Sensitive homogeneous fluorescent detection of DNA glycosylase by target-triggering ligation-dependent tricyclic...

Abnormal DNA glycosylases are concerned with the aging process as well as numerous pathologies in humans. Herein, a sensitive fluorescence method utilizing target-induced ligation-dependent tricyclic cascade amplification reaction was developed for the detecting DNA glycosylase activity. The presence of DNA glycosylase triggered the cleavage of damaged base in hairpin substrate, successively activating ligation-dependent strand displacement amplification (SDA) and exponential amplification reaction (EXPAR) for the generation of large amount of reporter probes. The resultant reporter probes bound with the signal probes to form stable dsDNA duplexes. And then the signal probes could be digested circularly in the dsDNA duplexes by T7 exonuclease, leading to the generation of an enhanced fluorescence signal. Due to the high efficiency of tricyclic cascade amplification and the low background signal deriving from the inhibition of nonspecific amplification, this method exhibited a detection limit of 0.14 U/mL and a dynamic range from 0.16 to 8.0 U/mL. Moreover, it could be applied for detecting DNA glycosylase activity in human serum with good selectivity and high sensitivity, and even quantifying other types of enzyme with 5'-PO₄ residue cleavage product by rationally designing the corresponding substrate. Importantly, this method could be performed in homogenous solution without any complicated separation steps, providing a new strategy for DNA glycosylase-related biomedical research.