Online Capillary Electrophoresis Reaction for Interaction Study of Amino Acid Modified Peptide Nucleic Acid and Proteins

Peptide nucleic acid (PNA) is a nucleic acid analog which consists of purines, pyrimidines bases, and a neutrally charged peptide backbone. The PNA has the potential as a very useful biological probe for protein analysis since it has more in vivo biological stability as compared to DNA- or RNA-based aptamers. Usually, the addition of amino acids or peptide to the PNA backbone is used to improve its water-solubility and cell-permeability, but these modifications may affect the interaction between PNA and proteins. To date, the investigation of the interaction between PNA and proteins is rare, and there is no reported study about the effects of modifications. In this work, we designed two types of amino acid modified PNAs, (Lys)₂-PNA and (Glu)₂-PNA, which kept the same base sequence with 15-mer thrombin aptamer and had two basic lysine and two acidic glutamic acid residues on N-terminal of the peptide backbone, respectively. To rapidly assess the binding affinity and specificity of modified PNA and proteins, the online CE reaction method was developed to analyze the interactions of (Lys)₂-PNA/(Glu)₂-PNA and three proteins: thrombin (THB), single-strand DNA-binding protein (SSB) and human serum albumin (HSA). Meanwhile, the interactions of (Lys)₂-PNA/(Glu)₂-PNA and thrombin were compared with that of the corresponding complementary base sequence (Lys)₂-cPNA/(Glu)₂-cPNA and thrombin. The online CE reaction results showed that the interaction of (Lys)₂-PNA and (Glu)₂-PNA with three proteins was in the order of THB > SSB > HSA. However, (Lys)₂-PNA and (Lys)₂-cPNA showed similar binding affinity with thrombin; while the binding affinity of (Glu)₂-PNA with thrombin was stronger than that of (Glu)₂-cPNA with thrombin. Moreover, the binding constant K_b of (Glu)₂-PNA and three proteins was determined by affinity capillary electrophoresis (ACE). The online CE reaction eliminates the requirement of incubation, and thus it is fast in detection, and easy to operate with minimum cost. The method is particularly suitable for the interaction studies of expensive modified PNAs and proteins, and can assist the design of PNA probe that binds to proteins.     

Electrochemiluminescent monomers for solid support syntheses of Ru(II)-PNA bioconjugates: multimodal biosensing tools with enhanced duplex stability

The feasibility of devising a solid support mediated approach to multimodal Ru(II)-peptide nucleic acid (PNA) oligomers is explored. Three Ru(II)-PNA-like monomers, [Ru(bpy)(2)(Cpp-L-PNA-OH)](2+) (M1), [Ru(phen)(2)(Cpp-L-PNA-OH)](2+) (M2), and [Ru(dppz)(2)(Cpp-L-PNA-OH)](2+) (M3) (bpy = 2,2'-bipyridine, phen = 1,10-phenanthroline, dppz = dipyrido[3,2-a:2',3'-c]phenazine, Cpp-L-PNA-OH = [2-(N-9-fluorenylmethoxycarbonyl)aminoethyl]-N-[6-(2-(pyridin-2yl)pyrimidine-4-carboxamido)hexanoyl]-glycine), have been synthesized as building blocks for Ru(II)-PNA oligomers and characterized by IR and (1)H NMR spectroscopy, mass spectrometry, electrochemistry and elemental analysis. As a proof of principle, M1 was incorporated on the solid phase within the PNA sequences H-g-c-a-a-t-a-a-a-a-Lys-NH(2) (PNA1) and H-P-K-K-K-R-K-V-g-c-a-a-t-a-a-a-a-lys-NH(2) (PNA4) to give PNA2 (H-g-c-a-a-t-a-a-a-a-M1-lys-NH(2)) and PNA3 (H-P-K-K-K-R-K-V-g-c-a-a-t-a-a-a-a-M1-lys-NH(2)), respectively. The two Ru(II)-PNA oligomers, PNA2 and PNA3, displayed a metal to ligand charge transfer (MLCT) transition band centered around 445 nm and an emission maximum at about 680 nm following 450 nm excitation in aqueous solutions (10 mM PBS, pH 7.4). The absorption and emission response of the duplexes formed with the cDNA strand (DNA: 5'-T-T-T-T-T-T-T-A-T-T-G-C-T-T-T-3') showed no major variations, suggesting that the electronic properties of the Ru(II) complexes are largely unaffected by hybridization. The thermal stability of the PNA·DNA duplexes, as evaluated from UV melting experiments, is enhanced compared to the corresponding nonmetalated duplexes. The melting temperature (T(m)) was almost 8 °C higher for PNA2·DNA duplex, and 4 °C for PNA3·DNA duplex, with the stabilization attributed to the electrostatic interaction between the cationic residues (Ru(II) unit and positively charged lysine/arginine) and the polyanionic DNA backbone. In presence of tripropylamine (TPA) as co-reactant, PNA2, PNA3, PNA2·DNA and PNA3·DNA displayed strong electrochemiluminescence (ECL) signals even at submicromolar concentrations. Importantly, the combination of spectrochemical, thermal and ECL properties possessed by the Ru(II)-PNA sequences offer an elegant approach for the design of highly sensitive multimodal biosensing tools.     

