A molecular gated HRCA quick sensing system intelligently controlled by APE1

Apurinic/apyrimidinic endonuclease 1 (APE1), as a bifunctional enzyme, can not only remove apurinic/apyrimidinic sites (AP-sites) to maintain gene stability but can also regulate the activity of transcription factors. It is an important biomarker of malignant tumours and a new anticancer drug target. However, rapid, simple, and sensitive APE1 detection strategies are still lacking. For this purpose, we designed a molecular gated hyperbranched rolling circle amplification (HRCA) quick sensing system. The identification probe consists of a padlock probe and a ligation probe with an AP-site, which neatly integrates the processes of circular template connection and target-specific recognition. This prevents the Exonuclease III (Exo III) from interfering with the signal. APE1 would swiftly unlock the closed mode and trigger HRCA after excising the AP-site on the identification probe. This would activate the entire system and cause the signal to exhibit exponential amplification in a short period. Under optimized conditions, the system exhibited a favourable linear relationship within the range of 0.0001 to 0.2 U/mL, with a detection limit up to 0.0001 U/mL. Additionally, the technique also performed well in complex human serum and human cell protein extracts, and it may be used to screen APE1 inhibitors and assess the effectiveness of inhibition. The molecular gated HRCA quick sensing method presented herein would help in the development of new anticancer medications and has potential therapeutic applications.