Identifying Antisense Oligonucleotides to Disrupt Small RNA Regulated Antibiotic Resistance via a Cell-Free Transcription–Translation Platform

[Image: see text] Bacterial small RNAs (sRNAs) regulate many important physiological processes in cells, including antibiotic resistance and virulence genes, through base-pairing interactions with mRNAs. Antisense oligonucleotides (ASOs) have great potential as therapeutics against bacterial pathogens by targeting sRNAs such as MicF, which regulates outer membrane protein OmpF expression and limits the permeability of antibiotics. Here we devised a cell-free transcription–translation (TX–TL) assay to identify ASO designs that sufficiently sequester MicF. ASOs were then ordered as peptide nucleic acids conjugated to cell-penetrating peptides (CPP-PNA) to allow for effective delivery into bacteria. Subsequent minimum inhibitory concentration (MIC) assays demonstrated that simultaneously targeting the regions of MicF responsible for sequestering the start codon and the Shine–Dalgarno sequence of ompF with two different CPP-PNAs synergistically reduced the MIC for a set of antibiotics. This investigation offers a TX–TL-based approach to identify novel therapeutic candidates to combat intrinsic sRNA-mediated antibiotic resistance mechanisms.