A novel biosensor based on tetrahedral DNA nanostructure and terminal deoxynucleotidyl transferase-assisted amplification strategy for fluorescence analysis of uracil-DNA glycosylase activity

Tetrahedral DNA nanostructure (TDN), as a classical bionanomaterial, which not only has excellent structural stability and rigidity, but also possesses high programmability due to strict base-pairs complementation, is widely used in various biosensing and bioanalysis fields. In this study, we first constructed a novel biosensor based on Uracil DNA glycosylase (UDG) -triggered collapse of TDN and terminal deoxynucleotidyl transferase (TDT)-induced insertion of copper nanoparticles (CuNPs) for fluorescence and visual analysis of UDG activity. In the presence of the target enzyme UDG, the uracil base modified on the TDN were specifically identified and removed to produce an abasic site (AP site). Endonuclease IV (Endo.IV) could cleave the AP site, making the TDN collapse and generating 3'-hydroxy (3'-OH), which were then elongated under the assistance of TDT to produce poly (T) sequences. Finally, Copper (II) sulfate (Cu2+) and l-Ascorbic acid (AA) were added to form CuNPs using poly (T) sequences as templates (T-CuNPs), resulting in a strong fluorescence signal. This method exhibited good selectivity and high sensitivity with a detection limit of 8.6 × 10-5 U/mL. Moreover, the strategy has been successfully applied to the screening of UDG inhibitors and the detection of UDG activity in complex cell lysates, which means that it has promising applications in clinical diagnosis and biomedical research.