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

4区生物学 Q3 Biochemistry, Genetics and Molecular Biology

Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-08-27 DOI:10.1016/bs.mie.2024.08.001

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

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引用次数: 0

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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RADD：基于实时 FRET 的 DNA 脱氨酶生化测定。

近年来，APOBEC3胞嘧啶脱氨酶与癌症诱变之间的联系变得越来越明显。由于人们对这一问题的认识不断提高，同时又缺乏抑制性药物，因此，人们对可用于识别和鉴定该酶家族潜在抑制剂的生化工具有了明显的需求。为了应对这一挑战，我们开发了一种实时 APOBEC3 介导的 DNA 脱氨（RADD）检测方法。RADD 检测法提供了 APOBEC3 DNA 脱氨基的快速、实时荧光读数，是对现有 APOBEC3 生化和细胞工具包的重要补充。该方法改进了现有的 DNA 脱氨基检测方法，提供了更快速、更可量化的读数，并提供了一个可随时适应高通量抑制剂发现的平台。在本章中，我们将详细介绍如何使用 RADD 检测法鉴定 APOBEC3 酶和潜在抑制剂。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Methods in enzymology 生物-生化研究方法

CiteScore

2.90

自引率

0.00%

发文量

308

审稿时长

3-6 weeks

期刊介绍： The critically acclaimed laboratory standard for almost 50 years, Methods in Enzymology is one of the most highly respected publications in the field of biochemistry. Each volume is eagerly awaited, frequently consulted, and praised by researchers and reviewers alike. Now with over 500 volumes the series contains much material still relevant today and is truly an essential publication for researchers in all fields of life sciences, including microbiology, biochemistry, cancer research and genetics-just to name a few. Five of the 2013 Nobel Laureates have edited or contributed to volumes of MIE.