Base dependent pyrrolidine ring pucker in aep-PNA monomers: NMR and PSEUROT analysis

The aep-PNA is a chiral and cyclic PNA analogue, which has a stronger and base dependent binding affinity with complementary DNA. To understand the base dependent properties at monomer level, the structural studies of aep-PNA-(T/C/A) monomers have been carried out focussing on the conformational analysis of pyrrolidine ring pucker in aep-PNA by ¹H NMR and the coupling constant data fitted into PSEUROT software. The results indicate that the type of pyrrolidine pucker depends on the electronic nature of substituent, implying the effect of pyrimidine or purine substituents in determining the ring pucker in monomers. This may consequently influence the aep-PNA oligomer conformation. Since pyrrolidine nucleic acids have emerged as an important class of PNA analogues, present results may have importance for their future development.     

PNAs grafted with (α/γ, R/S)-aminomethylene pendants: regio and stereo specific effects on DNA binding and improved cell uptake

PNAs grafted with cationic aminomethylene (am) pendants on the backbone at the glycyl (α) or ethylenediamine (γ) segments show regio (α/γ) and stereochemistry (R/S) dependent binding with complementary DNA. These are efficiently taken up by cells, with γ(S-am) aeg-PNA being the best in all properties.     

MD simulations of homomorphous PNA, DNA, and RNA single strands: characterization and comparison of conformations and dynamics

MD simulations of homomorphous single-stranded PNA, DNA, and RNA with the same base sequence have been performed in aqueous solvent. For each strand two separate simulations were performed starting from a (i) helical conformation and (ii) random coiled state. Comparisons of the simulations with the single-stranded helices (case i) show that the differences in the covalent nature of the backbones cause significant differences in the structural and dynamical properties of the strands. It is found that the PNA strand maintains its nice base-stacked initial helical structure throughout the 1.5-ns MD simulation at 300 K, while DNA/RNA show relatively larger fluctuations in the structures with a few local unstacking events during -ns MD simulation each. It seems that the weak physical coupling between the bases and the backbone in PNA causes a loss of correlation between the dynamics of the bases and the backbone compared to the DNA/RNA and helps maintain the base-stacked helical conformation. The global flexibility of a single-stranded PNA helix was also found to be lowest, while RNA appears to be the most flexible single-stranded helix. The sugar pucker of several nucleotides in single-stranded DNA and RNA was found to adopt both C2'-endo and C3'-endo conformations for significant times. This effect is more pronounced for single strands in completely coiled states. The simulations with single-stranded coils as the initial structure also indicate that a PNA can adopt a more compact globular structure, while DNA/RNA of the same size adopts a more extended coil structure. This allows even a short PNA in the coiled state to form a significantly stable nonsequentially base-stacked globular structure in solution. Due to the hydrophobic nature of the PNA backbone, it interacts with surrounding water rather weakly compared to DNA/RNA.     

