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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Braasch DA, Liu Y, Corey DR (2002) Antisense inhibition of gene expression in cells by oligonucleotides incorporating locked nucleic acids: effect of mRNA target sequence and chimera design. Nucl Acids Res 30:5160–5167
Deere J, Iversen P, Geller BL (2005) Antisense phosphorodiamidate morpholino oligomer length and target position effects on gene-specific inhibition in Escherichia coli. Antimicrob Agents Chemother 49:249–255
Demidov VV, Potaman VN, Frank-Kamenetskii MD, Egholm M, Buchard O, Sonnichsen SH, Nielsen PE (1994) Stability of peptide nucleic acids in human serum and cellular extracts. Biochem Pharmacol 48:1310–1313
Dryselius R, Aswasti SK, Rajarao GK, Nielsen PE, Good L (2003) The translation start codon region is sensitive to antisense PNA inhibition in Escherichia coli. Oligonucleotides 13:427–433
Geller BL, Deere JD, Stein DA, Kroeker AD, Moulton HM, Iversen PL (2003) Inhibition of gene expression in Escherichia coli by antisense phosphorodiamidate morpholino oligomers. Antimicrob Agents Chemother 47:3233–3239
Good L, Nielsen PE (1998) Inhibition of translation and bacterial growth by peptide nucleic acid targeted to ribosomal RNA. Proc Natl Acad Sci USA 95:2073–2076
Good L, Nielsen PE (1999) Peptide nucleic acid (PNA) antisense effects in Escherichia coli. Curr Issues Mol Biol 1:111–116
Good L, Sandberg R, Larsson O, Nielsen PE, Wahlestedt C (2000) Antisense PNA effects in Escherichia coli are limited by the outer-membrane LPS layer. Microbiology 146:2665–2670
Good L, Awasthi SK, Dryselius R, Larsson O, Nielsen PE (2001) Bactericidal antisense effects of peptide–PNA conjugates. Nat Biotechnol 19:360–364
Good L, Dryselius R, Nielsen PE (2004) Antisense effects in Escherichia coli. In: Nielsen PE (ed) Peptide nucleic acids. Protocols and applications. Horizon Bioscience, Norfolk, pp 291–304
Gruegelsiepe H, Brandt O, Hartmann RK (2006) Antisense inhibition of RNase P: mechanistic aspects and application to live bacteria. J Biol Chem 281:30613–30620
Hancock REW (1997) Peptide antibiotics. The Lancet 349:418–422
Janson CG, During MJ (eds) (2006) Peptide nucleic acids, morpholinos and related antisense biomolecules. Kluwer Academic, New York
Jayaraman K, McParland K, Miller P, Ts’o POP (1981) Selective inhibition of Escherichia coli protein synthesis and growth by nonionic oligonucleotides complementary to the 3′ end of 16S rRNA. Proc Natl Acad Sci USA 78:1537–1541
Knudsen H, Nielsen PE (1996) Antisense properties of duplex- and triplex-forming PNAs. Nucl Acids Res 24:494–500
Kulyte A, Nekhotiaeva N, Awasthi SK, Good L (2005) Inhibition of Mycobacterium smegmatis gene expression and growth using antisense peptide nucleic acids. J Mol Microbiol Biotechnol 9:101–109
Kurupati P, Tan KSW, Kumarasinghe G, Poh CL (2007) Inhibition of gene expression and growth by antisense peptide nucleic acids in a multiresistant ß-lactamase-producing Klebsiella pneumoniae strain. Antimicrob Agents Chemother 51:805–811
Lee LK, Roth CM (2003) Antisense technology in molecular and cellular bioengineering. Curr Opin Biotechnol 14:505–511
Nekhotiaeva N, Awasthi SK, Nielsen PE, Good L (2004) Inhibition of Staphylococcus aureus gene expression and growth using antisense peptide nucleic acids. Mol Ther 10:652–659
Nielsen PE, Egholm M (2004) An introduction to PNA. In: Nielsen PE (ed) Peptide nucleic acids. Protocols and applications. Horizon Bioscience, Norfolk, pp 1–36
Nikaido H (1994) Prevention of drug access to bacterial targets: permeability barriers and active efflux. Science 264:382–388
Ogle JM, Brodersen DE, Clemons WM Jr, Tarry MJ, Carter AP, Ramakrishnan V (2001) Recognition of cognate transfer RNA by the 30S ribosomal subunit. Science 292:897–902
Rajarao GK, Nekhotiaeva N, Good L (2002) Peptide-mediated delivery of green fluorescent protein into yeasts and bacteria. FEMS Microbiol Lett 215:267–272
Rappé MS, Giovannoni SJ (2003) The uncultured microbial majority. Annu Rev Microbiol 57:369–394
Rasmussen L, Sperling-Petersen H, Mortensen K (2007) Hitting bacteria at the heart of the central dogma: sequence-specific inhibition. Microbial Cell Factories 6:24
Rockwell P, O’Connor WJ, King K, Goldstein NI, Zhang LM, Stein CA (1997) Cell-surface perturbations of the epidermal growth factor and vascular endothelial growth factor receptors by phosphorothioate oligodeoxynucleotides. Proc Natl Acad Sci USA 94:6523–6528
Rodriguez-Correa D, Dahlberg AE (2004) Genetic evidence against the 16S ribosomal RNA helix 27 conformational switch model. RNA 10:28–33
Tan XX, Actor JK, Chen Y (2005) Peptide nucleic acid antisense oligomer as a therapeutic strategy against bacterial infection: proof of principle using mouse intraperitoneal infection. Antimicrob Agents Chemother 49:3203–3207
Tilley LD, Hine OS, Kellogg JA, Hassinger JN, Weller DD, Iversen PL, Geller BL (2006) Gene-specific effects of antisense phosphorodiamidate morpholino oligomer-peptide conjugates on Escherichia coli and Salmonella enterica serovar Typhimurium in pure culture and in tissue culture. Antimicrob Agents Chemother 50:2789–2796
Vaara M, Porro M (1996) Group of peptides that act synergistically with hydrophobic antibiotics against gram-negative enteric bacteria. Antimicrob Agents Chemother 40:1801–1805
Wahlestedt C, Salmi P, Good L, Kela J, Johnsson T, Hokfelt T et al (2000) Potent and nontoxic antisense oligonucleotides containing locked nucleic acids. Proc Natl Acad Sci USA 97:5633–5638
Xue-Wen H, Jie P, Xian-Yuan A, Hong-Xiang Z (2007) Inhibition of bacterial translation and growth by peptide nucleic acids targeted to domain II of 23S rRNA. J Pept Sci 13:220–226
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.
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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
- Peptide nucleic acid (PNA)
- 16S rRNA
- mRNA binding site
- Cell wall-permeablizing peptide