Nanopore detection of 8-oxo-7,8-dihydro-2'-deoxyguanosine in immobilized single-stranded DNA via adduct formation to the DNA damage site.
Nanopore detection of 8-oxo-7,8-dihydro-2'-deoxyguanosine in immobilized single-stranded DNA via adduct formation to the DNA damage site.
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DOI：
10.1021/ja109501x
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发表时间：
2010-12-29
影响因子：
15
通讯作者：
White HS
中科院分区：
文献类型：
--
作者：
Schibel AE;An N;Jin Q;Fleming AM;Burrows CJ;White HS
检测周围序列中DNA损伤的能力是医学诊断和治疗中的一个重要目标，但目前还没有令人满意的方法来检测受损的碱基，同时提供序列信息。最常见的碱基损伤之一是8-氧代-7，8-二氢鸟嘌呤，发生在鸟嘌呤氧化过程中。在这里提出的工作中，我们展示了使用离子通道纳米孔方法检测单个氧化损伤位置的方法，该方法使用α-溶血素。通过生物素连接将受损的DNA与链霉亲和素连接，并在α-溶血素离子通道内捕获DNA，检测了8-氧代-7，8-二氢鸟嘌呤进一步氧化产生的海因损伤，以及通过将伯胺共价连接到螺旋亚胺二海因损伤产生的螺旋环加合物。在均聚和杂聚背景的单链DNA序列中，螺环加合物产生的电流阻断水平与天然碱基电流阻断水平几乎不同10%。这些初步研究表明，离子通道记录不仅适用于最近受到极大关注的DNA测序，而且还适用于检测DNA损伤，这将是任何测序工作的重要组成部分。
The ability to detect DNA damage within the context of the surrounding sequence is an important goal in medical diagnosis and therapies, but there are no satisfactory methods available to detect a damaged base while providing sequence information. One of the most common base lesions is 8-oxo-7,8-dihydroguanine that occurs during oxidation of guanine. In the work presented here, we demonstrate the detection of a single oxidative damage site using ion channel nanopore methods employing α-hemolysin. Hydantoin lesions produced from further oxidation of 8-oxo-7,8-dihydroguanine, as well as spirocyclic adducts produced from covalently attaching a primary amine to the spiroiminodihydantoin lesion, were detected by tethering the damaged DNA to streptavidin via a biotin linkage, and capturing the DNA inside an α-hemolysin ion channel. Spirocyclic adducts, in both homo- and hetero-polymer background single-stranded DNA sequences, produced current blockage levels differing by almost 10% from those of native base current blockage levels. These preliminary studies show the applicability of ion channel recordings not only for DNA sequencing, which has recently received much attention, but also to detecting DNA damage, which will be an important component to any sequencing efforts.
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