized by facile design and shows good selectivity for Exo III detection. More importantly, it also shows its potential as a platform for Exo III inhibitor screening.