Jasiński Maciej, Kulik Marta, Wojciechowska Monika, Stolarski Ryszard, Trylska Joanna
Centre of New Technologies, University of Warsaw, Warsaw, Poland.
College of Inter-Faculty Individual Studies in Mathematics and Natural Sciences, University of Warsaw, Warsaw, Poland.
PLoS One. 2018 Jan 19;13(1):e0191138. doi: 10.1371/journal.pone.0191138. eCollection 2018.
Synthetic oligonucleotides targeting functional regions of the prokaryotic rRNA could be promising antimicrobial agents. Indeed, such oligonucleotides were proven to inhibit bacterial growth. 2'-O-methylated (2'-O-Me) oligoribonucleotides with a sequence complementary to the decoding site in 16S rRNA were reported as inhibitors of bacterial translation. However, the binding mode and structures of the formed complexes, as well as the level of selectivity of the oligonucleotides between the prokaryotic and eukaryotic target, were not determined. We have analyzed three 2'-O-Me oligoribonucleotides designed to hybridize with the models of the prokaryotic rRNA containing two neighboring aminoglycoside binding pockets. One pocket is the paromomycin/kanamycin binding site corresponding to the decoding site in the small ribosomal subunit and the other one is the close-by hygromycin B binding site whose dynamics has not been previously reported. Molecular dynamics (MD) simulations, as well as isothermal titration calorimetry, gel electrophoresis and spectroscopic studies have shown that the eukaryotic rRNA model is less conformationally stable (in terms of hydrogen bonds and stacking interactions) than the corresponding prokaryotic one. In MD simulations of the eukaryotic construct, the nucleotide U1498, which plays an important role in correct positioning of mRNA during translation, is flexible and spontaneously flips out into the solvent. In solution studies, the 2'-O-Me oligoribonucleotides did not interact with the double stranded rRNA models but all formed stable complexes with the single-stranded prokaryotic target. 2'-O-Me oligoribonucleotides with one and two mismatches bound less tightly to the eukaryotic target. This shows that at least three mismatches between the 2'-O-Me oligoribonucleotide and eukaryotic rRNA are required to ensure target selectivity. The results also suggest that, in the ribosome environment, the strand invasion is the preferred binding mode of 2'-O-Me oligoribonucleotides targeting the aminoglycoside binding sites in 16S rRNA.
靶向原核生物rRNA功能区域的合成寡核苷酸可能是很有前景的抗菌剂。事实上，这类寡核苷酸已被证明能抑制细菌生长。据报道，与16S rRNA解码位点序列互补的2'-O-甲基化（2'-O-Me）寡核糖核苷酸是细菌翻译的抑制剂。然而，所形成复合物的结合模式和结构，以及寡核苷酸在原核和真核靶点之间的选择性水平尚未确定。我们分析了三种设计用于与包含两个相邻氨基糖苷类结合口袋的原核rRNA模型杂交的2'-O-Me寡核糖核苷酸。一个口袋是对应于小核糖体亚基解码位点的巴龙霉素/卡那霉素结合位点，另一个是附近的潮霉素B结合位点，其动力学此前尚未见报道。分子动力学（MD）模拟以及等温滴定量热法、凝胶电泳和光谱研究表明，真核rRNA模型在构象稳定性（就氢键和堆积相互作用而言）上低于相应的原核模型。在真核构建体的MD模拟中，在翻译过程中对mRNA正确定位起重要作用的核苷酸U1498是灵活的，并自发翻转到溶剂中。在溶液研究中，2'-O-Me寡核糖核苷酸不与双链rRNA模型相互作用，但都与单链原核靶点形成稳定复合物。有一个和两个错配的2'-O-Me寡核糖核苷酸与真核靶点的结合较松散。这表明2'-O-Me寡核糖核苷酸与真核rRNA之间至少需要三个错配才能确保靶点选择性。结果还表明，在核糖体环境中，链侵入是靶向16S rRNA中氨基糖苷类结合位点的2'-O-Me寡核糖核苷酸的首选结合模式。
