Modular nucleic acid surrogates. Solid phase synthesis of alpha-helical peptide nucleic acids (alpha PNAs)

[formula: see text] The synthesis and characterization of prototype alpha-helical peptide nucleic acid (alpha PNA) modules 1-3 as well as disulfide dimers 4 and 5 are reported. These molecules combine an alpha-helical peptidyl scaffold with well-defined nucleobase molecular recognition patterns and could serve as a basis for novel antisense and/or antigene agents. Structure assignments for these alpha PNAs were supported by MALDI-TOF mass spectrometry, and the alpha-helical nature of 4 in water was confirmed by circular dichroism (CD) spectroscopy.     

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.     

Metal-containing peptide nucleic acid conjugates

Peptide Nucleic Acids (PNAs) are non-natural DNA/RNA analogues with favourable physico-chemical properties and promising applications. Discovered nearly 20 years ago, PNAs have recently re-gained quite a lot of attention. In this Perspective article, we discuss the latest advances on the preparation and utilisation of PNA monomers and oligomers containing metal complexes. These metal- conjugates have found applications in various research fields such as in the sequence-specific detection of nucleic acids, in the hydrolysis of nucleic acids and peptides, as radioactive probes or as modulators of PNA·DNA hybrid stability, and last but not least as probes for molecular and cell biology.     

Improved nucleic acid triggered probe activation through the use of a 5-thiomethyluracil peptide nucleic acid building block

To improve the efficiency of a nucleic acid triggered probe activation (NATPA) system a 5-thiomethyluracil peptide nucleic acid (PNA) building block has been synthesized. Attachment of imidazole and a coumarin ester to uracils at the ends of two PNAs resulted in a 550 000-fold acceleration of DNA-triggered coumarin release relative to imidazole and a 6-fold increase in k(cat) relative to a system which had these groups attached to the amino and carboxy ends of PNAs. [structure: see text]     

Rapid Synthesis of Propargyl-γ-Modified Peptide Nucleic Acid Monomers for Late-Stage Functionalization of Oligomers

The exceptional hybridization properties of peptide nucleic acids (PNAs) coupled with the ease of their synthesis has made this artificial nucleic acid mimetic a desirable platform for diagnostics, therapeutics and supramolecular engineering. PNA backbone modifications have been extensively explored to finetune physicochemical properties and for conjugation of functional molecules. Here, we detail the synthesis of a universal γ-propargyl-PNA backbone from serine, and its acylation with the four DNA canonical nucleobases. The availability of serine as d or l enantiomer provide simple accesses to PNA oligomers for hybridization with natural oligonucleotides or for orthogonal hybridization circuitry. We show that late-stage conjugation enables optimization of the physicochemical properties. This approach is appealing due to its orthogonality to Fmoc-SPPS, its flexibility and ease for introducing diversity by on-resin copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC). We exemplified the utility of these novel monomers with PNA based hybridization chain reactions (HCRs).     

Sequence-specific binding of DNA to liposomes containing di-alkyl peptide nucleic acid (PNA) amphiphiles

We present a method to covalently attach peptide nucleic acid (PNA) to liposomes by conjugation of PNA peptide to charged amino acids and synthetic di-alkyl lipids ("PNA amphiphile," PNAA) followed by co-extrusion with disteroylphosphatidylcholine (DSPC) and cholesterol. Attachment of four Glu residues and two ethylene oxide spacers to the PNAA was required to confer proper hydration for extrusion and presentation for DNA hybridization. The extent of DNA oligomer binding to 10-mer PNAA liposomes was assessed using capillary zone electrophoresis. Nearly all PNAs on the liposome surface are complexed with a stoichiometric amount of complementary DNA 10-mers after 3-h incubation in pH 8.0 Tris buffer. No binding to PNAA liposomes was observed using DNA 10-mers with a single mismatch. Longer DNA showed a greatly attenuated binding efficiency, likely because of electrostatic repulsion between the PNAA liposome double layer and the DNA backbone. Langmuir isotherms of PNAA:DSPC:chol monolayers indicate miscibility of these components at the compositions used for liposome preparation. PNAA liposomes preserve the high sequence-selectivity of PNAs and emerge as a useful sequence tag for highly sensitive bioanalytical devices.     

