Fluorinated peptide nucleic acid

The fluorinated olefinic peptide nucleic acid analogue (F-OPA) monomer containing the base thymine was synthesised in 13 steps. PNAs containing this unit were prepared and their pairing properties assessed by means of UV-melting experiments.     

Facile synthesis of peptide nucleic acids and peptide nucleic acid‐peptide conjugates on an automated peptide synthesizer

Peptide nucleic acids (PNAs) are DNA mimics with a neutral peptide backbone instead of the negatively charged sugar phosphates. PNAs exhibit several attractive features such as high chemical and thermal stability, resistance to enzymatic degradation, and stable binding to their RNA or DNA targets in a sequence‐specific manner. Therefore, they are widely used in molecular diagnosis of antisense‐targeted therapeutic drugs or probes and in pharmaceutical applications. However, the main hindrance to the effective use of PNAs is their poor uptake by cells as well as the difficult and laborious chemical synthesis. In order to achieve an efficient delivery of PNAs into cells, there are already many published reports of peptides being used for transport across the cell membrane. In this protocol, we describe the automated as well as cost‐effective semi‐automated synthesis of PNAs and PNA‐peptide constructs on an automated peptide synthesizer. The facile synthesis of PNAs will be helpful in generating PNA libraries usable, e.g. for high‐throughput screening in biomolecular studies. Efficient synthetic schemes, the automated procedure, the reduced consumption of costly reagents, and the high purity of the products are attractive features of the reported procedure. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Nucleobase modified peptide nucleic acid

The Pd0/Cu1 catalyzed cross-coupling of terminal alkynes onto peptide nucleic acid monomers or submonomers bearing iodinated nucleobases has been utilized as a route to base-modified oligomers. Both 5-iodouracil and 5-iodocytosine derivatives undergo the cross-coupling to give the expected products in moderate to good yields. However, depending on the particular substrates and reaction conditions, the cross-coupling may be followed by a ring closing reaction to give the fluorescent furano- and pyrrolo-fused uracil and cytosine derivatives, respectively.     

Applications of peptide nucleic acids

Several exciting new developments in the applications of the DNA mimic peptide nucleic acid (PNA) have been published recently. A possible breakthrough may have come in efforts to develop PNA into gene therapeutic drugs. In eukaryotic systems, antisense activity of PNAs (as peptide conjugates) has been reported in nerve cells and even in rats upon injection into the brain, and antisense activity has also been demonstrated in Escherichia coli. PNA hybridization technology has developed rapidly within in situ hybridization, and exciting new methods based on MALDI-TOF detection have also been presented.     

An introduction to peptide nucleic acid

Peptide Nucleic Acid (PNA) is a powerful new biomolecular tool with a wide range of important applications. PNA mimics the behaviour of DNA and binds complementary nucleic acid strands. The unique chemical, physical and biological properties of PNA have been exploited to produce powerful biomolecular tools, antisense and antigene agents, molecular probes and biosensors.     

Solid-Phase synthesis of peptide nucleic acids

Peptide nucleic acids (PNA) were synthesized by a modified Merrifield method using several improvements. Activation by O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate in combination with in situ neutralization of the resin allowed efficient coupling of all four Boc-protected PNA monomers within 30 min. HPLC analysis of the crude product obtained from a fully automated synthesis of the model PNA oligomer H-CGGACTAAGTCCATTGC-Gly-NH₂, indicated an average yield per synthetic cycle of 97.1%. N¹-benzyloxycarbonyl-N6³-methylimidazole triflate substantially outperformed acetic anhydride as a capping reagent. The resin-bound PNAs were successfully cleaved by the ‘low–high’ trifluoromethanesulphonic acid procedure.     

