| Autor: |
Goddard LR; 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., Mardle CE; School of Biological Sciences and Institute of Biological & Biomedical Sciences, University of Portsmouth, Portsmouth PO1 2DY, UK., Gneid H; RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA 01609, USA.; Department of Chemistry, University of Southampton, Southampton SO17 1BJ, UK., Ball CG; 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., Gowers DM; School of Biological Sciences and Institute of Biological & Biomedical Sciences, University of Portsmouth, Portsmouth PO1 2DY, UK., Atkins HS; Defence Science and Technology Laboratory, Porton Down, Salisbury SP4 0JQ, UK.; College of Life and Environmental Sciences, University of Exeter, Exeter EX4 4QD, UK., Butt LE; School of Biological Sciences and Institute of Biological & Biomedical Sciences, University of Portsmouth, Portsmouth PO1 2DY, UK., Watts JK; RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA 01609, USA., Vincent HA; 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., Callaghan AJ; 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. |
| Abstrakt: |
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. |
