Comparison of thermodynamic stabilities between PNA (peptide nucleic acid)/DNA hybrid duplexes and DNA/DNA duplexes.

We have examined quantitatively stabilities of PNA/DNA hybrid duplexes with identical nearest-neighbor base pairs and compared stabilities between PNA/DNA and DNA/DNA. The average difference of stabilization energy of the short PNA/DNA was 0.9 kcal mol(-1), which suggests that the stability of the hybrids with identical nearest-neighbor base pairs can be predicted with the nearest-neighbor model as well as those of nucleic acid duplexes.     

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.     

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.     

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.     

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.     

In vitro membrane penetration of modified peptide nucleic acid (PNA).

Efficient cellular uptake is crucial for the success of any drug directed towards targets inside cells. Peptide nucleic acid (PNA), a DNA analog with a promising potential as a gene-directed drug, has been shown to display slow membrane penetration in cell cultures. We here used liposomes as an in vitro model of cell membranes to investigate the effect on penetration of a PNA molecule colvalently modified with a lipophilic group, an adamantyl moiety. The adamantyl attachment was found to increase the membrane-penetration rate of PNA three-fold, as compared to corresponding unmodified PNA. From the penetration behaviour of a number of small and large molecules we could conclude that passive diffusion is the mechanism for liposome-membrane passage. Flow linear dichroism (LD) of the modified PNA in presence of rod-shaped micelles, together with octanol-water distribution experiments, showed that the adamantyl-modified PNA is amphiphilic; the driving force behind the observed increased membrane-penetration rate appears to be an accumulation of the PNA in the lipid double layer.     

Transition metal derivatives of peptide nucleic acid (PNA) oligomers-synthesis, characterization, and DNA binding

This work describes the first automated solid-phase synthesis of metal derivatives of peptide nucleic acid (PNA) oligomers and their interaction with DNA and PNA. PNA constitutes a relatively young and very promising class of DNA analogues with excellent DNA and RNA binding properties. However, PNA lacks a suitable handle that would permit its sensitive detection on its own as well as when hybridized with complementary oligonucleotides. Metal complexes, on the other hand, offer high potential as markers for biomolecules. In this paper, we describe the synthesis of PNA heptamers (tggatcg-gly, where gly is a C-terminal glycine carboxylic acid amide) with two covalently attached metal complexes at the PNA N-terminus, namely a ferrocene carboxylic acid derivative and a tris(bipyridine)ruthenium(II) derivative. We show how all synthesis steps may be carried out with high yield on a DNA synthesizer, including attachment of the metal complexes. The conjugates were characterized by HPLC (>90% purity) and ESI-MS. Binding studies of the purified Ru-PNA heptamer to complementary DNA and PNA and comparison to the isosequential metal-free acetyl PNA heptamer proves that the attached metal complex has an influence on the stability (UV-T(m)) and structure (CD spectroscopy) of the conjugates, possibly by disruption of the nearby A:T base pair.     

Sequence selective double strand DNA cleavage by peptide nucleic acid (PNA) targeting using nuclease S1.

A novel method for sequence specific double strand DNA cleavage using PNA (peptide nucleic acid) targeting is described. Nuclease S1 digestion of double stranded DNA gives rise to double strand cleavage at an occupied PNA strand displacement binding site, and under optimized conditions complete cleavage can be obtained. The efficiency of this cleavage is more than 10 fold enhanced when a tandem PNA site is targeted, and additionally enhanced if this site is in trans rather than in cis orientation. Thus in effect, the PNA targeting makes the single strand specific nuclease S1 behave like a pseudo restriction endonuclease.     

Synthesis of a monocharged peptide nucleic acid (PNA) analog and its recognition as substrate by DNA polymerases.

The preparation of a novel phosphoramidite monomer based on thyminyl acetic acid coupled to the secondary nitrogen of 2-(2-amino-ethylamino)ethanol is described. This monomer can be used to attach a deoxynucleotide to the carboxy terminus of a PNA oligomer by solid-phase synthesis. The resulting PNA primer is recognized as a substrate by various DNA polymerases.     

