Nucleobase Recognition by Truncated α-Hemolysin Pores.
Nucleobase Recognition by Truncated α-Hemolysin Pores.
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DOI：
10.1021/nn5060317
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发表时间：
2015-08-25
期刊：
影响因子：
17.1
通讯作者：
Bayley H
中科院分区：
文献类型：
--
作者：
Ayub M;Stoddart D;Bayley H
α-溶血素 (αHL) 蛋白纳米孔之前已被研究作为 DNA 和 RNA 链测序的碱基检测器。最近的研究结果表明，较短的毛孔可能会改善碱基辨别力。新的工作还表明，αHL 的截短桶形突变体 (TBM) 在脂质双层中形成功能性孔。因此，我们测试了 TBM 孔识别固定在其中的 DNA 链碱基的能力。在 TBMΔ6 的情况下，桶缩短了约 16 A，野生型孔中发现的三个识别位点之一 R1 几乎被消除。随着进一步的诱变（Met113→Gly），R1被完全去除，这表明TBM孔可以介导锐化识别。值得注意的是，TBMΔ6 (Met113→Phe) 的第二个突变体能够结合带正电荷的 β-环糊精 am7βCD，异常紧密地允许单个核苷单磷酸的连续识别，这对于由纳米孔碱基识别介导的核酸外切酶测序是必需的。
The α-hemolysin (αHL) protein nanopore has been investigated previously as a base detector for the strand sequencing of DNA and RNA. Recent findings have suggested that shorter pores might provide improved base discrimination. New work has also shown that truncated-barrel mutants (TBM) of αHL form functional pores in lipid bilayers. Therefore, we tested TBM pores for the ability to recognize bases in DNA strands immobilized within them. In the case of TBMΔ6, in which the barrel is shortened by ~16 A, one of the three recognition sites found in the wild-type pore, R1, was almost eliminated. With further mutagenesis (Met113→Gly), R1 was completely removed, demonstrating that TBM pores can mediate sharpened recognition. Remarkably, a second mutant of TBMΔ6 (Met113→Phe) was able to bind the positively charged β-cyclodextrin, am7βCD, unusually tightly permitting the continuous recognition of individual nucleoside monophosphates, which would be required for exonuclease sequencing mediated by nanopore base identification.