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Sequence-specific bacterial growth inhibition by peptide nucleic acid targeted to the mRNA binding site of 16S rRNA

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Abstract

Peptide nucleic acid (PNA) targeted to the functional domains of 23S rRNA can inhibit translation and cell growth. However, effective inhibition of translation and cell growth using 16S rRNA-targeted PNA has still not been achieved. Here, we report that PNA targeted to the functional site of 16S rRNA could inhibit both gene expression in vitro and bacterial growth in pure culture with sequence specificity. We used 10-mer PNAs conjugated with a cell-penetrating peptide, which targeted the mRNA binding site at the 3′ end of 16S rRNA. Using 0.6 µM of the peptide–PNAs, cell-free ß-galactosidase production decreased by 50%, whereas peptide–PNAs with one or two mismatches to the target sequence showed much weaker inhibition effects. To determine the growth inhibition and bactericidal effects of the peptide–PNA conjugate, we performed OD measurement and viable cell counting. We observed dose- and sequence-dependent inhibition of cell growth and bactericidal effects. These growth inhibitory effects are observed both in the Gram-negative bacterium of Escherichia coli and the Gram-positive bacteria Bacillus subtilis and Corynebacterium efficiens, although inhibitory concentrations were different for each bacterial species. These results present possibilities for 16S rRNA sequence-based specific bacterial growth inhibition using a peptide–PNA conjugate.

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Acknowledgment

This research was financially supported by the Sumitomo Foundation, the Japan Society for the Promotion of Science, and the Ministry of Education, Culture, Sports, Science and Technology, Japan.

Author information

Correspondence to Hiroyuki Imachi.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Fig. S1

Target sits of antiribosomal peptide–PNAs in secondary structure of three E. coli 16S rRNA. Highlighted sequences are the target of peptide–PNAs used in this study 4 (Table 1). Secondary structure model was obtained from the Comparative RNA Web Site 5 (http://www.rna.icmb.utexas.edu). (PDF 143 kb)

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Hatamoto, M., Nakai, K., Ohashi, A. et al. Sequence-specific bacterial growth inhibition by peptide nucleic acid targeted to the mRNA binding site of 16S rRNA. Appl Microbiol Biotechnol 84, 1161–1168 (2009). https://doi.org/10.1007/s00253-009-2099-0

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Keywords

  • Peptide nucleic acid (PNA)
  • 16S rRNA
  • mRNA binding site
  • Cell wall-permeablizing peptide