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

Synthesis of PAMAM dendrimers and investigations of their interaction with POPC/POPG lipids

PAMAM dendrimers are three dimensional organic polymers synthesised by repetitive steps to achieve a controlled size and shape with a choice of surface functional groups. One of the potential applications of dendrimers is for drug/gene delivery which requires the dendrimer to interact with the cellular membranes. This study is designed to probe the interactions between PAMAM dendrimers and lipid bilayers. To investigate these interactions PAMAM dendrimers up to the third generation were synthesised. 31P and 1H-1H NOESY NMR studies between these dendrimer and POPC/POPG-derived lipids were then carried out. The results obtained from the NMR experiments were then compared with those from fluorescence studies using a surface labelled PAMAM dendrimer and the nature of the dendrimer-lipid bilayer interactions was also explored using molecular dynamics modelling. The solid-state NMR study in a controlled buffer at pH 7.2 revealed that the larger dendrimer (third generation) interacts strongly with a bilayer containing POPG, but not with a bilayer containing only POPC, and no interaction between the smaller dendrimer (zero generation) was observed with either POPC or POPG. This was confirmed with the fluorescence experiments, as changes in the emission intensity of a labelled dendrimer were mainly detected for negatively charged species (SDS and POPG) and rather less for zwitterionic, neutral or cationic species (1,2-dodecyldiol, CTAB and POPC). The coarsegrained molecular dynamic simulations showed that the 3rd generation PAMAM dendrimer can interact with the surface of the membrane when dendrimer is positively charged, but not when the dendrimer is uncharged. These studies demonstrate how the positive charges and size of the dendrimer influence the interaction with negatively charged lipid, which can have an impact on both the dendrimer’s cellular uptake and potential toxicity.

Development of broad-spectrum antimicrobials using modified antisense oligonucleotides

Antibiotics have formed a corner stone of modern medicine. However, bacteria can develop resistance against antibacterial drugs and new antibiotics have to be created at all times to compete with this resistance. One way of potentially generating new antibiotics is by using antisense oligonucleotides (ASOs) that can recruit enzymes to cleave the mRNA of essential genes. In chapter 2, we thus designed and synthesized a number of PNA-based ASOs targeting essential, reporter and virulence genes of E. coli. The cell penetrating peptide KFFKFFKFFK was covalently attached to the PNAs in order to facilitate the uptake of the oligonucleotides into the bacterial cells. Using a variety of antibacterial assays, we were able to show that PNAs functioning by sterically blocking the ribosome binding site are able to silence the activity of the ftsZ and katG gene. Unfortunately, PNAs that function by recruiting RNase P via the external guide sequence did not seem to have any gene silencing ability. In vitro studies suggested that this is because the PNA backbone cannot be recognized by the RNase P ribozyme. In chapter 3 we therefore tried to recruit a different enzyme, RNase H. A number of LNA-DNA-LNA gapmers targeting the ftsZ gene in E. coli were designed and synthesized. The standard RNase H recruitment assay showed that these oligonucleotides are able to induce cleavage of the target mRNA by the enzyme. Microscopy studies confirmed that the gapmers are also able to induce gene silencing in bacteria, presumably through the recruitment of RNase H. In Chapter 4, we systemically designed and screened libraries of chemically modified oligonucleotides containing the external guide sequence for their ability to recruit RNase P in vitro. We had a number of successful hits, mainly hybrids of DNA, LNA and to a lesser extent OMe. Due to the high DNA content of the hits, the potential dual recruiting of RNase P and RNase H was explored. A number of chemically modified oligonucleotides were identified that can induce in vitro cleavage of their target mRNA by both enzymes. Such compounds are expected to have superior gene silencing ability. Microscopy studies confirmed the in vitro findings, as we were able to detect elongated E. coli cells (indicative of successful ftsZ gene silencing) for those compounds that showed potential dual recruitment activity. The work presented in this thesis has shown a promising technology for the use of chemically modified antisense oligonucleotides as antibiotics, especially when their gene silencing could be enhanced by the recruitment of RNase P, RNase H or both. However, the biggest challenge for the development of any oligonucleotide based therapeutic remains their problematic delivery into the target cells or bacteria. Future efforts will thus have to focus on identifying more efficient ways of delivering oligonucleotides into bacterial cells

Dataset for: Development of Broad-Spectrum Antimicrobials Using Modified Antisense Oligonucleotides

This dataset supports the PhD thesis entitled &#39;Development of Broad-Spectrum Antimicrobials Using Modified Antisense Oligonucleotides&#39; by Gneid.</span