One-step sensitive detection of UDG activity based on nicking enzyme-assisted signal amplification coupled with APE1 and triggered reporter.

Background: UDG (Uracil-DNA glycosylase) is a pivotal enzyme in the base excision repair (BER) mechanism, and it is widely distributed across most organisms. Its primary function is to identify and excise uracil bases from DNA, thereby facilitating the repair of DNA and the creation of apurinic/apyrimidinic (AP) sites. Furthermore, abnormal expression or dysregulation of UDG activity has been closely associated with ageing, cancer, and other diseases such as immunodeficiency and lymphoma. Consequently, the detection of UDG activity is critical for clinical diagnostics. However, there is currently a deficiency in simple and sensitive methods for UDG detection.

Results: To overcome this limitation,a one-step strategy for the sensitive detection of UDG activity was devised, combining nicking enzyme-assisted amplification (NEAA), APE1, and triggered reporter. Following treatment with UDG, the detection probe initiates NEAA, which amplifies a substantial quantity of single-stranded DNA (ssDNA) that is complementary to the triggered reporter. In the presence of APE1 and the amplified triggered probes, the triggered reporter is subjected to continuous cleavage, leading to an enhanced fluorescent signal output. The approach permits more convenient and sensitive UDG detection, with a detection limit of 1 × 10ˆ-5 U/mL and a linear range from 1 × 10ˆ-3 to 1 × 10ˆ-5 U/mL.

Significance: The biosensor described in this strategy detects UDG activity as a one-pot simple reaction without cumbersome assay steps. And it has excellent detection limit and linear range in the detection of biological samples. It will provide a simple and fast solution in the field of UDG activity detection.