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

Abstract

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.

Keywords: BSA; ExoIII; Exonuclease; G-quadruplex; Iridium; Luminescence; Metal complex; T4 PNK; T4 polynucleotide kinase; T7 Exo; T7 exonuclease; UDG; bovine serum albumin; calf-thymus DNA; ctDNA; double-stranded DNA; dsDNA; exonuclease III; lambda exonuclease; single-stranded DNA; ssDNA; uracil-DNA glycosylase; λ Exo.