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

Helen A Vincent,Hassan Gneid,Layla R Goddard,Jonathan K Watts,Helen S Atkins,Anastasia J Callaghan,Charlotte E Mardle,Louise E Butt,Ciara G Ball,Darren M Gowers

doi:10.3390/molecules26113414

Abstract

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.

Highlights

The emergence of both natural and engineered antimicrobial resistant strains of bacteria poses a significant challenge to the health and defense sectors
In the current study we explored the potential of targeting rne mRNA with antisense oligonucleotides as a possible alternative antibacterial strategy
The first step in investigating an antisense approach to potentially down-regulate rne gene expression was to identify a region of the E. coli rne mRNA to target and rationally design antisense oligonucleotides against it

Summary

Introduction

The emergence of both natural and engineered antimicrobial resistant strains of bacteria poses a significant challenge to the health and defense sectors. Traditional drug development programmes, that successfully provided the antibiotics of the 20th century, are failing to keep pace with emerging resistance [1,2]. There is a growing need for the development of novel antibacterial strategies that target alternative pathways and/or have unconventional mechanisms of action. RNA degradation pathways and ribonucleases (RNases), the enzymes responsible for RNA turnover, have recently been identified as targets that could be exploited for antibiotic development [3,4]. The endoribonuclease RNase E, and the rne gene that encodes it, are ideal candidates for antibacterial targeting for a number of reasons [3,4]. RNase E/rne is implicated in bacterial virulence of the pathogens Salmonella enterica and Yersinia pestis [9,10]

Methods

Results

Conclusion