Cell Permeabilization and Uptake of Antisense Peptide-Peptide Nucleic Acid (PNA) into Escherichia coli

Abstract

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.Antisense agents are designed to inhibit gene expression through sequence-specific nucleic acid binding, and this normally requires the formation of 10 or more base pairs. Oligonucleotides of such lengths are typically too large for efficient passive cellular uptake by diffusion across lipid bilayers (1), and cellular outer membranes can pose additional barriers. Recently we introduced antisense peptide nucleic acids (PNAs) 1 as antisense agents for bacterial applications. The early experiments indicated very encouraging sequence specificity against reporter genes; however, uptake appeared to be limited by bacterial cell barriers (2, 3). This limitation is not surprising as Escherichia coli and other Gram-negative bacteria have outer and inner bilayer membranes. In addition, the outer membrane contains a lipopolysaccharide (LPS) layer that stringently restricts the entry of foreign molecules (4). Nevertheless, there are opportunities to overcome these cellular barriers and even exploit membrane charge characteristics to enhance delivery.PNA is a DNA mimic with (standard) nucleobases attached to a pseudo-peptide backbone (see Fig. 1) (5). The PNA molecule provides improved hybridization affinity and specificity, chemical and enzymatic stability, and low toxicity. Antisense PNAs have outperformed DNA analogues in diverse systems (2, 6). The main limitation to wider applications in vivo appears to be poor cellular uptake; however, there are several recent examples showing improved delivery and antisense effects for PNAs attached to carrier peptides. For delivery into eukaryotic cells, PNAs have been attached to a peptide derived from the third helix domain of the Drosophila an...

Inhibition of Gene Expression inEscherichia coliby Antisense Phosphorodiamidate Morpholino Oligomers

“…It is inferred from the results that covalently attached KFC peptide enables the KFC-PMO conjugate to traverse the outer membrane. Indeed, the KFC peptide conjugated to peptide nucleic acids has been shown to increase antisense efficacy by increasing the kinetics of outer membrane permeability (4). Regardless of the mechanism, the conjugated KFC-PMO was necessary for inhibition of the target in the cytoplasm of E. coli with an intact outer membrane.…”

“…The resin was washed with 300 l of cleavage solution three times immediately followed by addition of 4 ml of concentrated ammonia hydroxide and incubation for 16 h at 45°C to remove base protection groups. PMO was precipitated out of the NMP-NH 4 OH solution with 8 volumes of acetone and precipitated by centrifugation (14,000 ϫ g, 5 min). The pellet was washed with 15 ml of CH 3 CN.…”

Antisense Antibacterials: From Proof-Of-Concept to Therapeutic Perspectives

“…In order to improve cellular uptake of PNA into bacterial cells, Good L and Nielsen PE, who first established CPP conjugating to the end of PNA in chemical synthesis, have realized efficient delivery of PNA through bacterial out membrane by observing its potent bacteriocidal antisense effects at micromolar ratio (Eriksson & et al, 2002). Further evidence demonstrates that introducing spacers or linkers between PNA and CPP in the direct covalent conjugate may increase its antisense efficacy and antibacterial potency.…”

Inhibition of aac(6′)-Ib -mediated amikacin resistance by nuclease-resistant external guide sequences in bacteria

“…Several groups have experimented with a variety of strategies to induce uptake of oligonucleotides and analogs into the cytosol. These strategies included liposome encapsulation, modification of the oligonucleotide by introduction of structures like hairpins, or attachment of cell-permeabilizing peptides (18,(24)(25)(26)(27)(28)(29)(30). We are presently carrying out assays using these approaches to induce penetration of LNA/DNA cooligomers into E. coli and other bacteria.…”