Control of electron transfer in DNA by peptide nucleic acids (PNA).

The one-electron oxidation of PNA-DNA hybrid containing G-triplet sequence was examined. In DNA duplex G-triplet was selectively cleaved by oxidation, whereas in PNA-DNA hybrid cleavage efficiency was extremely lowered. These result suggested that cleavage efficiency of PNA-DNA hybrid was different from that of B-form DNA duplex.     

An experimental study of mechanism and specificity of peptide nucleic acid (PNA) binding to duplex DNA

We investigated the mechanism and kinetic specificity of binding of peptide nucleic acid clamps (bis-PNAs) to double-stranded DNA (dsDNA). Kinetic specificity is defined as a ratio of initial rates of PNA binding to matched and mismatched targets on dsDNA. Bis-PNAs consist of two homopyrimidine PNA oligomers connected by a flexible linker. While complexing with dsDNA, they are known to form P-loops, which consist of a [PNA]2-DNA triplex and the displaced DNA strand. We report here a very strong pH-dependence, within the neutral pH range, of binding rates and kinetic specificity for a bis-PNA consisting of only C and T bases. The specificity of binding reaches a very sharp and high maximum at pH 6.9. In contrast, if all the cytosine bases in one of the two PNA oligomers within the bis-PNA are replaced by pseudoisocytosine bases (J bases), which do not require protonation to form triplexes, a weak dependence on pH of the rates and specificity of the P-loop formation is observed. A theoretical analysis of the data suggests that for (C+T)-containing bis-PNA the first, intermediate step of PNA binding to dsDNA occurs via Hoogsteen pairing between the duplex target and one oligomer of bis-PNA. After that, the strand invasion occurs via Watson-Crick pairing between the second bis-PNA oligomer and the homopurine strand of the target DNA, thus resulting in the ultimate formation of the P-loop. The data for the (C/J+T)-containing bis-PNA show that its high affinity to dsDNA at neutral pH does not seriously compromise the kinetic specificity of binding. These findings support the earlier expectation that (C/J+T)-containing PNA constructions may be advantageous for use in vivo.     

Modulation of remote DNA oxidation by hybridization with peptide nucleic acids (PNA)

We have examined the efficiency of DNA photooxidation in DNA/PNA duplex and DNA/(PNA)(2) triplex for the first time. DNA/PNA duplex was cleaved at GG steps by external riboflavin with high efficiency like specific GG cleavage in DNA/DNA duplex. However, the 5'G selectivity of the GG oxidation in DNA/PNA duplex was much lower than that observed in DNA/DNA duplex. Remote DNA oxidation of oxidant-tethered DNA/PNA duplex was considerably suppressed. In contrast, the formation of DNA/(PNA)(2) triplex by hybridization with two PNA strands completely inhibited the remote GG oxidation, indicating that PNA acts as an inhibition for remote oxidative DNA damage.     

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.     

Strand displacement recognition of mixed adenine-cytosine sequences in double stranded DNA by thymine-guanine PNA (peptide nucleic acid).

Mixed pyrimidine-purine peptide nucleic acids (PNAs) composed of thymines and guanines are shown to form a PNA(2)-DNA triplex with Watson-Crick complementary adenine-cytosine oligonucleotides and to bind complementary adenine-cytosine targets in double stranded DNA by helix invasion. These results for the first time demonstrate binding of an unmodified PNA oligomer to a mixed pyrimidine-purine target in double stranded DNA and illustrate a novel binding mode of PNA.     

