The dumbbell probe mediated triple cascade signal amplification strategy for sensitive and specific detection of uracil DNA glycosylase activity

Uracil DNA glycosylase (UDG) is a key base excision repair (BER) enzyme and its abnormal expression is nearly relevant to several diseases including cancer. The sensitive detection of UDG activity is beneficial for biomedical studies and clinic diagnosis. In this work, we proposed a dumbbell probe mediated triple cascade signal amplification strategy for sensitive and specific detection of UDG activity. The specially designed dumbbell probe contained two uracil bases, two recognition sites for nicking enzyme and a split sequence of DNAzyme. Unsealed dumbbell probes were first connected into sealed dumbbell probes by T4 DNA ligase, and then the unsealed probes were hydrolyzed by exonuclease to ensure the purity of probes. Under the influence of UDG, two uracil bases were removed to produce two apyrimidinic (AP) sites, which were subsequently cleaved by Endo.IV. The probes after cleavage acted as primers and templates for double nicking sites strand displacement amplification (SDA) to produce a mass of two products. The products of SDA continued to act as primers and templates for rolling circle amplification (RCA) to produce repeats containing complete DNAzyme sequences. The DNAzyme repeatedly cleaved multiple molecular beacons (MB), resulting in remarkable fluorescence enhancement. Benefiting from the triple cascade signal amplification, the sensitivity was improved and the detection limit was 7.2 × 10⁻⁵ U mL⁻¹. The method could well distinguish UDG from other interfering enzymes and detect UDG activity in real biological samples, showing good specificity. In addition, this method could be used for screening inhibitors. The above results suggested that the method provided a promising analytical means for UDG related biomedical research and clinic diagnosis.