Selective Detection of 5-Formyl-2′-deoxyuridine, an Oxidative Lesion of Thymidine, in DNA by a Fluorogenic Reagent
Selective Detection of 5-Formyl-2′-deoxyuridine, an Oxidative Lesion of Thymidine, in DNA by a Fluorogenic Reagent
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DOI：
10.1002/anie.201004087
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发表时间：
2010-01-01
影响因子：
16.6
通讯作者：
Matsuda, Akira
中科院分区：
文献类型：
--
作者：
Hirose, Wataru;Sato, Kousuke;Matsuda, Akira
活细胞中的DNA被来自紫外线、电离辐射和细胞呼吸的活性氧破坏。5-甲酰基-2 '-脱氧尿苷（fodUrd）是一种氧化的胸苷损伤，产生的产量与2'-脱氧-8-氧代鸟苷相当，[1]通过在DNA复制过程中电离形式的fodUrd与2 '-脱氧鸟苷的错配诱导DNA突变（AT至GC转变）。[2]似乎fodUrd的形成可能导致致癌性和/或细胞老化。[3]高度期望用于检测fodUrd的选择性和更方便的方法，因为现有方法复杂，涉及在靶DNA的完全酶促水解之后通过HPLC和/或质谱法的耗时分析，并且需要同位素标记的fodUrd（13 C和/或15 N）作为内标。[4]已经开发了一些其他方法来检测DNA损伤。[5]Gong和Sturla开发了通过在含有合成互补核碱基的寡核苷酸探针和O 6-苄基-dG之间形成碱基对的分子识别。[6]一种常用的方法是通过用含有报告基团的分子选择性标记来检测具有增强反应性的DNA损伤。Ide等人首先证明了通过使用醛反应性探针来检测脱碱基位点。[7]最近报道了使用化学试剂的其他选择性检测方法。[8]然而，信噪比（S/N）在这些方法中是一个问题，因为所使用的荧光和/或比色分子具有较高的背景信号。在这里，我们报告了一个新的概念，简单的检测fodUrd在受损的DNA与荧光试剂。试剂2-氨基-4，5-二甲氧基苯硫酚（3 '）在与DNA中的目标fodUrd反应之前未显示荧光。然而，在3 '与fodUrd反应时，fodUrd的5-位上的甲酰基转化为苯并噻唑-2-基，其直接与尿嘧啶基团缀合。该环系统类似于荧光素酶，其经历荧光素酶反应以产生发光。
DNA in living cells is damaged by reactive oxygen species derived from UV light, ionizing radiation, and cellular respiration. 5-Formyl-2’-deoxyuridine (fodUrd), an oxidized thymidine lesion generated in yields comparable to that of 2’-deoxy-8-oxoguanosine,[1] induces mutation in DNA (AT-to-GC transition) through the mispairing of an ionized form of fodUrd with 2’-deoxyguanosine during DNA replication.[2] It appears that the formation of fodUrd may cause carcinogenicity and/or aging of cells.[3] A selective and more convenient method for detecting fodUrd would be highly desirable, since the existing methods are complicated, involve time-consuming analysis by HPLC and/or mass spectrometry following complete enzymatic hydrolysis of the target DNA, and require isotope-labeled fodUrd (13C and/or 15N) as an internal standard.[4]Some other approaches have been developed for the detection of DNA damage.[5] Molecular recognition through the formation of a base pair between an oligonucleotide probe containing a synthetic complimentary nucleobase and O6-benzyl-dG was developed by Gong and Sturla.[6] A common method is to detect DNA damage with enhanced reactivity by selective tagging with a molecule that contains a reporter group. Ide et al. first demonstrated the detection of abasic sites by the use of an aldehyde reactive probe.[7] Other selective detection methods with chemical reagents have recently been reported.[8] However, the signal-to-noise (S/N) ratio is a problem in these methods, as the fluorescent and/or colorimetric molecules used have higher background signals. Herein, we report a new concept for the simple detection of fodUrd in damaged DNA with a fluorogenic reagent. The reagent 2-amino-4, 5-dimethoxythiophenol (3’) shows no fluorescence before reaction with the target fodUrd in DNA. However, upon the reaction of 3’with fodUrd, the formyl group at the 5-position of fodUrd is converted into a benzothiazol-2-yl group, which is directly conjugated with the uracil group. This ring system is similar to luciferin, which undergoes the luciferase reaction to produce luminescence.