Non-nearest-neighbor dependence of stability for group III RNA single nucleotide bulge loops

Thirty-five RNA duplexes containing single nucleotide bulge loops were optically melted and the thermodynamic parameters for each duplex determined. The bulge loops were of the group III variety, where the bulged nucleotide is either a AG/U or CU/G, leading to ambiguity to the exact position and identity of the bulge. All possible group III bulge loops with Watson–Crick nearest-neighbors were examined. The data were used to develop a model to predict the free energy of an RNA duplex containing a group III single nucleotide bulge loop. The destabilization of the duplex by the group III bulge could be modeled so that the bulge nucleotide leads to the formation of the Watson–Crick base pair rather than the wobble base pair. The destabilization of an RNA duplex caused by the insertion of a group III bulge is primarily dependent upon non-nearest-neighbor interactions and was shown to be dependent upon the stability of second least stable stem of the duplex. In-line structure probing of group III bulge loops embedded in a hairpin indicated that the bulged nucleotide is the one positioned further from the hairpin loop irrespective of whether the resulting stem formed a Watson–Crick or wobble base pair. Fourteen RNA hairpins containing group III bulge loops, either 3′ or 5′ of the hairpin loop, were optically melted and the thermodynamic parameters determined. The model developed to predict the influence of group III bulge loops on the stability of duplex formation was extended to predict the influence of bulge loops on hairpin stability.     

Evaluation of transfection protocols for unmodified and modified peptide nucleic acid (PNA) oligomers

We have compared the efficacy of different transfection protocols reported for peptide nucleic acid (PNA) oligomers. A precise evaluation of uptake efficacy was achieved by using a positive readout assay based on the ability of a PNA oligomer to correct aberrant splicing of a recombinant luciferase gene. The study comprised transfection of PNA conjugated to acridine, adamantyl, decanoic acid, and porphyrine (acr-PNA, ada-PNA, deca-PNA, and por-RNA, respectively) and unmodified PNA partially hybridized to a DNA oligomer (PNA/DNA cotransfection). Furthermore, the effect of conjugation to a nuclear localization signal (NLS) was evaluated as part of the PNA/DNA cotransfection protocol. Transfection of the tested PNAs was systematically optimized. PNA/DNA cotransfection was found to produce the highest luciferase activity, but only after careful selection of the DNA oligonucleotide. Both a cationic lipid, Lipofectamine, and a nonliposomal cationic polymer, polyethylenimine (PEI, ExGen 500), were efficient transfection reagents for the PNA/DNA complex. However, Lipofectamine, in contrast to PEI, showed severe side effects, such as cytotoxicity. acr-PNA, ada-PNA, and por-PNA were transfectable with efficacies between 5 and 10 times lower than that seen with PNA/DNA cotransfection. Conjugation of PNA to NLS had no effect on PNA/DNA cotransfection efficacy. An important lesson from the study was the finding that because of uncontrollable biologic variations, even optimal transfection conditions differed to a certain extend from experiment to experiment in an unpredictable way.     

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.     

Increased stability and specificity through combined hybridization of peptide nucleic acid (PNA) and locked nucleic acid (LNA) to supercoiled plasmids for PNA-anchored "Bioplex" formation

Low cellular uptake and poor nuclear transfer hamper the use of non-viral vectors in gene therapy. Addition of functional entities to plasmids using the Bioplex technology has the potential to improve the efficiency of transfer considerably. We have investigated the possibility of stabilizing sequence-specific binding of peptide nucleic acid (PNA) anchored functional peptides to plasmid DNA by hybridizing PNA and locked nucleic acid (LNA) oligomers as "openers" to partially overlapping sites on the opposite DNA strand. The PNA "opener" stabilized the binding of "linear" PNA anchors to mixed-base supercoiled DNA in saline. For higher stability under physiological conditions, bisPNA anchors were used. To reduce nonspecific interactions when hybridizing highly cationic constructs and to accommodate the need for increased amounts of bisPNA when the molecules are uncharged, or negatively charged, we used both PNA and LNA oligomers as "openers" to increase binding kinetics. To our knowledge, this is the first time that LNA has been used together with PNA to facilitate strand invasion. This procedure allows hybridization at reduced PNA-to-plasmid ratios, allowing greater than 80% hybridization even at ratios as low as 2:1. Using significantly lower amounts of PNA-peptides combined with shorter incubation times reduces unspecific binding and facilitates purification.