Antisense properties of peptide nucleic acid

Peptide nucleic acid (PNA) is a nucleic acid mimic in which the deoxyribose phosphate backbone has been replaced by a pseudo-peptide polymer to which the nucleobases are linked. PNA-oligomers can be synthesized in relatively large amounts, are highly stable in biological environments, and bind complementary DNA and RNA targets with remarkably high affinity and specificity. Thus PNA possesses many of the properties desired for a good antisense agent. Until recently, limited uptake of PNA into cells has been the major obstacle for applying PNA as an antisense agent in cell cultures and in vivo. Here, the antisense properties of PNA in vitro and in vivo will be reviewed. In particular, we will focus on recent observations indicating that PNA equipped with or without various uptake moieties may function as an efficient and gene-specific inhibitor of translation in Escherichia coli and in certain mammalian cell types.     

Structural pre-organization of peptide nucleic acids

Introduction of constraint via chemical bridging in the aegPNA leads to the five or six membered cyclic structures that may contribute towards maintaining the balance between rigidity and flexibility of the PNA backbone. The significant promise of our approach to use the naturally occurring trans-4-hydroxy-L-proline to arrive at different chirally pure cyclic PNA analogs and their DNA binding properties will be presented.     

Olefinic peptide nucleic acid (OPA)

The olefinic peptide nucleic acid analogues (OPA) monomers containing the bases thymine and adenine were synthesised in 11 steps. Fully modified oligomers containing these units were prepared and their pairing properties assessed by means of UV-melting experiments.     

Solid-phase synthesis of pseudo-complementary peptide nucleic acids

Pseudo-complementary peptide nucleic acid (pcPNA) is a DNA analog in which modified DNA bases 2,6-diaminopurine (D) and 2-thiouracil (U(s)) 'decorate' a poly[N-(2-aminoethyl)glycine] backbone, together with guanine (G) and cytosine (C). One of the most significant characteristics of pcPNA is its ability to effect double-duplex invasion of predetermined DNA sites inducing various changes in the biological and the physicochemical properties of the DNA. This protocol describes solid-phase synthesis of pcPNA. The monomers for G and C are commercially available, but the monomers for D and U(s) need to be synthesized (or can be ordered to custom synthesis companies). Otherwise, the procedure is the same as that employed for Boc-strategy synthesis of conventional PNA. This protocol, if the synthesis of D and U(s) monomers is not factored in, takes approximately 7 d to complete.     

Pretargeting with amplification using polymeric peptide nucleic acid

One goal of this investigation was to develop a polymer conjugated with multiple copies of peptide nucleic acid (PNA) and with pharmacokinetic properties suitable for applications in vivo. The second goal was to establish whether the multiple copies of PNA on the polymer could be targeted by hybridization in vitro and in vivo with (99m)Tc-labeled complementary PNA (cPNA). If successful, this approach could then be considered in further investigations as an alternative to existing pretargeting approaches because of the potential for signal amplification in the target. A 80 KDa poly(methyl vinyl ether-alt-maleic acid) (PA) polymer was conjugated with multiple copies of PNA and with multiple copies of poly(ethylene glycol) (PEG) by reacting the NHS derivative of PA with the amine derivatives of PNA and PEG. Using (99m)Tc-MAG(3)-cPNA, targeting of PNA-PA-PEG was studied in vitro and in vivo in inflammation and tumor mouse models, in both cases relying upon nonspecific diffusion for localization. In addition, cPNA-avidin was considered as a clearing agent with biotinylated PNA-PA-PEG. About 80 PNAs could be conjugated to PA provided that about 200 PEGs were also conjugated to raise the aqueous solubility of the PNA-PA-PEG polymer lowered by the addition of the PNAs. About 70% of the PNAs on this polymer in vitro either in solution or attached to beads could be successfully targeted with (99m)Tc-cPNA. In both the inflammation and tumor mouse models, between 35 and 60% of these PNAs could be targeted in the lesions. The advantage of amplification was evident when less favorable results were obtained with PNA-PA-PEG conjugated with only six PNAs. We conclude that amplification can be achieved in vivo using polymers of PNA followed by radiolabeled complementary PNA and that the application of pretargeting using polymers of PNA for amplification can improve localization.     

