Cell permeabilization and uptake of antisense peptide-peptide nucleic acid (PNA) into Escherichia coli

Peptide nucleic acid (PNA) is a DNA mimic with promising properties for the development of antisense agents. Antisense PNAs targeted to Escherichia coli genes can specifically inhibit gene expression, and attachment of PNA to the cell-permeabilizing peptide KFFKFFKFFK dramatically improves antisense potency. The improved potency observed earlier was suggested to be due to better cell uptake; however, the uptake kinetics of standard or modified PNAs into bacteria had not been investigated. Here we monitored outer and inner membrane permeabilization by using chemical probes that normally are excluded from cells but can gain access at points where membrane integrity is disturbed. Membrane permeabilization was much more rapid in the presence of peptide-PNA conjugates relative to the free components used alone or in combination. Indeed, peptide-PNAs permeabilized E. coli nearly as quickly as antimicrobial peptides. Furthermore, as expected for outer membrane-active compounds, added MgCl(2) reduced cell-permeabilization. Concurrent monitoring of outer and inner membrane permeabilization indicated that passage across the outer membrane is rate-limiting for uptake. The enhanced cell-permeation properties of peptide-PNAs can explain their potent antisense activity, and the results indicate an unanticipated synergy between the peptide and PNA components.