RNA toehold switch-based reporter assay to assess bacterial uptake of antisense oligomers

Antisense oligomers (ASOs) hold promise as antibiotics for the selective targeting of bacterial pathogens and as tools for the modulation of gene expression in microbes that are not amenable to genetic engineering. However, their efficient delivery across the complex bacterial envelope remains a major challenge. There are few methods to assess the efficiency of carrier-mediated ASO uptake by bacteria. Here, we have developed a "switch-on" reporter assay to measure ASO uptake efficiency in a semi-quantitative manner. The assay uses a synthetic RNA toehold switch fused to the mRNA of a fluorescent reporter protein, which is activated <i>in vivo</i> by a peptide nucleic acid (PNA)-based ASO upon delivery into the bacterial cytosol. We have used this assay to screen different cell-penetrating peptides (CPPs) as ASO carriers in <i>Escherichia coli</i> and <i>Salmonella enterica</i> and observed up to 60-fold activation, depending on the CPP and bacterial strain used. Our assay shows high dynamic range and sensitivity, which should enable high-throughput screens for bacterial ASO carriers. We also show that the reporter can be used to study routes of PNA uptake, as demonstrated by reduced reporter activity in the absence of the inner membrane protein SbmA. In summary, we present a tool for the discovery of species-specific and efficient ASO carriers that will also be useful for a broader investigation of cellular uptake mechanisms of antibacterial ASOs.IMPORTANCEThe rise of antimicrobial resistance presents a major global health challenge. If not addressed, the death toll from resistant infections is expected to rise dramatically in the coming years. As a result, it is essential to explore alternative antimicrobial therapies. One promising approach is to target bacterial mRNAs using antisense oligomers (ASOs) to silence genes involved in essential functions, virulence, or resistance. However, delivering ASOs across bacterial membranes remains a major challenge and effective methods to monitor their uptake are limited. In this study, we develop a reporter assay to facilitate the high-throughput discovery of bacterial ASO carriers. This research paves the way for developing novel precision antisense-based antibacterial therapies.