Sequence-specific bacterial growth inhibition by peptide nucleic acid targeted to the mRNA binding site of 16S rRNA
Sequence-specific bacterial growth inhibition by peptide nucleic acid targeted to the mRNA binding site of 16S rRNA
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DOI：
10.1007/s00253-009-2099-0
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发表时间：
2009-10-01
影响因子：
5
通讯作者：
Imachi, Hiroyuki
中科院分区：
文献类型：
--
作者：
Hatamoto, Masashi;Nakai, Kazufumi;Imachi, Hiroyuki
肽核酸（PNA）靶向23 S rRNA的功能域可以抑制翻译和细胞生长。然而，使用16S rRNA靶向PNA的翻译和细胞生长的有效抑制仍然没有实现。在这里，我们报告，PNA针对16S rRNA的功能位点可以抑制基因表达在体外和细菌生长在纯培养物中具有序列特异性。我们使用了与细胞穿透肽缀合的10-mer PNA，其靶向16S rRNA 3 '端的mRNA结合位点。使用0.6 μ M的肽-PNA，无细胞半乳糖苷酶的生产下降了50%，而肽-PNA与靶序列的一个或两个错配显示出弱得多的抑制作用。为了确定肽-PNA缀合物的生长抑制和杀菌作用，我们进行OD测量和活细胞计数。我们观察到细胞生长和杀菌作用的剂量和序列依赖性抑制。在革兰氏阴性细菌大肠杆菌和革兰氏阳性细菌枯草芽孢杆菌和高效棒状杆菌中均观察到这些生长抑制作用，尽管每种细菌的抑制浓度不同。这些结果提出了使用肽-PNA缀合物的基于16S rRNA序列的特异性细菌生长抑制的可能性。
Peptide nucleic acid (PNA) targeted to the functional domains of 23S rRNA can inhibit translation and cell growth. However, effective inhibition of translation and cell growth using 16S rRNA-targeted PNA has still not been achieved. Here, we report that PNA targeted to the functional site of 16S rRNA could inhibit both gene expression in vitro and bacterial growth in pure culture with sequence specificity. We used 10-mer PNAs conjugated with a cell-penetrating peptide, which targeted the mRNA binding site at the 3' end of 16S rRNA. Using 0.6 A mu M of the peptide-PNAs, cell-free -galactosidase production decreased by 50%, whereas peptide-PNAs with one or two mismatches to the target sequence showed much weaker inhibition effects. To determine the growth inhibition and bactericidal effects of the peptide-PNA conjugate, we performed OD measurement and viable cell counting. We observed dose- and sequence-dependent inhibition of cell growth and bactericidal effects. These growth inhibitory effects are observed both in the Gram-negative bacterium of Escherichia coli and the Gram-positive bacteria Bacillus subtilis and Corynebacterium efficiens, although inhibitory concentrations were different for each bacterial species. These results present possibilities for 16S rRNA sequence-based specific bacterial growth inhibition using a peptide-PNA conjugate.