(S,S)-trans-cyclopentane-constrained peptide nucleic acids. a general backbone modification that improves binding affinity and sequence specificity

Replacing the ethylenediamine portion of aminoethylglycine peptide nucleic acids (aegPNAs) with one or more (S,S)-trans-cyclopentane diamine units significantly increases binding affinity and sequence specificity to complementary DNA, making these modified PNAs ideal for use as nucleic acid probes in genomic analysis. The synthesis and study of this new class of PNAs (tcypPNAs) is described in which trans-cyclopentane diamine has been incorporated into several positions, and in varying number, within PNA backbones of mixed-base sequences.     

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.     

Direction control in DNA binding of chiral D-lysine-based peptide nucleic acid (PNA) probed by electrospray mass spectrometry

The DNA binding abilities of peptide nucleic acids (PNAs), both achiral and bearing three adjacent D-lysine-based monomers in the middle of the strand ("chiral box" PNA), were studied by means of electrospray mass spectrometry (ESI-MS). In contrast with achiral PNA, "Chiral box" PNA was confirmed to exert high direction control (antiparallel vs. parallel DNA target) in DNA binding.     

New aspects in fragmentation of peptide nucleic acids: comparison of positive and negative ions by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

The use of peptide nucleic acids (PNAs) is steadily increasing in biochemistry and diagnostics. So far, PNAs have mostly been investigated using cationic conditions in mass spectrometry. Furthermore, the use of fragmentation techniques developed for peptides and proteins like infrared multiphoton dissociation (IRMPD) and electron capture dissociation (ECD) has barely been examined. However, especially the fragmentation behavior of PNA oligomers in negative ion mode is of high importance, due to the ability to interact with nucleic acids which are almost exclusively analyzed in the negatively charged state. In the current study PNA fragmentations under cationic and anionic conditions were investigated and different fragmentation techniques like collision‐induced dissociation (CID), IRMPD and ECD were applied. Especially when using CID and IRMPD, amide bonds were broken, whereas ECD resulted in the elimination of nucleobases. Differences were also observed between positive and negative ionization, while the sequence coverage for the negative ions was superior to positive ions. The fragmentation behavior using IRMPD led to almost complete sequence coverage. Additionally, in anions the interesting effect of multiple eliminations of HNCO was found. Copyright © 2009 John Wiley & Sons, Ltd.     

Synthesis and oligodeoxynucleotide binding properties of pyrrolidinyl peptide nucleic acids bearing prolyl-2-aminocyclopentanecarboxylic acid (ACPC) backbones

A series of novel conformationally rigid pyrrolidinyl peptide nucleic acids (PNA) based on d-prolyl-2-aminocyclopentanecarboxylic acid (ACPC) backbones has been synthesized. Investigation of the binding properties of four stereoisomeric PNAs possessing different stereochemistry at the ACPC part with DNA revealed that a precise stereochemistry of the backbone is very important in determining the binding properties. Only the PNA containing (1S,2S)-ACPC can form a very stable 1:1 complex with the complementary DNA in a sequence-specific manner.     

Fluorinated olefinic peptide nucleic acid: synthesis and pairing properties with complementary DNA

The fluorinated olefinic peptide nucleic acid (F-OPA) system was designed as a peptide nucleic acid (PNA) analogue in which the base carrying amide moiety was replaced by an isostructural and isoelectrostatic fluorinated C-C double bond, locking the nucleobases in one of the two possible rotameric forms. By comparison of the base-pairing properties of this analogue with its nonfluorinated analogue OPA and PNA, we aimed at a closer understanding of the role of this amide function in complementary DNA recognition. Here we present the synthesis of the F-OPA monomer building blocks containing the nucleobases A, T, and G according to the MMTr/Acyl protecting group scheme. Key steps are a selective desymmetrization of the double bond in the monomer precursor via lactonization as well as a highly regioselective Mitsunobu reaction for the introduction of the bases. PNA decamers containing single F-OPA mutations and fully modified F-OPA decamers and pentadecamers containing the bases A and T were synthesized by solid-phase peptide chemistry, and their hybridization properties with complementary parallel and antiparallel DNA were assessed by UV melting curves and CD spectroscopic methods. The stability of the duplexes formed by the decamers containing single (Z)-F-OPA modifications with parallel and antiparallel DNA was found to be strongly dependent on their position in the sequence with T(m) values ranging from +2.4 to -8.1 degrees C/modification as compared to PNA. Fully modified F-OPA decamers and pentadecamers were found to form parallel duplexes with complementary DNA with reduced stability compared to PNA or OPA. An asymmetric F-OPA pentadecamer was found to form a stable self-complex (T(m) approximately 65 degrees C) of unknown structure. The generally reduced affinity to DNA may therefore be due to an increased propensity for self-aggregation.     

