Quantitative and in situ detection of oxidatively generated DNA damage 8,5’-cyclo-2’-deoxyadenosine using an immunoassay with a novel monoclonal antibody
Quantitative and in situ detection of oxidatively generated DNA damage 8,5’-cyclo-2’-deoxyadenosine using an immunoassay with a novel monoclonal antibody
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使用新型单克隆抗体进行免疫测定，定量原位检测氧化产生的 DNA 损伤 8,5-环-2-脱氧腺苷
DOI：
10.1111/php.12239
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发表时间：
2014
期刊：
影响因子：
--
通讯作者：
S. Sugiura and T. Mori.
中科院分区：
文献类型：
--
作者：
T. Iwamoto;P.J. Brooks;T. Nishiwaki;K. Nishimura;N. Kobayashi;S. Sugiura and T. Mori.
着色性干皮病(XP)是一种与核苷酸切除修复缺陷有关的遗传性疾病，它消除了包括阳光诱导的嘧啶二聚体在内的各种螺旋扭曲类型的DNA损伤。除了皮肤病，大约30%的XP患者还会发展成进行性神经系统疾病，这种疾病被认为与一种称为嘌呤8，5‘-环-2’-脱氧核苷(嘌呤环核苷)的特定类型的氧化产生的DNA损伤的积累有关。然而，目前还没有可用的方法来检测DNA中的嘌呤环核苷，而不需要DNA水解。在这项研究中，我们制备了一种新型的针对单链DNA中嘌呤环核苷的单抗(CDA-1)，它可以识别8，5‘-环-2’-脱氧腺苷(cleco-da)。使用CDA-1进行的免疫分析显示，寡核苷酸中已知量的环核苷酸与与其结合的抗体之间存在线性剂量反应。定量免疫分析表明，Fenton型试剂(CuCl2/H2O2/抗坏血酸)处理可有效地产生DNA中的环状dA，并呈剂量依赖关系。此外，使用CDA-1的免疫荧光分析能够在人骨肉瘤细胞中观察到Cyclo-da，该细胞已被转染了含有Cyclo-da的寡核苷酸。因此，CDA-1抗体对于检测和定量DNA中的Cyclo-da是一种有价值的工具，并可能有助于表征XP神经系统疾病发生的机制(S)。
Xeroderma pigmentosum (XP) is a genetic disorder associated with defects in nucleotide excision repair, which eliminates a wide variety of helix‐distorting types of DNA damage including sunlight‐induced pyrimidine dimers. In addition to skin disease, approximately 30% of XP patients develop progressive neurological disease, which has been hypothesized to be associated with the accumulation of a particular type of oxidatively generated DNA damage called purine 8,5′‐cyclo‐2′‐deoxynucleosides (purine cyclonucleosides). However, there are no currently available methods to detect purine cyclonucleosides in DNA without the need for DNA hydrolysis. In this study, we generated a novel monoclonal antibody (CdA‐1) specific for purine cyclonucleosides in single‐stranded DNA that recognizes 8,5′‐cyclo‐2′‐deoxyadenosine (cyclo‐dA). An immunoassay using CdA‐1 revealed a linear dose response between known amounts of cyclo‐dA in oligonucleotides and the antibody binding to them. The quantitative immunoassay revealed that treatment with Fenton‐type reagents (CuCl2/H2O2/ascorbate) efficiently produces cyclo‐dA in DNA in a dose‐dependent manner. Moreover, immunofluorescent analysis using CdA‐1 enabled the visualization of cyclo‐dA in human osteosarcoma cells, which had been transfected with oligonucleotides containing cyclo‐dA. Thus, the CdA‐1 antibody is a valuable tool for the detection and quantification of cyclo‐dA in DNA, and may be useful for characterizing the mechanism(s) underlying the development of XP neurological disease.