G-quadruplex-based fluorometric biosensor for label-free and homogenous detection of protein acetylation-related enzymes activities

Abstract Reversible protein acetylation, one of the key types of post-translational modifications, is composed of histone acetylation and deacetylation, which is typically catalyzed by histone acetyltransferases (HATs) and histone deacetylases (HDACs) respectively. Herein, a label-free fluorescent method has been established for the homogeneous bioassay of HAT/HDAC activity and respective inhibitors. The proposed approach is primarily based on the electrostatic interaction between G-quadruplexes (G4s) and acetylation-related peptides, which results in marked change of fluorescent intensity of G4/Thioflavin T (ThT) complexes. This HAT (p300) activity assay is exceedingly sensitive and selective, with a linear range from 0.1 to 120 nM and a detection limit of 0.05 nM. Moreover, this biosensor is feasible to detect the HDAC (Sirt1) activity with a linear range from 1 to 450 nM and a detection limit of 1 nM. The potency of this assay is further demonstrated by detecting HAT/HDAC activity in cell lysates and evaluating HAT and HDAC-targeted inhibitors, C464 and EX 527, respectively. The proposed assay is convenient, label-free and cost-efficient, which is promising for HAT/HDAC-targeted epigenetic research and pharmaceutical development.