Kulik Marta, Markowska-Zagrajek Agnieszka, Wojciechowska Monika, Grzela Renata, Wituła Tomasz, Trylska Joanna
Centre of New Technologies, University of Warsaw, Banacha 2c, 02-097, Warsaw, Poland; Faculty of Chemistry, University of Warsaw, Pasteura 1, 02-093, Warsaw, Poland.
Centre of New Technologies, University of Warsaw, Banacha 2c, 02-097, Warsaw, Poland; Faculty of Biology, University of Warsaw, Miecznikowa 1, 02-096, Warsaw, Poland.
Biochimie. 2017 Jul;138:32-42. doi: 10.1016/j.biochi.2017.04.001. Epub 2017 Apr 7.
A fragment of 23S ribosomal RNA (nucleotides 1906-1924 in E. coli), termed Helix 69, forms a hairpin that is essential for ribosome function. Helix 69 forms a conformationally flexible inter-subunit connection with helix 44 of 16S ribosomal RNA, and the nucleotide A1913 of Helix 69 influences decoding accuracy. Nucleotides U1911 and U1917 are post-transcriptionally modified with pseudouridines (Ψ) and U1915 with 3-methyl-Ψ. We investigated Helix 69 as a target for a complementary synthetic oligonucleotide - peptide nucleic acid (PNA). We determined thermodynamic properties of Helix 69 and its complexes with PNA and tested the performance of PNA targeted at Helix 69 in inhibiting translation in cell-free extracts and growth of E. coli cells. First, we examined the interactions of a PNA oligomer complementary to the G1907-A1919 fragment of Helix 69 with the sequences corresponding to human and bacterial species (with or without pseudouridine modifications). PNA invades the Helix 69 hairpin creating stable complexes and PNA binding to the pseudouridylated bacterial sequence is stronger than to Helix 69 without any modifications. Second, we confirmed the binding of PNA to 23S rRNA and 70S ribosomes. Third, we verified the efficiency of translation inhibition of these PNA oligomers in the cell-free translation/transcription E. coli system, which were in a similar range as tetracycline. Next, we confirmed that PNA conjugated to the (KFF)K transporter peptide inhibited E. coli growth in micromolar concentrations. Overall, targeting Helix 69 with PNA or other sequence-specific oligomers could be a promising way to inhibit bacterial translation.
一段23S核糖体RNA片段（大肠杆菌中核苷酸1906 - 1924），称为螺旋69，形成一个对核糖体功能至关重要的发夹结构。螺旋69与16S核糖体RNA的螺旋44形成构象灵活的亚基间连接，且螺旋69的核苷酸A1913影响解码准确性。核苷酸U1911和U1917在转录后被假尿苷（Ψ）修饰，U1915被3 - 甲基 - Ψ修饰。我们研究了将螺旋69作为互补合成寡核苷酸——肽核酸（PNA）的靶标。我们测定了螺旋69及其与PNA复合物的热力学性质，并测试了靶向螺旋69的PNA在无细胞提取物中抑制翻译以及在大肠杆菌细胞生长中的性能。首先，我们研究了与螺旋69的G1907 - A1919片段互补的PNA寡聚物与对应人类和细菌物种序列（有或无假尿苷修饰）的相互作用。PNA侵入螺旋69发夹结构形成稳定复合物，且PNA与经假尿苷修饰的细菌序列的结合强于与未修饰的螺旋69的结合。其次，我们证实了PNA与23S rRNA和70S核糖体的结合。第三，我们验证了这些PNA寡聚物在大肠杆菌无细胞翻译/转录系统中抑制翻译的效率，其与四环素的抑制效率范围相似。接下来，我们证实与（KFF）K转运肽偶联的PNA在微摩尔浓度下抑制大肠杆菌生长。总体而言，用PNA或其他序列特异性寡聚物靶向螺旋69可能是抑制细菌翻译的一种有前景的方法。
