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Updated: Dec 10, 2025
Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging ESI
Published on: September 24, 2015
Rolling circle amplification-driven encoding of different fluorescent molecules for simultaneous detection of
Chen-Chen Li1, Hui-Yan Chen1, Juan Hu1
1College of Chemistry , Chemical Engineering and Materials Science , Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong , Key Laboratory of Molecular and Nano Probes , Ministry of Education , Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals , Shandong Normal University , Jinan 250014 , China . Email: cyzhang@sdnu.edu.cn ; Email: juanhu@sdnu.edu.cn ; ; Tel: +86 0531-86186033.
Abstract:
DNA repair enzymes (e.g., DNA glycosylases) play a critical role in the repair of DNA lesions, and their aberrant levels are associated with various diseases. Herein, we develop a sensitive method for simultaneous detection of multiple DNA repair enzymes based on the integration of single-molecule detection with rolling circle amplification (RCA)-driven encoding of different fluorescent molecules. We use human alkyladenine DNA glycosylase (hAAG) and uracil DNA glycosylase (UDG) as the target analytes. We design a bifunctional double-stranded DNA (dsDNA) substrate with a hypoxanthine base (I) in one strand for hAAG recognition and an uracil (U) base in the other strand for UDG recognition, whose cleavage by APE1 generates two corresponding primers. The resultant two primers can hybridize with their respective circular templates to initiate RCA, resulting in the incorporation of multiple Cy3-dCTP and Cy5-dGTP nucleotides into the amplified products. After magnetic separation and exonuclease cleavage, the Cy3 and Cy5 fluorescent molecules in the amplified products are released into the solution and subsequently quantified by total internal reflection fluorescence (TIRF)-based single-molecule detection, with Cy3 indicating the presence of hAAG and Cy5 indicating the presence of UDG. This strategy greatly increases the number of fluorescent molecules per concatemer through the introduction of RCA-driven encoding of different fluorescent molecules, without the requirement of any specially labeled detection probes for simultaneous detection. Due to the high amplification efficiency of RCA and the high signal-to-ratio of single-molecule detection, this method can achieve a detection limit of 6.10 × 10-9 U mL-1 for hAAG and 1.54 × 10-9 U mL-1 for UDG. It can be further applied for simultaneous detection of multiple DNA glycosylases in cancer cells at the single-cell level and the screening of DNA glycosylase inhibitors, holding great potential in early clinical diagnosis and drug discovery.
