Mechanistic Studies of Antibacterial PNAs by RNA-Seq Analysis.

RNA sequencing (RNA-seq) has become the principal method for bacterial transcriptome analysis, enabling investigation of antibiotic mechanisms of action, stress responses, and resistance development. It has also played a key role in advancing antibacterial antisense oligomers (ASOs), particularly peptide nucleic acid (PNA)-based compounds. When conjugated to cell-penetrating peptides (CPPs) to enable intracellular delivery, PNAs provide a sequence-specific strategy to modulate bacterial gene expression. While growth-based assays can demonstrate bacterial killing by PNA-CPPs targeting essential genes, RNA-seq is required to elucidate the global transcriptional response to PNA-CPP exposure. This includes downregulation of PNA-targeted mRNAs, identification of cellular pathways linked to the targeted gene, assessment of off-target effects, and characterization of broader stress and membrane damage responses. Together, these insights provide a mechanistic understanding of PNA delivery, activity, and bacterial killing. This chapter describes optimized RNA-seq workflows for studying PNA-CPPs in Escherichia coli and Salmonella enterica; discusses considerations for RNA isolation, sequencing strategies, and data analysis; and emphasizes the importance of robust, reproducible pipelines for reliable data interpretation.