Enhanced strand invasion by peptide nucleic acid-peptide conjugates

Efficient and selective recognition of DNA by proteins is due to sequence-specific interactions with a target site and nonselective electrostatic interactions that promote the target's rapid location. If synthetic molecules could mimic these functions, they would render a wide range of chromosome sequences accessible to rationally designed probes. Here we describe conjugates between bispeptide nucleic acids (bisPNAs) designed to specifically recognize duplex DNA and peptides that have been designed to promote rapid sequence recognition. Peptide design was based on the surface of staphylococcal nuclease, a cationic DNA binding protein with low sequence selectivity. We observe that attachment of the designed peptide increases rates of strand invasion by 100-fold relative to unmodified bisPNA. The peptide can contain D-amino acids, increasing the likelihood that it will be stable in cell extract and inside cells. Binding of the conjugate containing the D-amino acid peptide occurred over a broad range of experimental conditions and was sensitive to a single mismatch. Strand invasion was efficient at neutral to basic pH, a wide range of temperatures (0-65 degrees C), and in the presence of up to 7 mM Mg(2+) and 100 mM Na(+) or K(+). Our data suggest that attachment of peptides that mimic cationic protein surfaces to PNAs can afford conjugates that mimic the rapid and selective binding that characterizes native DNA binding proteins. Rapid strand invasion over a wide range of experimental conditions should further expand the utility of strand invasion by PNAs.     

Membrane-based hybridization capture of intracellular peptide nucleic acid

Peptide nucleic acid (PNA) is a chimeric oligonucleotide with nucleotide-derived bases and a peptide backbone. Compared with natural nucleotides, PNA has several advantages, including improved stability and high sequence discrimination during duplex formation. Despite its potential for therapeutic application, analysis technologies have not been generalized, mainly due to ambiguous physiochemical properties resembling those of nucleic acids as well as protein. Here we present a PNA detection method: sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by electrotransfer to a Western blotting membrane and then hybridization with a radiolabeled oligonucleotide probe. This method is useful for evaluating the quality of synthetic PNA and determining its intracellular localization.     

Cellular uptake of adamantyl conjugated peptide nucleic acids

Peptide nucleic acids (PNA) (15-mers) conjugated to adamantyl acetic acid and labeled with fluorescein have been prepared, and their (liposome mediated) uptake in human cells in culture (HeLa, IMR-90 and MDA-MB-453) has been studied by confocal fluorescence microscopy. It is found that adamantyl-PNAs show greatly improved (endosomal) cellular uptake, but that this uptake is dependent on the cell line. Cellular uptake of such lipophilic PNAs is further mediated by cationic liposomes, and in some cases, the intracellular localization is diffuse cytoplasmic or nuclear, again cell-type dependent. The results show that this simple PNA modification can indeed greatly improve cellular uptake, but the effect appears strongly cell-type as well as PNA-sequence dependent.     

Receptor-specific targeting with complementary peptide nucleic acids conjugated to peptide analogs and radionuclides

Summary Genomic sequencing makes it possible to identify all the genes of an organism, now including Homo sapiens. Yet measurement of the expression of each gene of interest still presents a daunting prospect. Northern blots, RNase protection assays, as well as microarrays and related technologies permit measurement of gene expression in total RNA extracted from cultured cells or tissue samples. It would be most valuable, however, to quantitate gene expression noninvasively in living cells and tissues. Unfortunately, no reliable method has been available to measure levels of specific mRNAs in vivo. Peptide nucleic acids (PNAs) display superior ruggedness and hybridization properties as a diagnostic tool for gene expression, and could be used for this purpose. On the down side, they are negligibly internalized by normal or malignant cells in the absence of conjugated ligands. Nevertheless, we have observed that Tc-99m-peptides can delineate tumors, and PNA-peptides designed to bind to IGF-1 receptors on malignant cells are taken up specifically and concentrated in nuclei. We have postulated that antisense Tc-99m-PNA-peptides will be taken up by human cancer cells, will hybridize to complementary mRNA targets, and will permit scintigraphic imaging of oncogene mRNAs in human cancer xenografts in a mouse model. The oncogenes cyclin D1, ERBB2, c-MYC, K-RAS, and tumor suppressor p53 are being probed initially. These experiments provide a proof-of-principle for noninvasive detection of oncogene expression in living cells and tissues. This scintigraphic imaging technique should be applicable to any particular gene of interest in a cell or tissue type with characteristic receptors.     

