Synthesis of gamma-substituted peptide nucleic acids: a new place to attach fluorophores without affecting DNA binding

Molecular beacon strategies using PNA are currently restricted to fluorophore attachment to the ends of the PNA. We report the synthesis of PNA oligomers wherein fluorophores can be attached to the PNA backbone from novel gamma-lysine PNA monomers. Oligomers incorporating the modified PNA showed comparable thermal stability to the corresponding aegPNA oligomer with DNA. When the modified PNA oligomer was annealed with complementary DNA, the fluorescence intensity increased 4-fold over the unbound PNA. [structure: see text]     

Synthesis of a C-linked glycosylated thymine-based PNA monomer and its incorporation into a PNA oligomer

Backbone modification of peptide nucleic acids (PNAs) by glycosylation has been shown to enhance selective biodistribution and cellular targeting of PNA oligomers based on sugar and cell surface lectin interactions. Here we report the synthesis of a new backbone-glycosylated thymine-based PNA monomer (T(gal)). The sugar residue was attached to the backbone of PNA via a stable carbon-carbon linkage between the sugar and the PNA monomers. Also, incorporation of the modified monomer into a PNA decamer (H-Ala(gal)-G-G-G-T(gal)-C-A-G-C-T(gal)-T-Lys-NH2) was successfully performed. Melting temperature (UV-Tm) of the modified PNA against the complementary DNA was only slightly lower than unmodified PNA.     

Cyclic PNA-based compound directed against HIV-1 TAR RNA: modelling, liquid-phase synthesis and TAR binding

A cyclic molecule including a hexameric PNA sequence has been designed and synthesized in order to target the TAR RNA loop of HIV-1 through the formation of a "kissing complex". For comparison, its linear analogue has also been investigated. The synthesis of the cyclic and linear PNA has been accomplished following a liquid-phase strategy using mixed PNA and fully N-protected (aminoethylglycinamide) fragments. The interactions of this cyclic PNA and its linear analogue with TAR RNA have been studied and the results indicate clearly that no interaction occurs between the cyclic antisense PNA and TAR RNA, whereas a tenuous interaction has been detected with its linear PNA analogue.     

Coordination-driven inversion of handedness in ligand-modified PNA

Peptide nucleic acid (PNA) is a synthetic analogue of DNA, which has the same nucleobases as DNA but typically has a backbone based on aminoethyl glycine (Aeg). PNA forms duplexes by Watson Crick hybridization. The Aeg-based PNA duplexes adopt a chiral helical structure but do not have a preferred handedness because they do not contain a chiral center. An L-lysine situated at the C-end of one or both strands of a PNA duplex causes the duplex to preferably adopt a left-handed structure. We have introduced into the PNA duplexes both a C-terminal L-lysine and one or two PNA monomers that have a γ-(S)-methyl-aminoethyl glycine backbone, which is known to induce a preference for a right-handed structure. Indeed, we found that in these duplexes the γ-methyl monomer exerts the dominant chiral induction effect causing the duplexes to adopt a right-handed structure. The chiral PNA monomer had a 2,2':6',2'-terpyridine (Tpy) ligand instead of a nucleobase and PNA duplexes that contained one or two Tpys formed [Cu(Tpy)(2)](2+) complexes in the presence of Cu(2+). The CD spectroscopy studies showed that these metal-coordinated duplexes were right-handed due to the chiral induction effect exerted by the S-Tpy PNA monomer(s) except for the cases when the [Cu(Tpy)(2)](2+) complex was formed with Tpy ligands from two different PNA duplexes. In the latter case, the metal complex bridged the two PNA duplexes and the duplexes were left-handed. The results of this study show that the preferred handedness of a ligand-modified PNA can be switched as a consequence of metal coordination to the ligand. This finding could be used as a tool in the design of functional nucleic-acid based nanostructures.     

含羟基和咪唑基团新手性肽核酸的设计与合成

以含丝氨酸和组氨酸残基的手性肽核酸单体替换经典肽核酸单体, 采用固相合成方法设计合成了五个新序列含羟基和咪唑基团的10聚体手性肽核酸, 经ESI-MS或MALDI-TOF-MS证实目标物结构正确.     

Novel binding and efficient cellular uptake of guanidine-based peptide nucleic acids (GPNA)

Incorporation of a guanidine functional group into the PNA backbone facilitates cellular uptake of PNA into mammalian cells with efficiency comparable to that of the TAT transduction domain. The modified PNA recognizes and binds to the complementary DNA strand in accordance with Watson-Crick recognition rules. However, unlike polypyrimidine PNA which binds to DNA in 2:1 stoichiometry, the modified PNA binds to complementary DNA in a 1:1 ratio to form a highly stable duplex.     

