Designer Fluorescent Adenines Enable Real-Time Monitoring of MUTYH Activity.
Designer Fluorescent Adenines Enable Real-Time Monitoring of MUTYH Activity.
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DOI：
10.1021/acscentsci.0c00369
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发表时间：
2020-10-28
影响因子：
18.2
通讯作者：
Kool ET
中科院分区：
文献类型：
--
作者：
Zhu RY;Majumdar C;Khuu C;De Rosa M;Opresko PL;David SS;Kool ET
人类DNA碱基切除修复酶MUTYH（MutY同源物DNA糖基化酶）切除未受损的腺嘌呤，所述未受损的腺嘌呤已被错误掺入氧化损伤的8-oxoG的对面，防止颠换突变并作为对抗这种损伤的有害作用的重要防御。MUTYH基因突变使患者易患MUTYH相关息肉病和结直肠癌，并且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这种酶作为潜在的治疗靶标至关重要。然而，目前用于测量MUTYH活性的方法依赖于间接电泳和放射性测定，其难以在生物学和临床环境中实施。在此，我们合成并鉴定了新的荧光腺嘌呤衍生物，其可以作为MUTYH以及细菌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）探针在体外以及在哺乳动物细胞和人血液中真实的时间内报告酶碱基切除活性。我们还采用探针，以确定几个有前途的小分子调节MUTYH通过采用FMART探针在体外筛选。DNA修复酶MUTYH（MutY DNA糖基化酶）的活性用含有设计的荧光DNA碱基的探针实时测量。这些探针有望用于评估和诊断MUTYH相关疾病，包括MUTYH相关息肉病、胰腺癌和胃癌。
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. The activity of DNA repair enzyme MUTYH (MutY DNA glycosylase) was measured in real-time with probes containing designer fluorescent DNA bases. The probes hold promise for evaluating and diagnosing MUTYH-associated diseases, including MUTYH-associated polyposis, pancreatic cancer, and gastric cancer.
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