Toward single molecule DNA sequencing:: Direct identification of ribonucleoside and deoxyribonucleoside 5′-monophosphates by using an engineered protein nanopore equipped with a molecular adapter
Toward single molecule DNA sequencing:: Direct identification of ribonucleoside and deoxyribonucleoside 5′-monophosphates by using an engineered protein nanopore equipped with a molecular adapter
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DOI：
10.1021/ja057123
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发表时间：
2006-02-08
影响因子：
15
通讯作者：
Bayley, H
中科院分区：
文献类型：
--
作者：
Astier, Y;Braha, O;Bayley, H
单个核酸分子可以通过鉴定核苷5 '-单磷酸来测序，因为它们是由进行性核酸外切酶释放的。在这里，我们表明，使用修饰的蛋白质纳米孔的单分子检测可用于鉴定核糖核苷和2 '-脱氧核糖核苷5'-单磷酸，从而沿着这条路径迈出沿着一步。当分子结合在突变体α-溶血素孔（M113 R）（7）内时，观察到一组核苷5 '-单磷酸的四个成员中的每一个的电流阻断水平不同。配备有分子适配器七-（6-脱氧-6-氨基）-β-环糊精。虽然我们的结果与替代方法相比是有利的，但需要进一步的工作来提高核酸碱基识别的准确性，将每个释放的核苷酸进料到孔中，并确保每个核苷酸被适配器捕获。
Individual nucleic acid molecules might be sequenced by the identification of nucleoside 5'-monophosphates as they are released by processive exonucleases. Here, we show that single molecule detection with a modified protein nanopore can be used to identify ribonucleoside and 2'-deoxyribonucleoside 5'-monophosphates, thereby taking a step along this path. Distinct levels of current block are observed for each of the four members of a set of nucleoside 5'-monophosphates when the molecules bind within a mutant alpha-hemolysin pore, (M113R)(7), equipped with the molecular adapter heptakis-(6-deoxy-6-amino)-beta-cyclodextrin. While our results compare favorably with alternative approaches, further work will be required to improve the accuracy of identification of the nucleic acid bases, to feed each released nucleotide into the pore, and to ensure that every nucleotide is captured by the adapter.