Peptide nucleic acid (PNA) antisense effects in Escherichia coli

Antisense peptide nucleic acid (PNA) can be used to control cell growth, gene expression and growth phenotypes in the bacteria Escherichia coli. PNAs targeted to the RNA components of the ribosome can inhibit translation and cell growth, and PNAs targeted to mRNA can limit gene expression with gene and sequence specificity. In an E. coli cell extract, efficient inhibition is observed when using PNA concentrations in the nanomolar range, whereas micromolar concentrations are required for inhibition in growing cells. A mutant strain of E. coli that is more permeable to antibiotics also is more susceptible to antisense PNAs than the wild type. This chapter details methods for testing the antisense activities of PNA in E. coli. As an example of the specific antisense inhibition possible, we show the effects of an anti-beta-galactosidase PNA in comparison to control PNAs. With improvements in cell uptake, antisense PNAs may find applications as antimicrobial agents and as tools for microbial functional genomics.     

An RNA chaperone activity of non-specific RNA binding proteins in hammerhead ribozyme catalysis.

We have previously shown that a protein derived from the p7 nucleocapsid (NC) protein of HIV type-1 increases kcat/Km and kcat for cleavage of a cognate substrate by a hammerhead ribozyme. Here we show directly that the increase in kcat/Km arises from catalysis of the annealing of the RNA substrate to the ribozyme and the increase in kcat arises from catalysis of dissociation of the RNA products from the ribozyme. A peptide polymer derived from the consensus sequence of the C-terminal domain of the hnRNP A1 protein (A1 CTD) provides similar enhancements. Although these effects apparently arise from non-specific interactions, not all non-specific binding interactions led to these enhancements. NC and A1 CTD exert their effects by accelerating attainment of the thermodynamically most stable species throughout the ribozyme catalytic cycle. In addition, NC protein is shown to resolve a misfolded ribozyme-RNA complex that is otherwise long lived. These in vitro results suggest that non-specific RNA binding proteins such as NC and hnRNP proteins may have a biological role as RNA chaperones that prevent misfolding of RNAs and resolve RNAs that have misfolded, thereby ensuring that RNA is accessible for its biological functions.     

Peptide nucleic acid (PNA) amphiphiles: synthesis, self-assembly, and duplex stability

Peptide amphiphiles comprising a class of conjugates of peptide nucleic acid (PNA), natural amino acids, and n-alkanes were synthesized and studied. These PNA amphiphiles (PNAA) self-assemble at concentrations between 10 and 50 muM and exhibit water solubilities above 500 muM. The highly specific, stable DNA binding properties of PNAs are preserved by these modifications, with no significant differences between the thermodynamics of DNA binding of the PNA peptide and the PNA amphiphile. Proper solubilization of the PNAA required the attachment of (Lys)(2) and (Glu)(4) peptides to PNAs, which affected the PNAA-DNA duplex stability by electrostatic interactions between these charged amino acids and the negatively charged DNA backbone. These electrostatic effects did not affect the specificity of DNA binding, however. Electrostatic effects are screened with added salt, in a manner consistent with previous studies of PNA-DNA duplex stability and predictions from a charged-cylinder model for the duplex.     

Peptide nucleic acid (PNA) and its applications in chemical biology,diagnostics, and therapeutics

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Effects of variations in length of hammerhead ribozyme antisense arms upon the cleavage of longer RNA substrates.

The efficacy of intracellular binding of hammerhead ribozyme to its cleavage site in target RNA is a major requirement for its use as a therapeutic agent. Such efficacy can be influenced by several factors, such as the length of the ribozyme antisense arms and mRNA secondary structures. Analysis of various IL-2 hammerhead ribozymes having different antisense arms but directed to the same site predicts that the hammerhead ribozyme target site is present within a double-stranded region that is flanked by single-stranded loops. Extension of the low cleaving hammerhead ribozyme antisense arms by nucleotides that base pair with the single-stranded regions facilitated the hammerhead ribozyme binding to longer RNA substrates (e.g. mRNA). In addition, a correlation between the in vitro and intracellular results was also found. Thus, the present study would facilitate the design of hammerhead ribozymes directed against higher order structured sites. Further, it emphasises the importance of detailed structural investigations of hammerhead ribozyme full-length target RNAs.     

Peptide nucleic acid (PNA)/DNA hybrid duplexes: intercalation by an internally linked anthraquinone.

