Antimicrobial resistance (AMR) is a global threat to human health, with an estimated 1.27 million deaths attributed to AMR in 20191. Antibiotic development in the last five years has shown a limited degree of innovation, with 80% of approved drugs being derived from existing classes with established resistance2. This highlights the urgent need for novel antimicrobial strategies. Antisense peptide nucleic acids (PNAs) offer a programmable, sequence-specific method for gene knockdown that can inhibit bacterial growth or re-sensitise bacteria to antibiotics. While promising, previous studies indicate that they are active at low micromolar concentrations, leaving significant room to improve their activity. Here, we aim to enhance the activity of PNAs by incorporating molecular ‘tags’ that improve RNase E recruitment to facilitate messenger RNA (mRNA) degradation. First, we established a baseline activity of the unmodified PNA in vitro and in vivo. We demonstrated that multiple pathotypes of Escherichia coli (E. coli) are susceptible to killing by PNAs targeting the essential fatty acid biosynthesis gene, acpP. Using GFP translational fusions, we confirmed that the antibacterial activity of these PNAs is mediated through complementary base-pairing with their target sequence. To test the activity of the enhanced PNAs, we have established an in vitro RNA-guided RNase E processing assay. Purified, recombinant RNase E catalytic domain (1-529) is used to process fluorescently labelled ompD using a 12-mer RNA or PNA to guide cleavage. Sequence and chemical modifications of the PNA will be tested to identify signals that enhance PNA-mRNA recruitment to the RNA degradosome. Overall, our results establish a baseline for the antibacterial activity of PNAs against E. coli. These findings underscore the potential of antisense RNAs to be a platform technology that can be rapidly adapted to new AMR threats.