Gene function studies in bacteria lag behind progress in genome sequencing, in part because current reverse genetics technology based on genome disruption does not allow subtle control of gene expression for all genes in a range of species. Essential genes and clustered regions are particularly problematic. Antisense technology offers an attractive alternative for microbial genomics. Unfortunately, bacteria lack RNAi mechanisms and conventional oligonucleotides are not taken up efficiently. However, in Escherichia coli, efficient and gene-specific antisense knock down is possible using antisense peptide nucleic acids (PNAs) attached to carrier peptides (KFFKFFKFFK). Carrier peptides can enter a range of microbial species, and in this study we asked whether peptide-PNAs could mediate antisense effects in Staphylococcus aureus. Using low micromolar concentrations we observed dose- and sequence-dependent inhibition of the reporter gene gfp and endogenous gene phoB. Also, antisense peptide-PNAs targeted to the essential genes fmhB, gyrA, and hmrB were growth inhibitory. Control peptide-PNAs were much less effective, and sequence alterations within the PNA and target mRNA sequences reduced or eliminated inhibition. Further development is needed to raise the antibacterial potential of PNAs, but the present results show that the approach can be used to study gene function and requirement in this important pathogen. Copyright © The American Society of Gene Therapy.
Nekhotiaeva, N., Awasthi, S. K., Nielsen, P. E., & Good, L. (2004). Inhibition of Staphylococcus aureus gene expression and growth using antisense peptide nucleic acids. Molecular Therapy, 10(4), 652–659. https://doi.org/10.1016/j.ymthe.2004.07.006