Global RNA profiles show target selectivity and physiological effects of peptide-delivered antisense antibiotics

Nucleic Acids Res. 2021 May 7;49(8):4705-4724. doi: 10.1093/nar/gkab242.


Antisense peptide nucleic acids (PNAs) inhibiting mRNAs of essential genes provide a straight-forward way to repurpose our knowledge of bacterial regulatory RNAs for development of programmable species-specific antibiotics. While there is ample proof of PNA efficacy, their target selectivity and impact on bacterial physiology are poorly understood. Moreover, while antibacterial PNAs are typically designed to block mRNA translation, effects on target mRNA levels are not well-investigated. Here, we pioneer the use of global RNA-seq analysis to decipher PNA activity in a transcriptome-wide manner. We find that PNA-based antisense oligomer conjugates robustly decrease mRNA levels of the widely-used target gene, acpP, in Salmonella enterica, with limited off-target effects. Systematic analysis of several different PNA-carrier peptides attached not only shows different bactericidal efficiency, but also activation of stress pathways. In particular, KFF-, RXR- and Tat-PNA conjugates especially induce the PhoP/Q response, whereas the latter two additionally trigger several distinct pathways. We show that constitutive activation of the PhoP/Q response can lead to Tat-PNA resistance, illustrating the utility of RNA-seq for understanding PNA antibacterial activity. In sum, our study establishes an experimental framework for the design and assessment of PNA antimicrobials in the long-term quest to use these for precision editing of microbiota.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anti-Bacterial Agents / pharmacology
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Cell Proliferation / drug effects
  • Cell Proliferation / genetics
  • Drug Resistance, Bacterial / genetics
  • Microbial Sensitivity Tests
  • Oligonucleotides, Antisense / pharmacology*
  • Oligopeptides / pharmacology
  • Peptide Fragments / pharmacology
  • Peptide Nucleic Acids / metabolism
  • Peptide Nucleic Acids / pharmacology*
  • Peptides / chemistry*
  • Peptides / metabolism
  • Peptides / pharmacology
  • RNA Stability / drug effects
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism*
  • RNA-Seq
  • Salmonella enterica / drug effects*
  • Salmonella enterica / genetics
  • Salmonella enterica / metabolism
  • Signal Transduction / drug effects
  • Signal Transduction / genetics
  • Stress, Physiological / genetics*
  • tat Gene Products, Human Immunodeficiency Virus / pharmacology


  • (lysyl-phenylalanyl-phenylalanyl)3-lysine
  • Anti-Bacterial Agents
  • Bacterial Proteins
  • Oligonucleotides, Antisense
  • Oligopeptides
  • Peptide Fragments
  • Peptide Nucleic Acids
  • Peptides
  • RNA, Messenger
  • tat Gene Products, Human Immunodeficiency Virus
  • tat peptide (1-9), Human immunodeficiency virus 1
  • PhoP protein, Bacteria