RADD: A real-time FRET-based biochemical assay for DNA deaminase studies

Christopher A Belica; Patricia C. Hernandez; Michael A Carpenter; Yanjun Chen; William L. Brown; Reuben Harris; Hideki Aihara

doi:10.1016/bs.mie.2024.08.001

RADD: A real-time FRET-based biochemical assay for DNA deaminase studies

Christopher A Belica, Patricia C. Hernandez, Michael A Carpenter, Yanjun Chen, William L. Brown, Reuben Harris, Hideki Aihara

Research output: Chapter in Book/Report/Conference proceeding › Chapter

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.

Original language	English (US)
Title of host publication	Chemical and Genomic Methods in Nucleic Acid Biology
Editors	Brandt F. Eichman
Publisher	Academic Press Inc.
Pages	311-345
Number of pages	35
ISBN (Print)	9780443297243
DOIs	https://doi.org/10.1016/bs.mie.2024.08.001
State	Published - Jan 2024

Publication series

Name	Methods in Enzymology
Volume	705
ISSN (Print)	0076-6879
ISSN (Electronic)	1557-7988

Bibliographical note

Publisher Copyright:
© 2024

Keywords

APOBEC3
Cancer
Cytidine deaminase
Fluorescence resonance energy transfer (FRET)
High-throughput screening (HTS)
Real-time assay
DNA/metabolism
Enzyme Assays/methods
Humans
Cytidine Deaminase/metabolism
Enzyme Inhibitors/pharmacology
Fluorescence Resonance Energy Transfer/methods
Deamination
APOBEC Deaminases/metabolism

PubMed: MeSH publication types

Journal Article

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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10.1016/bs.mie.2024.08.001

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Belica, C. A., Hernandez, P. C., Carpenter, M. A., Chen, Y., Brown, W. L., Harris, R., & Aihara, H. (2024). RADD: A real-time FRET-based biochemical assay for DNA deaminase studies. In B. F. Eichman (Ed.), Chemical and Genomic Methods in Nucleic Acid Biology (pp. 311-345). (Methods in Enzymology; Vol. 705). Academic Press Inc.. https://doi.org/10.1016/bs.mie.2024.08.001

RADD: A real-time FRET-based biochemical assay for DNA deaminase studies. / Belica, Christopher A; Hernandez, Patricia C.; Carpenter, Michael A et al.
Chemical and Genomic Methods in Nucleic Acid Biology. ed. / Brandt F. Eichman. Academic Press Inc., 2024. p. 311-345 (Methods in Enzymology; Vol. 705).

Research output: Chapter in Book/Report/Conference proceeding › Chapter

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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.",

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doi = "10.1016/bs.mie.2024.08.001",

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AB - 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.

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KW - Cancer

KW - Cytidine deaminase

KW - Fluorescence resonance energy transfer (FRET)

KW - High-throughput screening (HTS)

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KW - DNA/metabolism

KW - Enzyme Assays/methods

KW - Humans

KW - Cytidine Deaminase/metabolism

KW - Enzyme Inhibitors/pharmacology

KW - Fluorescence Resonance Energy Transfer/methods

KW - Deamination

KW - APOBEC Deaminases/metabolism

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RADD: A real-time FRET-based biochemical assay for DNA deaminase studies

Abstract

Publication series

Bibliographical note

Keywords

PubMed: MeSH publication types

UN SDGs

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