Influence of the Type of Junction in DNA-3′-Peptide Nucleic Acid (PNA) Chimeras on Their Binding Affinity to DNA and RNA

The automated on-line synthesis of DNA-3′-PNA (PNA=Polyamide Nucleic Acids) chimeras 1 – 3 is described, in which the 3′-terminal part of the oligonucleotide is linked to the aminoterminal part of the PNA either via a N-(2-mercaptoethyl)- (X=S), a N-(2-hydroxyethyl)- (X=O), or a N-(2-aminoethyl)- (X=NH) N-[(thymin-1-yl)acetyl]glycine unit. Furthermore, the DNA-3′-PNA chimera 4 without a nucleobase at the linking unit was prepared. The binding affinities of all chimeras were directly compared by determining their T_m values in the duplex with complementary DNA, RNA, or DNA containing a mismatch or abasic site opposite to the linker unit. We found that all investigated chimeras with a nucleobase at the junction form more stable duplexes with complementary DNA and RNA than the corresponding unmodified DNA. The influence of X on duplex stabilization was determined to be in the order O>S≈NH, rendering the phosphodiester bridge the most favored linkage at the DNA/PNA junction. The observed strong duplex-destabilizing effects, when base mismatches or non-basic sites were introduced opposite to the nucleobase at the DNA/PNA junction, suggest that the base at the linking unit contributes significantly to duplex stabilization.     

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).     

A simple gamma-backbone modification preorganizes peptide nucleic acid into a helical structure

Peptide nucleic acid (PNA) is a synthetic analogue of DNA and RNA, developed more than a decade ago in which the naturally occurring sugar phosphate backbone has been replaced by the N-(2-aminoethyl) glycine units. Unlike DNA or RNA in the unhybridized state (single strand) which can adopt a helical structure through base-stacking, although highly flexible, PNA does not have a well-defined conformational folding in solution. Herein, we show that a simple backbone modification at the gamma-position of the N-(2-aminoethyl) glycine unit can transform a randomly folded PNA into a helical structure. Spectroscopic studies showed that helical induction occurs in the C- to N-terminal direction and is sterically driven. This finding has important implication not only on the future design of nucleic acid mimics but also on the design of novel materials, where molecular organization and efficient electronic coupling are desired.     

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.     

Aminomethylene peptide nucleic acid (am-PNA): synthesis, regio-/stereospecific DNA binding, and differential cell uptake of (α/γ,R/S)am-PNA analogues

Inherently chiral, cationic am-PNAs having pendant aminomethylene groups at α(R/S) or γ(S) sites on PNA backbone have been synthesized. The modified PNAs are shown to stabilize duplexes with complementary cDNA in a regio- and stereo-preferred manner with γ(S)-am PNA superior to α(R/S)-am PNAs and α(R)-am PNA better than the α(S) isomer. The enhanced stabilization of am-PNA:DNA duplexes is accompanied by a greater discrimination of mismatched bases. This seems to be a combined result of both electrostatic interactions and conformational preorganization of backbone favoring the cDNA binding. The am-PNAs are demonstrated to effectively traverse the cell membrane, localize in the nucleus of HeLa cells, and exhibit low toxicity to cells.     

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.     

A cyclopentane conformational restraint for a peptide nucleic acid: design,asymmetric synthesis,and improved binding affinity to DNA and RNA

[structure: see text] A strategy to restrict the highly flexible backbone conformation of a peptide nucleic acid (PNA) by incorporation of a cyclopentane ring is proposed. An asymmetric synthesis of cyclopentane-modified PNA is reported, and its binding properties were determined. The cyclopentane ring leads to a significant improvement in the binding properties of the resulting PNA to DNA and RNA.     

