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

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

Probing of surface-immobilised biotinylated-sRNA with partner mRNA-Cy3.

<p>(a) Streptavidin-coated microarray slide with control spots of (1) biotin-OxyS, (2) blank surface, (3) sRNA MicA, and test spot of (4) biotin-MicA. The surface was probed with Cy3-labelled <i>ompA</i>. The specific <i>ompA i</i>nteraction with surface-immobilised biotin-MicA is shown by the green spot. (b) As for (a) but in this case the test spot (4) is biotin-Qrr1 and the control sRNA spot (3) is Qrr1. The surface was probed with Cy3-labelled <i>hapR</i>. The specific <i>hapR</i> interaction with surface-immobilised biotin-Qrr1 is seen by the green spot. Schematic illustrations of the interactions occurring in (a) and (b) are shown beneath the microarray slides with the streptavidin surface in yellow, sRNAs in brown and Cy3-labelled mRNA in green.</p

SPR analysis of Qrr3-mRNA interactions.

<p>(a) Surface-immobilised biotin-<i>hapR</i>. Example sensorgram of sequential injections of Qrr3 from 0–0.25 µM; data fit (black) with chi² = 0.28 RU². (b) Surface-immobilised biotin-<i>vca0939</i>. Example sensorgram of sequential injections of Qrr3 from 0–0.25 µM; data fit (black) with chi² = 0.41 RU².</p

Kinetic data for sRNA-mRNA interactions from SPR analyses.

<p>Kinetic data for sRNA-mRNA interactions from SPR analyses.</p

Analysis of the ligation reaction for Qrr2 sRNA (a) with and (b) without A-tails.

<p>Gels were stained with the SYBR-Gold, whereas blots were probed with streptavidin-HRP to detect biotin-labelled RNA. Schematic representations of RNA species identified on the gels/blots are shown. The sequences of the RNAs are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079142#pone.0079142.s001" target="_blank">Table S1 in File S1</a>.</p

SPR analysis of RNA-RNA interactions.

<p>(a) Surface-immobilised biotin-<i>ompA</i>. Example sensorgrams of sequential injections of MicA (red) or OxyS (blue) from 0–10 µM; MicA data fit (black) with chi² = 0.20 RU². (b) Control sensorgrams of sequential injections of MicA from 0–10 µM over surface-immobilised <i>rpoS</i> mRNA (orange) or U-biotin reagent (green).</p