Sequentially\nActivated-Dumbbell DNA Nanodevices for\nAccurate Detection of Uracil-DNA Glycosylase via PER-Based Orthogonal\nSignal Outputs

Accurate and reliable detection of uracil-DNA glycosylase\n(UDG)\nactivity is crucial for clinical diagnosis and prognosis assessment.\nHowever, current techniques for accurately monitoring UDG activity\nstill face significant challenges due to the single input or output\nsignal modes. Here, we develop a sequentially activated-dumbbell DNA\nnanodevice (SEAD) that enables precise and reliable evaluation of\nUDG activity through primer exchange reactions (PER)-based orthogonal\nsignal output. The SEAD incorporates a double-hairpin structure with\na stem containing two deoxyuridine (dU) sites for target recognition\nand two preblocked primer binding regions for target amplification\nand signal output. Upon UDG recognition of dU, the SEAD can be cleaved\nby apurinic/apyrimidinic endonuclease 1 (APE1), generating two different\nhairpins with exposed primer binding regions. These hairpins serve\nas templates to initiate the parallel PER, enabling the extending\nof two different amplification products: a long single-stranded DNA\n(ssDNA) with repetitive sequences and a short ferrocene-labeled ssDNA\nwith complementary sequences. These products further self-assemble\ninto DNA nano-strings in an orthogonal manner that act as an electrochemiluminescence\nsignal switch, enabling precise detection of low-abundance UDG. This\nwork develops a sequential input and orthogonal output strategy for\naccurately monitoring UDG activity, highlighting the significant potential\nin cancer diagnosis and treatment.