PNA/dsDNA complexes: site specific binding and dsDNA biosensor applications

The ability of peptide nucleic acids (PNA) to form specific higher-order (i.e., three- and four-stranded) complexes with DNA makes it an ideal structural probe for designing strand-specific dsDNA biosensors. Higher-order complexes are formed between a dye-labeled charge-neutral PNA probe and complementary dsDNA. Addition of a light-harvesting cationic conjugated polymer (CCP) yields supramolecular structures held together by electrostatic forces that incorporate the CCP and the dye-labeled PNA/DNA complexes. Optimization of optical properties allows for excitation of the CCP and subsequent fluorescence resonance energy transfer (FRET) to the PNA-bound dye. In the case of noncomplementary dsDNA, complexation between the probe and target does not occur, and dye emission is weak. The binding between PNA and noncomplementary and complementary dsDNA was examined by several methods. Gel electrophoresis confirms specificity of binding and the formation of higher-order complexes. Nano-electrospray mass spectrometry gives insight into the stoichiometric composition, including PNA/DNA, PNA(2)/DNA, PNA/DNA(2), and PNA(2)/DNA(2) complexes. Finally, structural characteristics and binding-site specificity were examined using ion mobility mass spectrometry in conjunction with molecular dynamics. These results give possible conformations for each of the higher-order complexes formed and show exclusive binding of PNA to the complementary stretch of DNA for all PNA/DNA complexes. Overall, the capability and specificity of binding indicates that the CCP/PNA assay is a feasible detection method for dsDNA and eliminates the need for thermal denaturing steps typically required for DNA hybridization probe assays.     

Synthesis of pyrrolidinone PNA: a novel conformationally restricted PNA analogue

To preorganize PNA for duplex formation, a new cyclic pyrrolidinone PNA analogue has been designed. In this analogue the aminoethylglycine backbone and the methylenecarbonyl linker are connected, introducing two chiral centers compared to PNA. The four stereoisomers of the adenine analogue were synthesized, and the hybridization properties of PNA decamers containing one analogue were measured against complementary DNA, RNA, and PNA strands. The (3S,5R) isomer was shown to have the highest affinity toward RNA, and to recognize RNA and PNA better than DNA. The (3S,5R) isomer was used to prepare a fully modified decamer which bound to rU10 with only a small decrease in Tm (delta Tm/mod = 1 degree C) relative to aminoethylglycine PNA.     

Synthesis and characterization of novel poly(1-naphthylamine)/zinc oxide nanocomposites: Application in catalytic degradation of methylene blue dye

The organic–inorganic poly(1-naphthylamine)/ZnO (PNA/ZnO) nanocomposites were synthesized by in situ chemical oxidative polymerization of 1-naphthylamine monomer and ZnO nanomaterials for the photocatalytic degradation of methylene blue (MB) dye under visible light. The surface properties of PNA molecules were altered by adding the ZnO nanomaterials. The crystalline and the optical properties of PNA/ZnO nanocomposites were improved with the increased contents of ZnO nanomaterials in PNA molecules due to the effective interaction of PNA to the surface of ZnO nanomaterials. The prepared PNA/ZnO nanocomposites presented moderate photocatalytic MB dye degradation of ~22% under visible light. The occurrence of dye degradation under visible light might attribute to high-charge separation of ē-h⁺ pairs at the interfaces of PNA and ZnO nanomaterials in its excited state under light irradiation.     

Peptide nucleic acid and amino acid modified peptide nucleic acid analysis by capillary zone electrophoresis

A rapid, high resolution, and low sample consumption CZE method is developed for peptide nucleic acid (PNA) analysis for the first time. 30% v/v acetonitrile in PNA sample and 20% v/v acetonitrile in 50 mM borax‐boric acid (pH 8.7) as BGE were employed after optimization. The calibration curves were linear for PNA concentration ranging from 1 to 50 μmol/L. LOD and LOQ of PNA were 0.2 and 1.0 μmol/L, respectively. Since the commercially available reagent gives rise to huge PNA peak and an apparent impurity peak, the purity of PNA was evaluated to be about 81.4% by CZE method, obviously lower than the supplier's purity value of 99.9% evaluated by RP–HPLC, and also lower than 94.8% determined with RP–HPLC by our research group. The CZE method takes only 5 min, needs only 90 nL PNA, much less than 20 min and 20 μL PNA in RP–HPLC method. Moreover, the CZE method is applicable for the analysis of glutamic acid modified and lysine modified PNAs, they show different migration time with their corresponding complementary PNAs. Our results show CZE provides a new choice for PNA and modified PNA analysis, also their purity or quality evaluation.     

