Construction of a self-directed replication system for label-free and real-time sensing of repair glycosylases with zero background

Abstract

Genomic DNA damage and repair are involved in multiple fundamental biological processes, including metabolism, disease, and aging. Inspired by the natural repair mechanism in vivo, we demonstrate for the first time the construction of a self-directed replication system for label-free and real-time sensing of repair glycosylases with zero background. The presence of DNA glycosylase can catalyze the excision repair of the damaged base, successively autostarting the self-directed replication through recycling polymerization extension and strand-displacement DNA synthesis for the generation of exponentially amplified dsDNAs. The resultant dsDNA products can be label-free and real-time monitored with SYBR Green I as the fluorescent indicator. Owing to the high efficiency of self-directed exponential replication and the absolute zero background resulting from the efficient inhibition of nonspecific amplification induced by multiple primer-dependent amplification, this strategy exhibits high sensitivity with a detection limit of 1 × 10^-8 U μL^-1 in vitro and 1 cell in vivo, and it can be further used to screen inhibitors, quantify DNA glycosylase from diverse cancer cells, and even monitor various repair enzymes by simply changing the specific damaged base in the DNA template. Importantly, this assay can be performed in a label-free, real-time and isothermal manner with the involvement of only a single type of polymerase, providing a simple, robust and universal platform for repair enzyme-related biomedical research and clinical therapeutics.

Fig. 1. Mechanism of DNA glycosylase-catalyzed damaged base-excision repair through the BER pathway. DNA glycosylase can excise the specific damaged base (red color) to generate an AP site (pink color). The AP site will be cleaved by AP endonuclease to generate the 5′-PO₄ and 3′-OH termini.

Scheme 1. Schematic illustration of the construction of a self-directed replication system for the repair glycosylase assay. This strategy contains two consecutive steps: (A) DNA repairing-driven formation of double-stem-loop DNAs for initiating the self-directed exponential replication, (B) self-directed exponential replication for the generation of dsDNA products.

Fig. 2. (A) PAGE analysis of the products of hOGG1-catalyzed 8-oxoG-excision reaction under different conditions. Lane 1, in the presence of DNA template; lane 2, in the presence of hOGG1 + DNA template; lane 3, the synthesized excision product. (B) PAGE analysis of the products of self-directed exponential replication reaction. Lane 1, the synthesized BIP; lane 2, the synthesized FIP; lane 3, in the presence of hOGG1; lane 4, without hOGG1; lane 5, the synthesized primers FOP and BOP. (C) Real-time fluorescence monitoring of the amplification reaction in the presence (red line) and absence (black line) of hOGG1. The color images marked in (A) and (B) are the fragments in Scheme 1 corresponding to the bands. 0.25 U μL^–1 hOGG1 was used in this experiment.

Fig. 3. (A) Real-time fluorescence curves in response to different concentrations of hOGG1. (B) Linear relationship between the POI value and the logarithm of hOGG1 concentration. (C) Real-time fluorescence curves in response to 0.1 U μL^–1 hOGG1 (red line), 0.1 U μL^–1 hAAG (green line), 0.1 U μL^–1 UDG (pink line), 0.1 g L^–1 BSA (blue line), and the control group with only reaction buffer (black line), respectively. The error bars represent standard deviations of three independent experiments.

Fig. 4. (A) Real-time fluorescence curves in response to different concentrations of CdCl₂. (B) Variance of the relative activity of hOGG1 with the logarithm of CdCl₂ concentration. Inset shows the POI values of the real-time fluorescence curves in response to the different concentrations of CdCl₂. The error bars represent standard deviations of three independent experiments.

Fig. 5. (A) ELISA analysis of hOGG1 in A549 cells. Color changes in response to the control (I), cytoplasm (II), nucleus (III) and whole cell extracts (IV), respectively, and the variance of O.D. in response to the control, cytoplasm, nucleus and whole cell extracts, respectively. Inset shows the relative O.D. values in response to the cytoplasm, nucleus and whole cell extracts, respectively. (B) Real-time fluorescence curves in response to the control, cytoplasm and nucleus extracts from 1000 A549 cells, respectively. (C) Real-time fluorescence curves in response to different numbers of A549 cells. (D) Linear relationship between the POI value and the logarithm of the A549 cell number. Each curve represents the average measurement of three independent experiments. The error bars represent standard deviations of three independent experiments.

Fig. 6. (A) Real-time fluorescence curves in response to 5000 cancer cells including A549 cells, HeLa cells, MCF-7 cells, A549 + MCF-10A cells, MCF-10A cells and control groups (the nuclear extracts from the respective cancer cell without hOGG1), respectively. (B) Measurement of POI values of the real-time fluorescence curves in (A). (C) Real-time fluorescence curves in response to 0.25 U μL^–1 hOGG1, 0.25 U μL^–1 hAAG, 0.25 U μL^–1 UDG, 0.25 U μL^–1 TDG and control groups (the reaction solution with the specific DNA template), respectively. (D) Measurement of POI values of the real-time fluorescence curves in (C). Each curve represents the average measurement of three independent experiments. The error bars represent standard deviations of three independent experiments.