Label-free luminescence switch-on detection of hepatitis C virus NS3 helicase activity using a G-quadruplex-selective probe

Abstract

A series of luminescent Ir(iii) complexes were synthesised and evaluated for their ability to act as luminescent G-quadruplex-selective probes. The Ir(iii) complex 9, [Ir(phq)₂(phen)]PF₆ (where phq = 2-phenylquinoline; phen = 1,10-phenanthroline), exhibited high luminescence in the presence of G-quadruplex DNA compared to dsDNA and ssDNA, and was employed to construct a label-free G-quadruplex-based assay for hepatitis C virus NS3 helicase activity in aqueous solution. Moreover, the application of the assay for screening potential helicase inhibitors was demonstrated. To our knowledge, this is the first G-quadruplex-based assay for helicase activity.

Fig. 1. Chemical structures of the luminescent Ir(iii) complexes 1–17 that were synthesised and evaluated in this study.

Scheme 1. Schematic diagram of the luminescent switch-on assay to monitor the duplex-DNA unwinding activity of helicase using a G-quadruplex-selective probe.

Fig. 2. (a) Chemical structure of complex 9; (b) G4-FID titration curves of DNA duplex ds17 and Pu27 G-quadruplex in the presence of increasing concentrations of complex 9 in Tris–HCl buffer. The DC₅₀ value is equal to the half-maximal concentration of compound required to displace 50% TO from DNA.

Fig. 3. (a) Luminescence spectra of the 9/G4-quadruplex system in response to increasing concentrations of helicase: 0, 0.09, 0.18, 0.27, 0.36, 0.45, 0.54, 0.72, and 0.9 μM. (b) The relationship between luminescence intensity at λ = 571 nm and helicase concentration. Inset: linear plot of the change in luminescence intensity at λ = 571 nm vs. helicase concentration.

Fig. 4. (a) Luminescence response of the system in the presence of helicase, S1, Endo, DpnI, ExoI, EcoRI, RNase, DNase and SSB. (b) Luminescence spectra of the 9/G-quadruplex system in a reaction system containing 0.5% (v/v) cell extract in response to various concentrations of helicase: 0, 0.18, 0.36, 0.45, 0.54, 0.72, and 0.9 μM. (c) Relative luminescence intensities of the system in the presence of increasing concentrations of ciprofloxacin: 0, 1, 2.5, 5, 10, and 20 μM. (d) Emission spectra of complex 9 in the absence and presence of 20 μM ciprofloxacin. (e) Relative luminescence response of the 9/G-quadruplex ensemble upon the addition of 20 μM ciprofloxacin. (f) Luminescence response of the system treated with 0.8 μM helicase and 10 μM of suramin, TBBT and ciprofloxacin.