Double-stranded endonuclease activity exploration through scanning substrate assay and its biosensing application

Double-stranded endonucleases (DENs) are widely used in the life sciences, and their catalytic activities are susceptible to the substrates and consequently impact the system outcomes. Traditionally, it has been believed that the maximum hydrolytic activity of DENs could be activated by their "well-defined" substrates when other experimental conditions were constant. In this work, we proposed that DEN's catalytic activity might be regulated by altering the number of complementary base pairs near the cleavage site. Firstly, taking APE1 as a proof of concept, the cleavage activity of APE1 with different substrates was evaluated by measuring the system fluorescence increase rate. An approximately one order of magnitude cleavage activity enhancement was obtained with the optimized substrate, compared to the "well-defined" substrate. Besides, we also found that the catalytic activity of APE1 was more susceptible to the number of base pairs following the 3'-end of the AP site. Furthermore, this enzyme catalytic activity scanning method was applied to the other five representative endonucleases (Nt.BsmAI, Nt. BstNBI, Nt. BbvCI, BsmAI, and Lba Cas12a nuclease). They also preferred non-canonical substrates, indicating the potential for widespread application of this strategy. Finally, the APE1 detection performance with the optimized substrate was determined in the buffer system and the cell lysate samples for biosensing applications. The detection sensitivity of APE1 was enhanced by approximately fivefold, reaching 0.01 U/mL. The APE1 level in normal and cancer cell lysate samples could also be distinguished using the "optimized" substrate.