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RADD: A real-time FRET-based biochemical assay for DNA deaminase studies
Christopher A Belica1, Patricia C Hernandez1, Michael A Carpenter2
1Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, United States; Institute for Molecular Virology, University of Minnesota, Minneapolis, MN, United States; Masonic Cancer Center, University of Minnesota, Minneapolis, MN, United States.
Abstract:
In recent years, the connection between APOBEC3 cytosine deaminases and cancer mutagenesis has become ever more apparent. This growing awareness and lack of inhibitory drugs has created a distinct need for biochemical tools that can be used to identify and characterize potential inhibitors of this family of enzymes. In response to this challenge, we have developed a Real-time APOBEC3-mediated DNA Deamination (RADD) assay. The RADD assay provides a rapid, real-time fluorescence readout of APOBEC3 DNA deamination and serves as a crucial addition to the existing APOBEC3 biochemical and cellular toolkit. This method improves upon contemporary DNA deamination assays by offering a more rapid and quantifiable readout as well as providing a platform that is readily adaptable to a high-throughput format for inhibitor discovery. In this chapter we provide a detailed guide for the usage of the RADD assay for the characterization of APOBEC3 enzymes and potential inhibitors.
Insights
Researchers developed a Real-time APOBEC3-mediated DNA Deamination (RADD) assay to identify inhibitors of APOBEC3 enzymes, which are linked to cancer mutagenesis. This new assay offers a rapid, quantifiable method for characterizing these enzymes and discovering potential drugs.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- APOBEC3 cytosine deaminases are increasingly recognized for their role in cancer mutagenesis.
- There is a significant need for biochemical tools to identify and characterize inhibitors of these enzymes due to their link to cancer.
- Existing assays for DNA deamination lack the speed and quantifiability required for efficient inhibitor discovery.
Purpose of the Study:
- To develop a novel biochemical assay for studying APOBEC3 enzymes.
- To provide a rapid, real-time fluorescence readout for APOBEC3-mediated DNA deamination.
- To establish a platform for high-throughput screening of potential APOBEC3 inhibitors.
Main Methods:
- Development of a Real-time APOBEC3-mediated DNA Deamination (RADD) assay.
- Utilizing fluorescence detection for real-time monitoring of DNA deamination activity.
- Adaptation of the assay for high-throughput screening.
Main Results:
- The RADD assay provides a rapid and quantifiable fluorescence readout of APOBEC3 DNA deamination.
- The assay is a valuable addition to the existing APOBEC3 biochemical and cellular toolkit.
- The RADD assay is adaptable to a high-throughput format for inhibitor discovery.
Conclusions:
- The RADD assay offers significant improvements over contemporary DNA deamination assays.
- This assay facilitates the characterization of APOBEC3 enzymes and the discovery of potential inhibitors.
- The RADD assay is a crucial tool for advancing research in APOBEC3-related cancer mutagenesis and drug development.
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