Background Antisense peptide nucleic acids (PNAs) constitute an alternative to traditional antibiotics, by their ability to silence essential genes. Objectives To evaluate the antibacterial effects of antisense PNA-peptide conjugates that target the gene encoding the alpha subunit (NrdA) of the Escherichia coli ribonucleotide reductase (RNR). Methods Bacterial susceptibility of a series of NrdA-targeting PNAs was studied by MIC determination and time–kill analysis. Western-blot analysis, gene complementation and synergy with hydroxyurea were employed to determine the efficiency of NrdA-PNA antisense treatment. The effect on chromosome replication was addressed by determining the DNA synthesis rate, by flow cytometry analysis, by quantitative PCR and by fluorescence microscopy. The use of DNA repair mutants provided insight into the bactericidal action of NrdA-PNA. Results Treatment with NrdA-PNA specifically inhibited growth of E. coli, as well as NrdA protein translation at 4 μM. Also, the DNA synthesis rate was reduced, preventing completion of chromosome replication and resulting in formation of double-stranded DNA breaks and cell death. Conclusions These data present subunits of the NrdAB RNR as a target for future antisense microbial agents and provide insight into the bacterial physiological response to RNR-targeting antimicrobials.

中文翻译：

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由于诱导 DNA 链断裂，大肠杆菌 NrdAB 需氧核糖核苷酸还原酶的反义抑制具有杀菌作用

背景 反义肽核酸 (PNA) 通过沉默必需基因的能力构成传统抗生素的替代品。目的 评估靶向编码大肠杆菌核糖核苷酸还原酶 (RNR) α 亚基 (NrdA) 基因的反义 PNA-肽偶联物的抗菌作用。方法通过MIC测定和时间-杀伤分析研究了一系列NrdA靶向PNA的细菌敏感性。采用蛋白质印迹分析、基因互补和与羟基脲的协同作用来确定 NrdA-PNA 反义处理的效率。通过确定 DNA 合成速率、流式细胞术分析、定量 PCR 和荧光显微镜来解决对染色体复制的影响。DNA 修复突变体的使用提供了对 NrdA-PNA 杀菌作用的深入了解。结果 NrdA-PNA 处理特异性抑制大肠杆菌的生长，以及 4 μM 的 NrdA 蛋白翻译。此外，DNA 合成速率降低，阻止了染色体复制的完成，并导致双链 DNA 断裂和细胞死亡的形成。结论 这些数据将 NrdAB RNR 的亚基作为未来反义微生物剂的靶标，并提供对 RNR 靶向抗微生物剂的细菌生理反应的深入了解。阻止染色体复制的完成并导致双链DNA断裂和细胞死亡的形成。结论 这些数据将 NrdAB RNR 的亚基作为未来反义微生物剂的靶标，并提供对 RNR 靶向抗微生物剂的细菌生理反应的深入了解。阻止染色体复制的完成并导致双链DNA断裂和细胞死亡的形成。结论 这些数据将 NrdAB RNR 的亚基作为未来反义微生物剂的靶标，并提供对 RNR 靶向抗微生物剂的细菌生理反应的深入了解。