Abstract
Short modified oligonucleotides targeted at bacterial DNA or RNA could serve as antibacterial agents provided that they are efficiently taken up by bacterial cells. However, the uptake of such oligonucleotides is hindered by the bacterial cell wall. To overcome this problem, oligomers have been attached to cell-penetrating peptides, but the efficiency of delivery remains poor. Thus, we have investigated the ability of vitamin B12 to transport peptide nucleic acid (PNA) oligomers into cells of Escherichia coli and Salmonella Typhimurium. Vitamin B12 was covalently linked to a PNA oligomer targeted at the mRNA of a reporter gene expressing Red Fluorescent Protein. Cu-catalyzed 1,3-dipolar cycloaddition was employed for the synthesis of PNA-vitamin B12 conjugates; namely the vitamin B12 azide was reacted with PNA possessing the terminal alkyne group. Different types of linkers and spacers between vitamin B12 and PNA were tested, including a disulfide bond. We found that vitamin B12 transports antisense PNA into E. coli cells more efficiently than the most widely used cell-penetrating peptide (KFF)3K. We also determined that the structure of the linker impacts the antisense effect. The results of this study provide the foundation for developing vitamin B12 as a carrier of PNA oligonucleotides into bacterial cells.
Highlights
We have investigated the ability of the non-peptidic carrier vitamin B12 to transport peptide nucleic acid (PNA) oligomers into E. coli and S. typhimurium cells
Typhimurium, we constructed pBBR(rfp), an Red Fluorescent Protein (RFP) reporter vector optimized for expression in Enterobacteriaceae (Fig. 2a)
The anti-mrfp[1] PNA was designed to target the region of the mRNA overlapping the translation start codon, which was shown to be sensitive to antisense inhibition[37], plus part of the ribosome binding site (RBS) B0034 (Fig. 2b)
Summary
System to monitor inhibition of mrfp[1] mRNA translation. To provide a convenient system for comparative studies of the effect of antisense PNA in E. coli and S. Similar results were obtained for both E. coli and S Typhimurium, in the latter bacterium, the inhibition of RFP production by all compounds was less effective (~75% at most). As a control for these experiments, we used the scrambled PNA (Fig. 2c) attached to vitamin B12 via –(CH2)[12] (Fig. 5d), i.e. the linker that inhibited RFP production most effectively when conjugated to the complementary PNA. In E. coli, at concentrations between 4 and 16 μM, the decrease in fluorescence produced by the most effective vitamin B12-(CH2)12-PNA constructs was greater than for (KFF)3K-PNA (Figure S2). The (KFF)3K-PNA and vitamin B12-PNA conjugates showed comparable dose-dependent inhibition of RFP fluorescence in this bacterium (Figs 4 and 6). In the future we plan to investigate whether vitamin B12 can be used to transport PNA into the cells of Gram-positive bacteria
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