Peptide nucleic acids (PNA) mimic DNA and RNA by forming complementary duplex structures following Watson-Crick base pairing. A set of reporter compounds that bind to DNA by intercalation are known, but these compounds do not intercalate in PNA/DNA hybrid duplexes. Analysis of the hybrid PNA duplexes requires development of reporter compounds that probe their chemical and physical properties. We prepared a series of anthraquinone (AQ) derivatives that are linked to internal positions of a PNA oligomer. These are the first non-nucleobase functional groups that have been incorporated into a PNA. The resulting PNA(AQ) conjugates form stable hybrids with complementary DNA oligomers. We find that when the AQ groups are covalently bound to PNA that they stabilize the hybrid duplex and are, at least partially, intercalated.     

Divalent hammerhead ribozyme targeting template region of human telomerase RNA has potent cleavage activity,but less inhibitory activity on telomerase

The template region of human telomerase RNA is a crucial area for regulating telomerase activity and would be a good target for ribozymes. In fact, potent telomerase inhibitory activity of the ribozyme targeting the GUC sequence of the 5(') end of this region (36-ribosome) has been well demonstrated. To search for a more potent ribozyme, we designed a divalent ribozyme to cleave both the phosphodiester bonds following the GUC and the 23 nucleotides downstream of GUA. An in vitro cleavage study showed that this divalent ribozyme cleaved telomerase RNA more efficiently than the 36-ribozyme or the 59-ribozyme to target the GUA. When this ribozyme was introduced into the carcinoma cells, its inhibitory effect on telomerase activity was less than that of the 36-ribozyme. The 59-ribozyme showed minimum activity on telomerase. This implies that, although the divalent ribozyme possesses a potent cleavage activity on hTR in vitro, the 36-ribozyme is most potent to suppress telomerase activity.     

Synthesis of new chiral peptide nucleic acid (PNA) monomers

We have synthesised a series of new chiral type I peptide nucleic acid monomers in total yields of 36-53%, derived from Val, Ile, Ser(Bzl), Pro, and Trp, employing convenient procedure.     

Synthesis and characterization of new chiral peptide nucleic acid (PNA) monomers.

PNAs are DNA analogues in which the nucleic acid's backbone is replaced by a chiral or achiral pseudopeptide backbone and nucleobases are attached to the backbone by methylene carbonyl linkers. The easy to modify PNA structure gives the possibility to obtain monomers, and subsequently oligomers, with improved properties. We have synthesised several new PNA monomers, starting from a series of 2'-substituted methyl N-(2-Boc-aminoethyl)glycinates. The pseudodipeptides were obtained using modified Kosynkina's method, based on the reductive amination of N-Boc-protected alpha-amino aldehydes [glycinal, isoleucinal, valinal, tryptophanal, serinal(Bzl), prolinal] with methyl glycinate. The compounds were then acylated with nucleic acid base derivatives by simplified procedure, and the purification was limited to the last step of the synthesis. The applied procedure is useful in synthesis of various chiral PNA monomers.     

Hybridisation based DNA screening on peptide nucleic acid (PNA) oligomer arrays.

Arrays of up to some 1000 PNA oligomers of individual sequence were synthesised on polymer membranes using a robotic device originally designed for peptide synthesis. At approximately 96%, the stepwise synthesis efficiency was comparable to standard PNA synthesis procedures. Optionally, the individual, fully deprotected PNA oligomers could be removed from the support for further use, because an enzymatically cleavable but otherwise stable linker was used. Since PNA arrays could form powerful tools for hybridisation based DNA screening assays due to some favourable features of the PNA molecules, the hybridisation behaviour of DNA probes to PNA arrays was investigated for a precise understanding of PNA-DNA interactions on solid support. Hybridisation followed the Watson-Crick base pairing rules with higher duplex stabilities than on corresponding DNA oligonucleotide sensors. Both the affinity and specificity of DNA hybridisation to the PNA oligomers depended on the hybridisation conditions more than expected. Successful discrimination between hybridisation to full complementary PNA sequences and truncated or mismatched versions was possible at salt concentrations down to 10 mM Na+and below, although an increasing tendency to unspecific DNA binding and few strong mismatch hybridisation events were observed.     

Cationic nucleopeptides as novel non-covalent carriers for the delivery of peptide nucleic acid (PNA) and RNA oligomers

Cationic nucleopeptides belong to a family of synthetic oligomers composed by amino acids and nucleobases. Their capability to recognize nucleic acid targets and to cross cellular membranes provided the basis for considering them as novel non-covalent delivery agents for nucleic acid pharmaceuticals. Herein, starting from a 12-mer nucleopeptide model, the number of cationic residues was modulated in order to obtain new nucleopeptides endowed with high solubility in acqueous medium, acceptable bio-stability, low cytotoxicity and good capability to bind nucleic acid. Two candidates were selected to further investigate their potential as nucleic acid carriers, showing higher efficiency to deliver PNA in comparison with RNA. Noteworthy, this study encourages the development of nucleopeptides as new carriers to extend the known strategies for those nucleic acid analogues, especially PNA, that still remain difficult to drive into the cells.     

