School of Biological Sciences and Institute of Biological & Biomedical Sciences, University of Portsmouth, Portsmouth PO1 2DY, UK.
Centre for Enzyme Innovation, University of Portsmouth, Portsmouth PO1 2DY, UK.
Molecules. 2021 Jun 4;26(11):3414. doi: 10.3390/molecules26113414.
The increase in antibacterial resistance is a serious challenge for both the health and defence sectors and there is a need for both novel antibacterial targets and antibacterial strategies. RNA degradation and ribonucleases, such as the essential endoribonuclease RNase E, encoded by the gene, are emerging as potential antibacterial targets while antisense oligonucleotides may provide alternative antibacterial strategies. As mRNA has not been previously targeted using an antisense approach, we decided to explore using antisense oligonucleotides to target the translation initiation region of the mRNA. Antisense oligonucleotides were rationally designed and were synthesised as locked nucleic acid (LNA) gapmers to enable inhibition of mRNA translation through two mechanisms. Either LNA gapmer binding could sterically block translation and/or LNA gapmer binding could facilitate RNase H-mediated cleavage of the mRNA. This may prove to be an advantage over the majority of previous antibacterial antisense oligonucleotide approaches which used oligonucleotide chemistries that restrict the mode-of-action of the antisense oligonucleotide to steric blocking of translation. Using an electrophoretic mobility shift assay, we demonstrate that the LNA gapmers bind to the translation initiation region of mRNA. We then use a cell-free transcription translation reporter assay to show that this binding is capable of inhibiting translation. Finally, in an in vitro RNase H cleavage assay, the LNA gapmers facilitate RNase H-mediated mRNA cleavage. Although the challenges of antisense oligonucleotide delivery remain to be addressed, overall, this work lays the foundations for the development of a novel antibacterial strategy targeting mRNA with antisense oligonucleotides.
细菌耐药性的增加是卫生和国防部门面临的一个严重挑战，因此需要寻找新的抗菌靶点和抗菌策略。RNA 降解和核糖核酸酶（如由基因编码的必需内切核糖核酸酶 RNase E）正成为有潜力的抗菌靶点，而反义寡核苷酸可能提供替代的抗菌策略。由于之前没有使用反义方法靶向 mRNA，我们决定探索使用反义寡核苷酸靶向 mRNA 的翻译起始区。我们合理设计了反义寡核苷酸，并将其合成为锁核酸（LNA）gapmer，以通过两种机制抑制 mRNA 翻译。LNA gapmer 的结合可以通过空间位阻阻止翻译，或者 LNA gapmer 的结合可以促进 RNase H 介导的 mRNA 切割。与大多数之前使用限制反义寡核苷酸作用方式为翻译空间位阻的寡核苷酸化学的抗菌反义寡核苷酸方法相比，这可能是一个优势。通过电泳迁移率变动分析，我们证明 LNA gapmer 结合到 mRNA 的翻译起始区。然后，我们使用无细胞转录翻译报告基因测定来证明这种结合能够抑制翻译。最后，在体外 RNase H 切割测定中，LNA gapmer 促进了 RNase H 介导的 mRNA 切割。尽管反义寡核苷酸递送的挑战仍有待解决，但总的来说，这项工作为开发针对 mRNA 的新型抗菌反义寡核苷酸策略奠定了基础。