Receptor-specific targeting with complementary peptide nucleic acids conjugated to peptide analogs and radionuclides

Genomic sequencing makes it possible to identify all the genes of an organism, now including Homo sapiens. Yet measurement of the expression of each gene of interest still presents a dauntingprospect. Northern blots, RNase protection assays, as well as microarrays and related technologies permit measurement of gene expression in total RNA extracted from cultured cells or tissue samples. It would be most valuable, however, to quantitate gene expression noninvasively in living cells and tissues. Unfortunately,no reliable method has been available to measure levels of specificmRNAs in vivo. Peptide nucleic acids (PNAs) display superior ruggedness and hybridization properties as a diagnostic tool for gene expression, and could be used for this purpose. On the down side, they are negligibly internalized by normal or malignant cells in the absence of conjugated ligands. Nevertheless,we have observed that Tc-99m-peptides can delineate tumors, and PNA-peptides designed to bind to IGF-1 receptors on malignant cellsare taken up specifically and concentrated in nuclei. We have postulated that antisense Tc-99m-PNA-peptides will be taken up by human cancer cells, will hybridize to complementary mRNA targets, and will permit scintigraphic imaging of oncogene mRNAsin human cancer xenografts in a mouse model. The oncogenes cyclinD1, ERBB2, c-MYC, K-RAS, and tumor suppressor p53 are being probed initially. These experimentsprovide a proof-of-principle for noninvasive detection of oncogeneexpression in living cells and tissues. This scintigraphic imaging technique should be applicable to any particular gene of interest in a cell or tissue type with characteristic receptors.     

Receptor-specific targeting with complementary peptide nucleic acids conjugated to peptide analogs and radionuclides

Summary Genomic sequencing makes it possible to identify all the genes of an organism, now includingHomo sapiens. Yet measurement of the expression of each gene of interest still presents a daunting prospect. Northern blots, RNase protection assays, as well as microarrays and related technologies permit measurement of gene expression in total RNA extracted from cultured cells or tissue samples. It would be most valuable, however, to quantitate gene expression noninvasively in living cells and tissues. Unfortunately, no reliable method has been available to measure levels of specific mRNAsin vivo. Peptide nucleic acids (PNAs) display superior ruggedness and hybridization properties as a diagnostic tool for gene expression, and could be used for this purpose. On the down side, they are negligibly internalized by normal or malignant cells in the absence of conjugated ligands. Nevertheless, we have observed that Tc-99m-peptides can delineate tumors, and PNA-peptides designed to bind to IGF-1 receptors on malignant cells are taken up specifically and concentrated in nuclei. We have postulated that antisense Tc-99m-PNA-peptides will be taken up by human cancer cells, will hybridize to complementary mRNA targets, and will permit scintigraphic imaging of oncogene mRNAs in human cancer xenografts in a mouse model. The oncogenes cyclin D1,ERBB2, c-MYC, K-RAS, and tumor suppressor p53 are being probed initially. These experiments provide a proof-of-principle for noninvasive detection of oncogene expression in living cells and tissues. This scintigraphic imaging technique should be applicable to any particular gene of interest in a cell or tissue type with characteristic receptors.     

Imaging gene expression using oligonucleotides and peptide nucleic acids

The development of methods for non-invasive, real-time imaging of gene expression would provide powerful tools for biomedical research and medical diagnostics. A broadly applicable strategy for achieving this goal is the use of complementary oligonucleotide probes for recognition of mRNA. The major challenge for molecular imaging is the development of specific and efficient transducers for signaling probe-target interaction. This review summarizes the strengths and limitations of reported molecular approaches for imaging of mRNA expression and discusses the challenges to development of in vivo methods.     

Conformationally restricted chiral peptide nucleic acids derived from azetidines

The initial experiments towards the chemical synthesis of conformationally rigid peptide nucleic acid analogues with azetidine moieties have been described.