Enzymatic DNA repairing amplification-powered construction of an Au nanoparticle-based nanosensor for single-molecule monitoring of cytosine deaminase activity in cancer cells.

APOBEC3A (A3A) is a cytidine deaminase with critical roles in molecular diagnostics. Herein, we demonstrate the enzymatic DNA repairing amplification-powered construction of an Au nanoparticle-based nanosensor for single-molecule monitoring of A3A activity in cancer cells. Target A3A can convert cytosine (C) in substrate probe to uracil (U), and then the template binds with substrate probe to form a dsDNA containing U/A base pairs. Uracil DNA glycosylase (UDG) excises the U base to produce an apurinic/apyrimidinic (AP) site that can be cleaved by apurinic/apyrimidic endonuclease 1 (APE1) to obtain the substrate fragment with 3'-OH end. Subsequently, the substrate fragment initiates cyclic enzymatic repairing amplification (ERA), releasing trigger-1 and trigger-2. The resultant trigger-1 can act as the primer to induce multiple cycles of cyclic ERA, producing numerous trigger-1 and trigger-2. The hybridization of trigger-2 with signal probe forms the dsDNA duplexes with an AP site, inducing the cyclic cleavage of signal probes by APE1 to release abundant Cy5 molecules from the AuNPs. Released Cy5 molecules can be easily quantified by single-molecule imaging. This nanosensor allows for specific and sensitive detection of A3A activity with a detection limit of 0.855 aM, and it can further measure kinetic parameters, screen inhibitors, and quantify endogenous A3A activity at the single-cell level, with prospect application in disease diagnostics and therapy.