Hybridization and Melting Behavior of Peptide Nucleic Acid (PNA) Oligonucleotide Chimeras Conjugated to Gold Nanoparticles

Peptide nucleic acids (PNA) and PNA–DNA chimeras carrying thiol groups were used for surface functionalization of Au nanoparticles. Conjugation of PNA to citrate‐stabilized Au nanoparticles destabilized the nanoparticles causing them to precipitate. Addition of a tail of glutamic acid to the PNA prevented destabilization of the nanoparticles but resulted in loss of interaction with complementary sequences. Importantly, PNA–DNA chimeras gave stable conjugates with Au nanoparticles. The hybridization and melting properties of complexes formed from chimera–nanoparticle conjugates and oligonucleotide–nanoparticle conjugates are described for the first time. Similar to oligonucleotide–nanoparticle conjugates, conjugates with PNA–DNA chimeras gave sharper and more‐defined melting profiles than those obtained with unmodified oligonucleotides. In addition, mismatch discrimination was found to be more efficient than with unmodified oligonucleotides.     

Real Time Electrochemical Monitoring of DNA/PNA Dissociation by Melting Curve Analysis

An immobilization‐free electrochemical method is reported for real‐time monitoring of the DNA hybrid dissociation between a ferrocene labeled peptide nucleic acid (PNA) and a fully‐complementary or single‐base‐mismatched DNA. This method takes advantages of electrostatic charge characteristics and interactions among the neutrally charged PNA, the negatively charged DNA and the negatively charged electrode surface made of indium tin oxide (ITO). When a ferrocene labeled PNA (Fc‐PNA) sequence is hybridized to a complementary DNA strand, electrostatic repulsion between the negatively charged PNA/DNA hybrid and the negative ITO surface retards the diffusion of the electroactive Fc to the electrode, resulting in a much reduced electrochemical signal. On the other hand, when the Fc‐PNA is dissociated from the hybrid at elevated temperatures, the neutrally charged Fc‐PNA easily diffuses to the electrode with an enhanced electrochemical signal. Therefore, an electrochemical melting curve of the Fc‐PNA/DNA hybrid can be obtained by measuring the Fc signal with the increasing temperature. This strategy allows monitoring of the dissociation of the DNA hybrid in real time, which might lead to a simple detection method for single nucleotide polymorphism (SNP) analysis.     

The Crystal Structure of Non‐Modified and Bipyridine‐Modified PNA Duplexes

Peptide nucleic acid (PNA) is a synthetic analogue of DNA that commonly has an N‐aminoethyl glycine backbone. The crystal structures of two PNA duplexes, one containing eight standard nucleobase pairs (GGCATGCC)₂, and the other containing the same nucleobase pairs and a central pair of bipyridine ligands, have been solved with a resolution of 1.22 and 1.10 Å, respectively. The non‐modified PNA duplex adopts a P‐type helical structure similar to that of previously characterized PNAs. The atomic‐level resolution of the structures allowed us to observe for the first time specific modes of interaction between the terminal lysines of the PNA and the backbone and the nucleobases situated in the vicinity of the lysines, which are considered an important factor in the induction of a preferred handedness in PNA duplexes. Our results support the notion that whereas PNA typically adopts a P‐type helical structure, its flexibility is relatively high. For example, the base‐pair rise in the bipyridine‐containing PNA is the largest measured to date in a PNA homoduplex. The two bipyridines bulge out of the duplex and are aligned parallel to the major groove of the PNA. In addition, two bipyridines from adjacent PNA duplexes form a π‐stacked pair that relates the duplexes within the crystal. The bulging out of the bipyridines causes bending of the PNA duplex, which is in contrast to the structure previously reported for biphenyl‐modified DNA duplexes in solution, where the biphenyls are π stacked with adjacent nucleobase pairs and adopt an intrahelical geometry. This difference shows that relatively small perturbations can significantly impact the relative position of nucleobase analogues in nucleic acid duplexes.     

8-Vinylguanine nucleo amino acid: a fluorescent PNA building block

Attachment of a vinyl group at guanine position 8 provides fluorescent properties of the nucleobase. Therefore, 8-vinylguanine was introduced as a 2-aminoethylglycine peptide nucleic acid (PNA) building block. Incorporation of the guanine analog in short PNA sequences by Fmoc solid phase peptide synthesis allowed the differentiation between hybridization states of specific double strands with DNA, RNA, and PNA as well as quadruplex forming RNA/PNA oligomers based on fluorescence intensity.     

