Activity assay and intracellular imaging of APE1 assisted with tetrahedral DNA nanostructure modified-dnazyme and molecular beacon

摘要

Human apurinic/apyrimidinic endonuclease 1 (APE1) is an emerging target for sensitizing tumor cells to radiation and chemotherapy due to its critical function in DNA repair and reduction-oxidation regulation. Thus, accurate detection and intracellular imaging of APE1 hold great promise for drug screening, cancer diagnosis and prognosis evaluation. Here, a DNAzyme modified tetrahedral DNA nanostructure (DZ-TDN) and molecular beacon (MB) incorporated biosensing system was developed to reliably monitor intracellular APE1 activity. DZ-TDN was self-assembled from four single-stranded DNAs, one of which contained an extrude DNAzyme sequence on the 5'-end. At normal status, DNAzyme activity was plugged by performing a duplex with a blocker strand containing an AP site. The digestion of APE1 on this site can restore DNAzyme activity, which consequently trigger MB cleavage recycle to amplify detection signal. The results indicated that the proposed fluorescence method showed a linear response ranged for APE1 from 0.01 to 1 UmL~(-1) with a limit of detection (LOD)of 0.01 U mL~(-1) (S/N = 3). Moreover, the strategy was successfully used for effectors screening and intracellular imaging of APE1 with high reliability.