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Updated: Apr 10, 2026
A Simple, Rapid, and Quantitative Assay to Measure Repair of DNA-protein Crosslinks on Plasmids Transfected into Mammalian Cells
Published on: March 5, 2018
In Vitro Fluorogenic Real-Time Assay of the Repair of Oxidative DNA Damage
Sarah K Edwards1, Toshikazu Ono1,2, Shenliang Wang1
1Department of Chemistry, Stanford University, Stanford, CA 94305 (USA).
Abstract:
The repair of oxidative damage to DNA is essential to avoid mutations that lead to cancer. Oxidized DNA bases, such as 8-oxoguanine, are a main source of these mutations, and the enzyme 8-oxoguanine glycosylase 1 (OGG1) is the chief human enzyme that excises 8-oxoguanine from DNA. The activity of OGG1 has been linked to human inflammation responses and to cancer, and researchers are beginning to search for inhibitors of the enzyme. However, measuring the activity of the enzyme typically requires laborious gel-based measurements of radiolabeled DNAs. Here we report the design and properties of fluorogenic probes that directly report on the activity of OGG1 (and its bacterial homologue Fpg) in real time as the oxidized base is excised. The probes are short, modified DNA oligomers containing fluorescent DNA bases and are designed to utilize 8-oxoguanine itself as a fluorescence quencher. Screening of combinations of fluorophores and 8-oxoguanine revealed two fluorophores, pyrene and tCo, that are strongly quenched by the damaged base. We tested 42 potential probes containing these fluorophores: the optimum probe, OGR1, yields a 60-fold light-up signal in vitro with OGG1 and Fpg. It can report on oxidative repair activity in mammalian cell lysate and with bacterial cells overexpressing a repair enzyme. Such probes might prove useful in quantifying enzyme activity and performing competitive inhibition assays.
Insights
Researchers developed novel fluorogenic probes to directly measure DNA repair enzyme activity in real time. These probes enable efficient quantification of 8-oxoguanine glycosylase 1 (OGG1) activity, aiding cancer research.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Oxidative DNA damage, particularly 8-oxoguanine, is a significant mutagenic lesion linked to cancer.
- 8-oxoguanine glycosylase 1 (OGG1) is the primary human enzyme responsible for excising 8-oxoguanine.
- Current methods for measuring OGG1 activity are laborious and time-consuming.
Purpose of the Study:
- To design and characterize novel fluorogenic probes for real-time monitoring of OGG1 activity.
- To develop a more efficient and direct assay for measuring oxidative DNA repair.
- To facilitate the discovery of OGG1 inhibitors for cancer therapy.
Main Methods:
- Design of short, modified DNA oligomers incorporating fluorescent bases and 8-oxoguanine as a quencher.
- Screening of fluorophore-quencher combinations to optimize signal detection.
- In vitro and cellular assays using the developed probes to measure OGG1 and Fpg activity.
Main Results:
- Identified pyrene and tCo as effective fluorophores quenched by 8-oxoguanine.
- Developed an optimal probe (OGR1) yielding a 60-fold signal increase with OGG1 and Fpg.
- Demonstrated probe utility in mammalian cell lysates and bacterial cells expressing repair enzymes.
Conclusions:
- Fluorogenic probes offer a direct, real-time method for assessing oxidative DNA repair enzyme activity.
- The OGR1 probe is a sensitive tool for quantifying OGG1/Fpg activity and for inhibition assays.
- These probes have potential applications in drug discovery and understanding DNA repair mechanisms.
