An Investigation into the Potential of Targeting Escherichia coli rne mRNA with Locked Nucleic Acid (LNA) Gapmers as an Antibacterial Strategy

Molecules

Helen S. Atkins¹  Jonathan K. Watts²  Hassan Gneid²  Ciara G. Ball³  Layla R. Goddard³  Helen A. Vincent³  Louise E. Butt³  Anastasia J. Callaghan³  Darren M. Gowers³  Charlotte E. Mardle³ 

[1] Defence Science and Technology Laboratory, Porton Down, Salisbury SP4 0JQ, UK;RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA 01609, USA;School of Biological Sciences and Institute of Biological & Biomedical Sciences, University of Portsmouth, Portsmouth PO1 2DY, UK;

关键词: antibacterial;    antisense oligonucleotide;    gapmer;    locked nucleic acid (LNA);    RNase E;    RNase H;   

DOI  :  10.3390/molecules26113414

来源: DOAJ

【 摘 要 】

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 rne gene, are emerging as potential antibacterial targets while antisense oligonucleotides may provide alternative antibacterial strategies. As rne 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 Escherichia coli rne mRNA. Antisense oligonucleotides were rationally designed and were synthesised as locked nucleic acid (LNA) gapmers to enable inhibition of rne 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 rne 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 E. coli rne 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 rne mRNA with antisense oligonucleotides.

【 授权许可】

Unknown