Detection of nicking endonuclease activity using a G-quadruplex-selective luminescent switch-on probe

A series of luminescent Ir(III) complexes were synthesised and evaluated for their ability to act as G-quadruplex-selective probes. A novel Ir(III) complex was found to be highly selective for G-quadruplex DNA, and was employed in a label-free G-quadruplex-based detection assay for nicking endonuclease activity in aqueous solution. A proof-of-concept of this probe has been demonstrated by using Nb.BbvCI as a model enzyme. In this assay, a DNA substrate comprised of oligonucleotides ON1 (5'-GTG(3)TAG(3)CG(3)T(2)G(2)CTGAG(2)TGA-3') and ON2 (5'-TCAC(2)TCAGC(2)A(2)C(2)-3') initially exists in a duplex conformation, resulting in a low luminescence signal due to the weak interaction between the Ir(III) complex and duplex DNA. Upon cleavage by Nb.BbvCI, the guanine-rich sequence is released and folds into a G-quadruplex, which greatly enhances the luminescence of the Ir(III) probe. This method was highly sensitive for Nb.BbvCI over other DNA-modifying enzymes.