Subnanomolar antisense activity of phosphonate-peptide nucleic acid (PNA) conjugates delivered by cationic lipids to HeLa cells

In the search of facile and efficient methods for cellular delivery of peptide nucleic acids (PNA), we have synthesized PNAs conjugated to oligophosphonates via phosphonate glutamine and bis-phosphonate lysine amino acid derivatives thereby introducing up to twelve phosphonate moieties into a PNA oligomer. This modification of the PNA does not interfere with the nucleic acid target binding affinity based on thermal stability of the PNA/RNA duplexes. When delivered to cultured HeLa pLuc705 cells by Lipofectamine, the PNAs showed dose-dependent nuclear antisense activity in the nanomolar range as inferred from induced luciferase activity as a consequence of pre-mRNA splicing correction by the antisense-PNA. Antisense activity depended on the number of phosphonate moieties and the most potent hexa-bis-phosphonate-PNA showed at least 20-fold higher activity than that of an optimized PNA/DNA hetero-duplex. These results indicate that conjugation of phosphonate moieties to the PNA can dramatically improve cellular delivery mediated by cationic lipids without affecting on the binding affinity and sequence discrimination ability, exhibiting EC(50) values down to one nanomolar. Thus the intracellular efficacy of PNA oligomers rival that of siRNA and the results therefore emphasize that provided sufficient in vivo bioavailability of PNA can be achieved these molecules may be developed into potent gene therapeutic drugs.     

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.     

Application of Mitsunobu reaction to solid-phase peptide nucleic acid (PNA) monomer synthesis

PNA type I monomer backbone with a reduced peptide bond was synthesized on a Merrifield resin in Mitsunobu reaction of Boc-aminoethanol with resin-bound o-nitrobenzenesulfonylglycine. The pseudodipeptide secondary amine group was deprotected by thiolysis and acylated with thymin-1-ylacetic acid. The monomer was released as a methyl ester. The procedure seems to be of general applicability and allows various modifications of PNA structure by using diverse alcohols and amino acid esters.     

Inhibiting gene expression with peptide nucleic acid (PNA)--peptide conjugates that target chromosomal DNA

Peptide nucleic acids (PNAs) are nonionic DNA/RNA mimics that can recognize complementary sequences by Watson-Crick base pairing. The neutral PNA backbone facilitates the recognition of duplex DNA by strand invasion, suggesting that antigene PNAs (agPNAs) can be important tools for exploring the structure and function of chromosomal DNA inside cells. However, before agPNAs can enter wide use, it will be necessary to develop straightforward strategies for introducing them into cells. Here, we demonstrate that agPNA-peptide conjugates can target promoter DNA and block progesterone receptor (PR) gene expression inside cells. Thirty-six agPNA-peptide conjugates were synthesized and tested. We observed inhibition of gene expression using cationic peptides containing either arginine or lysine residues, with eight or more cationic amino acids being preferred. Both 13 and 19 base agPNA-peptide conjugates were inhibitory. Inhibition was observed in human cancer cell lines expressing either high or low levels of progesterone receptor. Modification of agPNA-peptide conjugates with hydrophobic amino acids or small molecule hydrophobic moieties yielded improved potency. Inhibition by agPNAs did not require cationic lipid or any other additive, but adding agents to cell growth media that promote endosomal release caused modest increases in agPNA potency. These data demonstrate that chromosomal DNA is accessible to agPNA-peptide conjugates and that chemical modifications can improve potency.     

Peptide nucleic acid (PNA) facilitates multistranded hybrid formation between linear double-stranded DNA targets and RecA protein-coated complementary single-stranded DNA probes

RecA protein-coated single-stranded DNA probes, known as RecA nucleoprotein filaments, bind specifically to homologous DNA sequences within double-stranded DNA targets, forming multistranded probe-target DNA hybrids. This DNA hybridization reaction can be used for sequence-specific gene capture, gene modification, and gene regulation. Thus, factors that enhance the efficiency of the hybridization reaction are of significant practical importance. We show here that the hybridization of a peptide nucleic acid (PNA) within or adjacent to the probe-target homology region significantly enhances the yield of hybrid DNA formed in the reaction between linear double-stranded DNA targets and RecA protein-coated complementary single-stranded (css)DNA probes. The possible mechanisms and the advantages of using RecA nucleoprotein filaments in combination with PNA for genomic DNA cloning and mutagenesis are presented.