Enantiomeric separation of chiral peptide nucleic acid monomers by capillary electrophoresis with charged cyclodextrins

Direct chiral separation of chiral peptide nucleic acid (PNA) monomers has been achieved for the first time by capillary electrophoresis (CE) with charged cyclodextrins as chiral selectors added to the electrophoretic buffer. Selectively modified 6-deoxy-6-N-histamino-beta-cyclodextrin and sulfobutyl ether-beta-CD were successfully used as chiral selectors for the enantiomeric separation of chiral monomers based on different aminoethylamino acids bearing thymine or adenine as nucleobases. Chiral separations were obtained at low selector concentrations (1-3 mM) with good enantioselectivity and resolution factors. Separations were optimized as a function of pH in order to exploit the effect of the electrostatic interactions between the oppositely charged selector and selectand. The method has been applied to the analysis of the enantiomeric excess of chiral monomers used for the solid phase synthesis of chiral PNA oligomers. CE chiral analysis showed that a very high enantiomeric purity was generally achieved in the synthesis of all monomers, except for histidine and aspartic acid based monomers in which ca. 10% of the "wrong" enantiomer was always present.     

Efficient and sequence-specific DNA-templated polymerization of peptide nucleic acid aldehydes

On the basis of the distance-dependence of DNA-templated reductive amination reactions and of recent findings of D. Lynn and co-workers, we developed DNA-templated polymerizations of synthetic peptide nucleic acid (PNA) aldehydes. The coupling reactions proceed in a highly efficient and sequence-specific manner, even in the presence of mixtures of PNA aldehydes of different sequence. Synthetic peptide nucleic acid polymers containing as many as 40 PNA units (representing 10 consecutive coupling reactions) were formed efficiently. The ease of preparing PNAs containing tailor-made functional groups together with these findings raises the possibility of evolving synthetic sequence-defined polymers by iterated cycles of translation, selection, PCR amplification, and diversification previously available only to biological macromolecules.     

Charge transfer through modified peptide nucleic acids

We studied the charge transfer properties of bipyridine-modified peptide nucleic acid (PNA) in the absence and presence of Zn(II). Characterization of the PNA in solution showed that Zn(II) interacts with the bipyridine ligands, but the stability of the duplexes was not affected significantly by the binding of Zn(II). The charge transfer properties of these molecules were examined by electrochemistry for self-assembled monolayers of ferrocene-terminated PNAs and by conductive probe atomic force microscopy for cysteine-terminated PNAs. Both electrochemical and single molecular studies showed that the bipyridine modification and Zn(II) binding do not affect significantly the charge transfer of the PNA duplexes.     

Metal binding to ligand-containing peptide nucleic acids

The substitution of nucleobases in nucleic acid duplexes with ligands that have high affinity for transition metal ions creates metal-binding sites at specific locations within the duplexes. Several studies on the incorporation of metal ions into DNA and peptide nucleic acid (PNA) duplexes have suggested that the stability constant of the metal complex formed within the duplexes is a primary determinant of the thermal stability of the duplexes. To understand this relationship, we have synthesized two PNA monomers that carry the same ligand, namely 8-hydroxyquinoline, but have this ligand attached differently to the PNA backbone. The PNA monomers have been incorporated into PNA duplexes. UV and CD spectroscopy and calorimetric studies of the 8-hydroxyquinoline-PNA duplexes showed that the effect of the stability of the metal complex on the PNA duplexes was significantly modulated by the steric relationship between the complex and the duplex. This information is useful for the construction of hybrid inorganic-nucleic acid nanostructures.     

