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迈向单分子DNA测序:通过使用配备分子适配器的工程化蛋白质纳米孔直接鉴定核糖核苷和脱氧核糖核苷5'-单磷酸。

Toward single molecule DNA sequencing: direct identification of ribonucleoside and deoxyribonucleoside 5'-monophosphates by using an engineered protein nanopore equipped with a molecular adapter.

作者信息

Astier Yann, Braha Orit, Bayley Hagan

机构信息

Department of Chemistry, University of Oxford, Chemistry Research Laboratory, Mansfield Road, OX1 3TA, Oxford, UK.

出版信息

J Am Chem Soc. 2006 Feb 8;128(5):1705-10. doi: 10.1021/ja057123+.

Abstract

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.

摘要

单个核酸分子可通过识别由连续性核酸外切酶释放的5'-单磷酸核苷来进行测序。在此,我们表明,使用经过修饰的蛋白质纳米孔进行单分子检测可用于识别核糖核苷和2'-脱氧核糖核苷5'-单磷酸,从而在此道路上迈出了一步。当一组5'-单磷酸核苷的四个成员中的每一个分子结合在配备分子适配器七(6-脱氧-6-氨基)-β-环糊精的突变α-溶血素孔(M113R)(7)内时,会观察到不同程度的电流阻断。虽然我们的结果与其他方法相比具有优势,但仍需要进一步开展工作,以提高核酸碱基识别的准确性,将每个释放的核苷酸送入孔中,并确保每个核苷酸都被适配器捕获。

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