Quadruplex formation by a guanine-rich PNA oligomer

A guanine-rich PNA dodecamer having the sequence H-G4T4G4-Lys-NH2 (G-PNA) hybridizes with a DNA dodecamer of homologous sequence to form a four-stranded quadruplex (Datta, B.; Schmitt, C.; Armitage, B. A. J. Am. Chem. Soc. 2003, 125, 4111-4118). This report describes quadruplex formation by the PNA alone. UV melting curves and fluorescence resonance energy transfer experiments reveal formation of a multistranded structure stabilized by guanine tetrads. The ion dependency of these structures is analogous to that reported for DNA quadruplexes. Electrospray ionization mass spectrometry indicates that both dimeric and tetrameric quadruplexes are formed by G4-PNA, with the dimeric form being preferred. These results have implications for the use of G-rich PNA for homologous hybridization to G-rich targets in chromosomal DNA and suggest additional applications in assembling quadruplex structures within lipid bilayer environments.     

Mono- and dinuclear ruthenium(II) and osmium(II) polypyridine complexes built around spiro-bridged bis(phenanthroline) ligands: synthesis, electrochemistry, and photophysics

Two new dyads have been synthesized in which terminal Ru(II) and Os(II) polypyridine complexes are separated by sterically constrained spiro bridges. The photophysical properties of the corresponding mononuclear complexes indicate the importance of the decay of the lowest-energy triplet states localized on the metallo fragments through the higher-energy metal-centered excited states. This effect is minimized at 77 K, where triplet lifetimes are relatively long, and for the Os(II)-based systems relative to their Ru(II)-based counterparts. Intramolecular triplet energy transfer takes place from the Ru(II)-based fragment to the appended Os(II)-based unit, the rate constant being dependent on the molecular structure and on temperature. In all cases, the experimental rate constant matches surprisingly well with the rate constant calculated for F?rster-type dipole-dipole energy transfer. As such, the disparate rates shown by the two compounds can be attributed to stereochemical factors. It is further concluded that the spiro bridging unit does not favor through-bond electron exchange interactions, a situation confirmed by cyclic voltammetry.     

The RNA2-PNA2 hybrid i-motif-a novel RNA-based building block

We report the formation of a hybrid RNA2-PNA2 i-motif comprised of two RNA and two PNA strands based on the sequence specific self assembly of RNA, with potential as a building block for structural RNA nanotechnology.     

Crystal structure of a partly self-complementary peptide nucleic acid (PNA) oligomer showing a duplex-triplex network

The X-ray structure of a partly self-complementary peptide nucleic acid (PNA) decamer (H-GTAGATCACT-l-Lys-NH(2)) to 2.60 A resolution is reported. The structure is mainly controlled by the canonical Watson-Crick base pairs formed by the self-complementary stretch of four bases in the middle of the decamer (G(4)A(5)T(6)C(7)). One right- and one left-handed Watson-Crick duplex are formed. The two PNA units C(9)T(10) change helical handedness, so that each PNA strand contains both a right- and a left-handed section. The changed handedness in C(9)T(10) allows formation of Hoogsteen hydrogen bonding between C(9)T(10) and G(4)A(5) of a PNA strand in an adjacent Watson-Crick double helix of the same handedness. Thereby, a PNA-PNA-PNA triplex is formed. The PNA unit A(3) forms a noncanonical base pair with A(8) in a symmetry-related strand of opposite handedness; the base pair is of the A-A reverse Hoogsteen type. The structural diversity of this PNA demonstrates how the PNA backbone is able to adapt to structures governed by the stacking and hydrogen-bonding interactions between the nucleobases. The crystal structure further shows how PNA oligomers containing limited sequence complementarity may form complex hydrogen-bonding networks.     

