Rolling circle transcription and CRISPR/Cas12a-assisted versatile bicyclic cascade amplification assay for sensitive uracil-DNA glycosylase detection

作者：Xia Cheng, Huahua Song, Dandan Ren, Mingcong Gao, Xinyi Xia, Ping Yu, Xiaolan Bian · 发表于：Talanta · 年份：2023 · DOI：10.1016/j.talanta.2023.124684 · 被引用次数：12 · 研究领域：Cytomegalovirus and herpesvirus research、CRISPR and Genetic Engineering、RNA and protein synthesis mechanisms

Uracil-DNA glycosylase (UDG) is pivotal in maintaining genome integrity and aberrant expressed UDG is highly relevant to numerous diseases. Sensitive and accurate detecting UDG is critically significant for early clinical diagnosis. In this research, we demonstrated a sensitive UDG fluorescent assay based on rolling circle transcription (RCT)/CRISPR/Cas12a-assisted bicyclic cascade amplification strategy. Target UDG catalyzed to remove uracil base of DNA dumbbell-shape substrate probe (Sub UDG ) to produce an apurinic/apyrimidinic (AP) site, at which Sub UDG was cleaved by apurinic/apyrimidinic endonuclease (APE1) subsequently. The exposed 5′-PO 4 was ligated with the free 3′-OH terminus to form an enclosed DNA dumbbell-shape substrate probe (E-Sub UDG ). E-Sub UDG functioned as a template can actuate T7 RNA polymerase-mediated RCT signal amplification, generating multitudes of crRNA repeats. The resultant Cas12a/crRNA/activator ternary complex activated the activity of Cas12a, causing a significantly enhanced fluorescence output. In this bicyclic cascade strategy, target UDG was amplified via RCT and CRISPR/Cas12a, and the whole reaction was completed without complex procedures. This method enabled sensitive and specific monitor UDG down to 0.0005 U/mL, screen corresponding inhibitors, and analyze endogenous UDG in A549 cells at single-cell level. Importantly, this assay can be extended to analyze other DNA glycosylase (hAAG and Fpg) by altering the recognition site in DNA s...