Sensitive label-free resonance Rayleigh scattering DNA machine-based dual amplification strategy for the active uracil-DNA glycosylase assay

Abstract

Uracil-DNA glycosylase (UDG), one of the base-excision repair enzymes, is closely relevant for the diseases of the organism. Here, we proposed a resonance Rayleigh scattering (RRS) strategy for sensitively detecting the active UDG based on a dual amplification DNA machine without label under the catalysis of exonuclease III (Exo III). In our study, a double-stranded DNA complex S1-S2 containing triggers and peculiar uracil bases was employed. To achieve the dual amplification strategy, we adopted hairpin probes HP1 and HP2. The unique hairpin probes can partially hybridize with S1 and S2, respectively. With the excision reaction of UDG, the uracil bases were excised from S2 in double-stranded S1-S2, resulting in a lower melting temperature of the S1-S2. Then, the S1-S2 was dissociated to single-stranded S1 and S2 with abasic sites. Subsequently, the liberated strands separately hybridized with hairpins to initiate the Exo III-catalyzed dual amplification. In the presence of K+, countless c-myc sequences which were produced by the amplification reaction formed G-quadruplex structures. Finally, with the addition of Mg2+, long and continuous G-wire structures were obtained, causing an obvious enhancement of RRS intensity. The proposed RRS DNA machine obtained the limit of detection as low as 1.0×10−5U/mL for the active UDG assay. Moreover, qualitative analysis of the active UDG was successfully achieved in HeLa cells lysate. The DNA machine is a potential tool to be used in UDG clinical diagnosis or functional study.