Isolated monohydrates of a model peptide chain: effect of a first water molecule on the secondary structure of a capped phenylalanine

The formation of monohydrates of capped phenylalanine model peptides, CH(3)-CO-Phe-NH(2) and CH(3)-CO-Phe-NH-CH(3), in a supersonic expansion has been investigated using laser spectroscopy and quantum chemistry methods. Conformational distributions of the monohydrates have been revealed by IR/UV double-resonance spectroscopy and their structures assigned by comparison with DFT-D calculations. A careful analysis of the final hydrate distribution together with a detailed theoretical investigation of the potential energy surface of the monohydrates demonstrates that solvation occurs from the conformational distribution of the isolated peptide monomers. The distribution of the monohydrates appears to be strongly dependent on both the initial monomer conformation (extended or folded backbone) and the solvation site initially occupied by the water molecule. The solvation processes taking place during the cooling can be categorized as follows: (a) solvation without significant structural changes of the peptide, (b) solvation inducing significant distortions of the backbone but retaining the secondary structure, and (c) solvation triggering backbone isomerizations, leading to a modification of the peptide secondary structure. It is observed that solvation by a single water molecule can fold a β-strand into a γ-turn structure (type c) or induce a significant opening of a γ-turn characterized by an elongated C(7) hydrogen bond (type b). These structural changes can be considered as a first step toward the polyproline II condensed-phase structure, illustrating the role played by the very first water molecule in the solvation process.     

Aryl-bridged 1-hydroxypyridin-2-one: sensitizer ligands for Eu(III)

The synthesis, crystal structure, solution stability, and photophysical properties of an aryl group bridging two 1-hydroxypyridin-2-one units complexed to Eu(III) are reported. The results show that this backbone unit increases the rigidity of the ensuing complex, and also the conjugation of the ligand. As a result of the latter, the singlet absorption energy is decreased, along with the energy of the lowest excited triplet state. The resulting efficiency of sensitization for the Eu(III) ion is influenced by these phenomena, yielding an overall quantum yield of 6.2% in aqueous solution. The kinetic parameters arising from the luminescence data reveal an enhanced nonradiative decay rate for this compound when compared to previously reported aliphatic bridges.     

Backbone modifications of aromatic peptide nucleic acid (APNA ) monomers and their hybridization properties with DNA and RNA

Aromatic peptide nucleic acid (APNA) monomers containing N-(2-aminobenzyl)-glycine, N-(2-aminobenzyl)-(R)- or -(S)-alanine, and N-(2-aminobenzyl)-beta-alanine moieties as part of their backbone were synthesized. These novel analogues were incorporated as a single "point mutation" in PNA hexamers, and their physicochemical properties were investigated by UV thermal denaturation and CD experiments. Destabilization in triplex formation between the PNA-APNA chimeras and complementary DNA or RNA oligomers was observed, as compared to the PNA control. The APNA monomer composed of the N-(2-aminobenzyl)-glycine backbone led to the smallest decrease in the thermal stability of the triplexes formed with DNA and RNA, while maintaining selectivity for base-pairing recognition. Since the PNA-APNA chimeras are more lipophilic than the corresponding PNA homopolymers, these oligomers may also exhibit better cell membrane permeability properties.     

A new triferrocenyl-tris(hydroxymethyl)aminomethane derivative as a highly sensitive electrochemical marker of biomolecules: application to the labelling of PNA monomers and their electrochemical characterization

We have designed and synthesised a new organometallic molecule containing three ferrocene groups for use as a highly sensitive electrochemical marker in biological assays. This trisferrocene derivative was conjugated to different PNA monomers, and the electrochemical activities of the conjugates were extensively investigated in organic solvents, in view of their potential diagnostic applications. The results showed that the introduction of a trisferrocene unit on the PNA monomer triples the current signal in comparison with the monoferrocene-labelled one. Despite their greater molecular complexity, trisferrocene-conjugated PNA monomers are even more electrochemically active than the reference ferrocene. By using differential pulse voltammetry (DPV), the detection limit can reach 10(-8) M in acetonitrile solution. These results are a good premise for the use of the trisferrocene unit as an effective electrochemical probe for biomolecules.