A novel self-feedback biosensor for label-free detection of APE1 by primer exchange reaction and rolling circle amplification/dimeric G-quadruplex

Abstract

Human apurinic/apyrimidinic endonuclease 1(APE1), a critical DNA base excision repair enzyme, plays a fundamental role in nucleotide damage repair and transcriptional regulation, emerging as a vital target for anticancer therapy. Therefore, sensitive and label-free detection of APE1 is necessary. Herein, a primer exchange reaction (PER) cascade rolling circle amplification (RCA)/ dimeric G-quadruplex (G4) self-feedback biosensor was developed to detect APE1. Initially, a gated hairpin (GHP) was meticulously crafted for the specific recognition of APE1, in which GHP is not only the recognition substrate but also acts as a catalytic hairpin (CHP) and a primer for PER. The target recognizes and cleaves the GHP, instigating the PER cycle and engendering extended single-strands DNA, thereby activating RCA reaction. Subsequently, the resulting protracted tandem G-rich ssDNA conformationally adapts into a pronounced G4 dimeric structure through affinity binding to K+ and thioxlavin T (ThT), culminating in a stronger fluorescence signal. The design of GHP and G4 dimer greatly improves the signal-to-noise ratio and detection sensitivity, with a wide linear range (0.02–30 U/mL) and a low limit of detection (0.001 U/mL). Moreover, this protocol exhibits robust resistance to interference and holds significant potential in pharmaceutical evaluation, which may provide a reliable analytical method for the detection of APE1 and the subsequent clinical assessment of associated malignancies.