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Designer Fluorescent Adenines Enable Real-Time Monitoring of MUTYH Activity
ACS Central Science ( IF 12.7 ) Pub Date : 2020-08-31 , DOI: 10.1021/acscentsci.0c00369 Ru-Yi Zhu 1 , Chandrima Majumdar 2 , Cindy Khuu 2 , Mariarosaria De Rosa 3, 4 , Patricia L. Opresko 3, 4 , Sheila S. David 2 , Eric T. Kool 1
ACS Central Science ( IF 12.7 ) Pub Date : 2020-08-31 , DOI: 10.1021/acscentsci.0c00369 Ru-Yi Zhu 1 , Chandrima Majumdar 2 , Cindy Khuu 2 , Mariarosaria De Rosa 3, 4 , Patricia L. Opresko 3, 4 , Sheila S. David 2 , Eric T. Kool 1
Affiliation
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The human DNA base excision repair enzyme MUTYH (MutY homolog DNA glycosylase) excises undamaged adenine that has been misincorporated opposite the oxidatively damaged 8-oxoG, preventing transversion mutations and serving as an important defense against the deleterious effects of this damage. Mutations in the MUTYH gene predispose patients to MUTYH-associated polyposis and colorectal cancer, and MUTYH expression has been documented as a biomarker for pancreatic cancer. Measuring MUTYH activity is therefore critical for evaluating and diagnosing disease states as well as for testing this enzyme as a potential therapeutic target. However, current methods for measuring MUTYH activity rely on indirect electrophoresis and radioactivity assays, which are difficult to implement in biological and clinical settings. Herein, we synthesize and identify novel fluorescent adenine derivatives that can act as direct substrates for excision by MUTYH as well as bacterial MutY. When incorporated into synthetic DNAs, the resulting fluorescently modified adenine-release turn-on (FMART) probes report on enzymatic base excision activity in real time, both in vitro and in mammalian cells and human blood. We also employ the probes to identify several promising small-molecule modulators of MUTYH by employing FMART probes for in vitro screening.
中文翻译：
Designer荧光腺嘌呤可实时监控MUTYH活动
人类DNA碱基切除修复酶MUTYH（MutY同源DNA糖基化酶）切除了未损坏的腺嘌呤，该腺嘌呤已与氧化受损的8-oxoG错掺入，防止了转位突变，并作为抵抗这种伤害的有害作用的重要防御手段。MUTYH中的突变该基因使患者易患MUTYH相关性息肉病和结肠直肠癌，MUTYH表达已被证明是胰腺癌的生物标志物。因此，测量MUTYH活性对于评估和诊断疾病状态以及测试该酶作为潜在的治疗靶点至关重要。然而，当前用于测量MUTYH活性的方法依赖于间接电泳和放射性测定，这在生物学和临床环境中难以实现。本文中，我们合成并鉴定了新型荧光腺嘌呤衍生物，它们可以作为MUTYH和细菌MutY切除的直接底物。当掺入合成DNA中时，所得的荧光修饰的腺嘌呤释放开启（FMART）探针可实时报告酶促碱基切除的活性，两者在体外以及在哺乳动物细胞和人类血液中。我们还使用探针，通过使用FMART探针进行体外筛选，来鉴定MUTYH的几种有希望的小分子调节剂。
更新日期：2020-10-29
中文翻译：
Designer荧光腺嘌呤可实时监控MUTYH活动
人类DNA碱基切除修复酶MUTYH（MutY同源DNA糖基化酶）切除了未损坏的腺嘌呤，该腺嘌呤已与氧化受损的8-oxoG错掺入，防止了转位突变，并作为抵抗这种伤害的有害作用的重要防御手段。MUTYH中的突变该基因使患者易患MUTYH相关性息肉病和结肠直肠癌，MUTYH表达已被证明是胰腺癌的生物标志物。因此，测量MUTYH活性对于评估和诊断疾病状态以及测试该酶作为潜在的治疗靶点至关重要。然而，当前用于测量MUTYH活性的方法依赖于间接电泳和放射性测定，这在生物学和临床环境中难以实现。本文中，我们合成并鉴定了新型荧光腺嘌呤衍生物，它们可以作为MUTYH和细菌MutY切除的直接底物。当掺入合成DNA中时，所得的荧光修饰的腺嘌呤释放开启（FMART）探针可实时报告酶促碱基切除的活性，两者在体外以及在哺乳动物细胞和人类血液中。我们还使用探针，通过使用FMART探针进行体外筛选，来鉴定MUTYH的几种有希望的小分子调节剂。
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