End-labeling of peptide nucleic acid with osmium complex. Voltammetry at carbon and mercury electrodes

Peptide nucleic acid (PNA), the DNA mimic with electrically neutral pseudopeptide backbone, is intensively used in biotechnologies and particularly in single-base mismatch detection in DNA hybridization sensors. We propose a simple method of covalent end-labeling of PNA with osmium tetroxide, 2,2′-bipyridine (Os,bipy). Os,bipy-modified PNA (PNA–Os,bipy) produces voltammetric stripping peaks at carbon and mercury electrodes. Peak potential (E_p) of one of the anodic peaks of PNA–Os,bipy at the pyrolytic graphite electrode (PGE) differs from E_p of the reagent, allowing PNA–Os,bipy analysis directly in the reaction mixture. At the hanging mercury drop electrode (HMDE) the PNA–Os,bipy yields a catalytic peak Cat_p, in addition to the redox couples. Using Cat_p it is possible to detect purified PNA–Os,bipy down to 1 pM concentration at accumulation time 60 s. To our knowledge this is the highest sensitivity of the electrochemical detection of PNA.     

Synthesis and photo activity of phenylazonaphthalene peptide nucleic acid monomers

Phenylazonaphthalene peptide nucleic acid （PNA） monomers were successfully synthesized, and their photoisomerization was examined. The new PNA monomers showed reversible trans-cis isomerization with UVand visible light irradiation, which might be the foundation of photo-regulating the hybridization between PNA containing phenylazonaphthalene unit and DNA. Simultaneously, the fluorescence of the new PNA monomers might make them especially useful as structural probes.     

Enhanced stability of G-quadruplexes from conformationally constrained aep-PNA backbone

Nucleic (DNA) acids having contiguous stretch of G sequence form quadruplex structure, which is very critical to control cell division. Recently the existence of G-quadruplex in RNA is also reported in presence of monovalent metal ion. PNA is a promising DNA analogue which binds strongly to DNA to form PNA:DNA duplex or PNA(2):DNA triplex. PNA also forms quadruplexes such G-quadruplex and i-motif in G and C-rich sequences respectively. aep-PNA containing a prolyl ring is one of several PNA analogues that provide rigidity and chirality in backbone and has binding affinity to natural DNA which is higher than that of PNA. Here we examine the ability of aep-PNA-G to form a quadruplex by UV, CD and mass spectroscopic techniques.     

Synthesis and characterization of (R)-miniPEG-containing chiral γ-peptide nucleic acids using the Fmoc strategy

Diethylene glycol (miniPEG)-containing chiral γPNA is considered to be one of the best PNA derivatives. Its preparation is mainly based on the Boc strategy for solid phase peptide synthesis (SPPS), requiring the repeated use of trifluoroacetic acid TFA, which is not suitable for the in situ synthesis of PNA arrays and some other applications under mild conditions. Herein, Fmoc/Cbz orthogonal protected miniPEG-containing chiral γPNA monomers were synthesized, and a 15mer γPNA was prepared using the Fmoc strategy under mild conditions.     

High-affinity peptide nucleic acid oligomers containing tricyclic cytosine analogues

[structure: see text] Peptide nucleic acid (PNA) monomers containing the tricyclic cytosine analogues phenoxazine, 9-(2-aminoethoxy)phenoxazine (G-clamp), and 9-(3-aminopropoxy)phenoxazine (propyl-G-clamp) have been synthesized. The modified nucleobases were incorporated into PNA oligomers using Boc-chemistry for solid-phase synthesis. PNAs containing single G-clamp modifications exhibit significantly enhanced affinity toward RNA and DNA targets relative to unmodified PNA while maintaining mismatch discrimination. These PNA G-clamp modifications exhibit the highest increase in affinity toward nucleic acid targets reported so far for PNA modifications.     

Strategies for the synthesis of fluorescently labelled PNA

A simple and effective strategy for preparing fluorophore-labelled PNA is described. A C-terminal S-t-butylmercaptocysteine-derivatized PNA was prepared on solid-phase using Fmoc chemistry. Selective deprotection of the S-t-butylmercapto group on-bead, allowed the free thiol to be reacted with a fluorophore derivatized via an iodoacetamido or maleimido linker. Subsequent cleavage and sidechain deprotection yielded C-terminal labelled PNA in good yield and purity. Dual labelled PNA was also prepared by using both C-terminal (-SH) and N-terminal (-NH(2)) labelling chemistries.