Towards RNA Antibiotics: Investigating Antisense Oligonucleotides in Escherichia coli

Abstract

The rise in antibiotic-resistant pathogens resulting from misuse and overuse of antibiotics, coupled with the shortage of new antibiotics to fight resistant infections has created an increasing need for new or alternative antibacterial therapeutics. Narrow-spectrum, species-specific antibiotics can target bacteria on a species-level and thus spare beneficial species of the microbiome as well as prevent the dissemination of resistance genes to non-targeted species. Narrow-spectrum antibiotics are therefore an attractive solution to rising antibiotic resistance but finding drugs that only target specific species has proven challenging. Towards this goal, programmable antisense oligonucleotides (ASOs) have been proposed as a way to target bacterial genes with species-specificity. If successful, this therapeutic has the potential to treat resistant bacteria on a species-level while avoiding harm to other species of the microbiome that are often indiscriminately targeted by broad-spectrum drugs leading to negative health outcomes. The present study thus aims to aid the design and discovery of novel antisense oligonucleotides and mRNA targets with growth inhibitory or killing capacity. Using a randomized synthetic RNA screen, we employ and evaluate a Next-Generation Sequencing (NGS)-based approach involving simultaneous induction of the complete synthetic RNA-expression library. We also evaluate another approach involving individual culturing and induction of single library strains. Our work yields two potential synthetic RNAs capable of growth inhibition. Additionally, we conduct peptide nucleic acid (PNA) studies to better understand the antisense mechanism of action. Using PNAs targeting the acyl carrier peptide gene (acpP), we demonstrate an antibacterial effect in clinically relevant UPEC CFT073, synergy with the membrane-permeabilizing antibiotic, Polymyxin B, and identify the involvement of SbmA, OppA, DppA peptide transporters in the bacterial uptake of PNA. Additionally, we show that the Hfq molecular chaperone, involved in the mediating small RNA-mRNA binding, is not required for peptide-conjugated PNA (PPNA)-killing, as was previously thought. Together these efforts define new ways of identifying ASOs and new understanding of the mechanisms of action of PNAs, which represent important steps towards ultimately generating RNA-based species-specific antibiotics.