Uracil removal-inhibited ligase reaction in combination with catalytic hairpin assembly for the sensitive and specific detection of uracil-DNA glycosylase activity

Abstract

Sensitive and specific detection of uracil-DNA glycosylase (UDG) activity is crucial in biomedical study and disease diagnosis. Here, we developed a uracil removal-inhibited ligase reaction in combination with catalytic hairpin assembly (CHA) for the sensitive and specific detection of UDG activity. A hairpin probe is specially designed, which contains two uracil bases in the loop and is extended with toehold and branch-migration domains at the ends of the stem. Two short oligonucleotides are separately hybridized to one-half of the loop of the hairpin probe to form a DNA complex with a nick. Under the action of UDG, two uracil bases in the hairpin-loop are removed to generate apurinic/apyrimidinic (AP) sites. The AP sites locating at the 3′-side of the nick inhibit the ligase reaction, leaving the toehold and branch-migration domains at the ends of the hairpin probe still adjacent. The adjacent toehold and branch-migration domains initiate CHA, producing numerous G-quadruplex (G4) structures, which interact with N-methyl-mesoporphyrin IX (NMM) to generate an enhanced fluorescence signal. The excessive probes would be masked by the ligase reaction that closes the nick and forms a long DNA strand fully complementary to the hairpin domain. The probes then get opened and the toehold/branch-migration domains are not associated, prohibiting the CHA reaction and minimizing false-positive interferences. The detection limit is as low as 0.00028 U mL⁻¹, and UDG can be well distinguished from other DNA glycosylases. Furthermore, this method is successfully applied for detecting UDG activity from HeLa cell lysates. Additionally, the inhibition of UDG activity is analyzed, which shows inhibitor dose-dependent activity suppression. This strategy will provide a promising tool for assaying UDG activity in biomedical study and disease diagnosis.