Cell permeabilization and uptake of antisense peptide-peptide nucleic acid (PNA) into Escherichia coli
Cell permeabilization and uptake of antisense peptide-peptide nucleic acid (PNA) into Escherichia coli
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DOI：
10.1074/jbc.m106624200
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发表时间：
2002-03-01
影响因子：
4.8
通讯作者：
Good, L
中科院分区：
文献类型：
--
作者：
Eriksson, M;Nielsen, PE;Good, L
肽核酸（PNA）是一种DNA模拟物，具有开发反义药物的潜力。针对大肠杆菌基因的反义PNA可以特异性地抑制基因表达，并且PNA与细胞透化肽KFFKFFK的连接显著地提高了反义效力。早期观察到的效力改善被认为是由于更好的细胞摄取;然而，尚未研究标准或修饰的PNA进入细菌的摄取动力学。在这里，我们监测外和内膜透化，通过使用化学探针，通常被排除在细胞外，但可以在膜完整性受到干扰的点获得访问。相对于单独或组合使用的游离组分，在肽-PNA缀合物的存在下膜透化快得多。事实上，肽-PNA透化E.大肠杆菌几乎和抗菌肽一样快。此外，正如对外膜活性化合物的预期，加入MgCl 2降低了细胞透化。外膜和内膜透化的同时监测表明，穿过外膜是摄取的速率限制。肽-PNA的增强的细胞渗透性质可以解释其有效的反义活性，并且结果表明肽和PNA组分之间的意外协同作用。
Peptide nucleic acid (PNA) is a DNA mimic with promising properties for the development of antisense agents. Antisense PNAs targeted to Escherichia coli genes can specifically inhibit gene expression, and attachment of PNA to the cell-permeabilizing peptide KFFKFFKFFK dramatically improves antisense potency. The improved potency observed earlier was suggested to be due to better cell uptake; however, the uptake kinetics of standard or modified PNAs into bacteria had not been investigated. Here we monitored outer and inner membrane permeabilization by using chemical probes that normally are excluded from cells but can gain access at points where membrane integrity is disturbed. Membrane permeabilization was much more rapid in the presence of peptide-PNA conjugates relative to the free components used alone or in combination. Indeed, peptide-PNAs permeabilized E. coli nearly as quickly as antimicrobial peptides. Furthermore, as expected for outer membrane-active compounds, added MgCl2 reduced cell-permeabilization. Concurrent monitoring of outer and inner membrane permeabilization indicated that passage across the outer membrane is rate-limiting for uptake. The enhanced cell-permeation properties of peptide-PNAs can explain their potent antisense activity, and the results indicate an unanticipated synergy between the peptide and PNA components.