Detection of 3'→5' exonuclease activity using a metal-based luminescent switch-on probe

A luminescent iridium(III) complex has been discovered to be selective for G-quadruplex DNA, and was employed in a label-free G-quadruplex-based detection assay for 3'→5' exonuclease activity in aqueous solution. A proof-of-concept of this assay has been demonstrated by using prokaryotic exonuclease III (ExoIII) as a model enzyme. In this assay, a G-quadruplex-forming hairpin oligonucleotide (hairpin-G4 DNA, 5'-GAG₃TG₄AG₃TG₄A₂GCAGA₂G₂ATA₂CT₂C₄AC₃TC₄AC₃TC-3') initially exists in a duplex conformation, resulting in a low luminescence signal due to the weak interaction between the iridium(III) complex and duplex DNA. Upon digestion by ExoIII, the guanine-rich sequence is released and folds into a G-quadruplex, which greatly enhances the luminescence emission of the iridium(III) probe. This method was highly sensitive for 3'→5' exonuclease over other DNA-modifying enzymes.