Kinetics of quadruplex to duplex conversion

Abstract

The equilibrium between G-quadruplex (G4) and duplex DNA structures is a fundamental question whenever G4 are considered within a genomic context. In this study, we performed a detailed thermodynamic and kinetic analysis of this equilibria using a new fast and high throughput technique for the screening of G4 structures. This assay examines the isothermal stability of G-quadruplexes in the presence of complementary strands monitoring the unwinding process by fluorescence techniques. Unlabelled G4 structures were used in order to avoid any thermodynamic effect that fluorophores could have on the G4 stability. The assay was applied to investigate the effect that flanking sequences can have on the thermodynamic stability of the quadruplex motifs. Interestingly, the presence of adjacent bases to the G4 structure facilitates the recognition of the complementary strand accelerating the G4 unfolding reaction. The simplicity of the systems employed and the use of fluorescence emission allowed the use of high throughput techniques and to monitor the opening reaction in real time in a "pseudo" label-free method. This G4 opening reaction may be easily implemented as a new isothermal assay for the screening of G4 structures, G4 ligands or G4-binding proteins.

Keywords: DNA kinetics; Duplex-quadruplex equilibrium; G4 unfolding; Screening assay.