Extended Peptide Nucleic Acid Nucleobases Based on Isoorotic Acid for the Recognition of A–U Base Pairs in Double-Stranded RNA

Peptide nucleic acids (PNA) with extended isoorotamide containing nucleobases ( I _o ) were designed for binding A–U base pairs in double-stranded RNA. Isothermal titration calorimetry and UV thermal melting experiments revealed improved affinity for A–U using the Io scaffold in PNA. PNAs having four sequential Io extended nucleobases maintained high binding affinity.     

Nanocrystals modified with peptide nucleic acids (PNAs) for selective self-assembly and DNA detection

Gold nanocrystals modified with peptide nucleic acids (PNAs) have been prepared and applied to self-assembly and DNA sensing. Experiments with different PNA structural motifs show that (1). the versatility in PNA synthetic design can be used to modulate the electrostatic surface properties of nanocrystals, presenting an opportunity to control assembly rate and aggregate size, (2). short (6 base) PNAs can hybridize effectively while attached to nanoparticles, providing a route to generating materials with small interparticle spacings, and (3). the superior base pair mismatch selectivity of PNAs is further enhanced on nanosurfaces, enabling PNA-modified nanoparticles to act as highly selective nanoscale sensors, as well as synthons for defect-free self-assembly. This last feature was coupled with a substantial change in colloidal stability upon DNA hybridization to develop a novel colorimetric DNA assay that detects the presence of single base imperfections within minutes. Various modes of PNA hybridization, including the first practical application of PNA-PNA interactions, were used to direct the assembly of nanoparticles into macroscopic arrangements. Shorter duplex interconnects and greater specificity in assembly were obtained compared to similar experiments with DNA-modified nanocrystals.     

An experimental and theoretical study of the gas-phase decomposition of monoprotonated peptide nucleic acids

Peptide nucleic acids (PNAs) are DNA/RNA mimics which have recently generated considerable interest due to their potential use as antisense and antigene therapeutics and as diagnostic and molecular biology tools. These synthetic biomolecules were designed with improved properties over corresponding oligonucleotides such as greater binding affinity to complementary nucleic acids, enhanced cellular uptake, and greater stability in biological systems. Because of the stability and unique structure of PNAs, traditional sequence confirmation methods are not effective. Alternatively, electrospray ionization coupled with Fourier transform ion cyclotron resonance mass spectrometry shows great potential as a tool for the characterization and structural elucidation of these oligonucleotide analogs. Extensive gas-phase fragmentation studies of a mixed nucleobase 4-mer (AACT) and a mixed nucleobase 4-mer with an acetylated N-terminus (N-acetylated AACT) have been performed. Gas-phase collision-induced dissociation of PNAs resulted in water loss, cleavage of the methylene carbonyl linker containing a nucleobase, cleavage of the peptide bond, and the loss of nucleobases. These studies show that the fragmentation behavior of PNAs resembles that of both peptides and oligonucleotides. Molecular mechanics (MM+), semiempirical (AM1), and ab initio (STO-3G) calculations were used to investigate the site of protonation and determine potential low energy conformations. Computational methods were also employed to study prospective intramolecular interactions and provide insight into potential fragmentation mechanisms.     

Side chain homologation of alanyl peptide nucleic acids: pairing selectivity and stacking

Alanyl peptide nucleic acids (alanyl-PNAs) are oligomers based on a regular peptide backbone with alternating configuration of the amino acids. All side chains are modified by covalently linked nucleobases. Alanyl-PNAs form very rigid, well defined, and linear double strands based on hydrogen bonding of complementary strands, stacking, and solvation. Side chain homology was examined by comparing a methylene linker (alanyl-PNA) with an ethylene linker (homoalanyl-PNA), a trimethylene linker (norvalyl-PNA), and PNA sequences with mixed linker length between nucleobase and backbone. Side chain homology in combination with a linear double strand topology turned out to be valuable in order to selectively manipulate pairing selectivity (pairing mode) and base pair stacking.