Enzymes-mediated facile biosynthesis of G-quadruplexes for sensitive detection of apurinic/apyrimidinic endonuclease 1 activity

Apurinic/apyrimidinic endonuclease 1 (APE1) plays an essential role in regulating physiological functions and is a critical node in tumor progression. However, conventional detection methods for discriminating APE1 activity are hampered by labor-intensive procedures, multiple operations, and limited detection sensitivity. Herein, we have developed a terminal deoxynucleotidyl transferase (TdT)-mediated randomly arrayed G-quadruplexes (G4s) biosynthesis assay (TGBA) for facile and sensitive detection of APE1 activity. In this assay, the recognition DNA hairpin probe (rDHP) with an AP-site in the stem and modification of the phosphate group in 3′-end (3′-P) has been well-designed to be specifically cleaved by APE1 and then expose the new 3′-OH for rDHP template-free elongation by TdT. Following the rational proportion of the G-rich dNTP pool, the amplified and well-formed G4s can be obtained to bind to the G4 fluorescent dye, such as Thioflavin T (ThT). The effective, specific, high-contrast, and rapid fluorescence readout of TGBA has been validated by the in vitro analysis of APE1 activity and the evaluation of APE1 inhibitor, which enables the sensitive detection of APE1 activity with a lower detection limit of 0.001 U/mL and the detection linear range from 0.005 to 1 U/mL. Inspired by these results, we seek to explore the analysis of APE1 activity in different scenarios, including human serum and cell lysates. The unambiguously distinguishing capability of TGBA is proved by both detecting endogenous APE1 activity in cell lysates and exogenous APE1 activity in human sera. Collectively, TGBA will exhibit promising potential in the diagnosis of APE1-related diseases and the evaluation of drug efficacy.