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.     

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.     

(SR/RS)-cyclohexanyl PNAs: conformationally preorganized PNA analogues with unprecedented preference for duplex formation with RNA

PNA oligomers H-GTAGATCAT-lys-NH2 with cis-(1S,2R/1R,2S)-cyclohexyl-T (III) in the backbone form PNA:RNA duplexes with Tm approximately 30-50 degrees C higher than that of PNA:DNA duplexes. In comparison, cis-(1S,2R/1R,2S)-cyclopentyl PNA-T (IV) form highly stable duplexes with both RNA and DNA without discrimination.     

Cyclopropane PNA: observable triplex melting in a PNA constrained with a 3-membered ring

The first peptide nucleic acid (PNA) with a cyclopropane in the backbone has been synthesized, and the effects of the ring on DNA/RNA binding properties of the PNA have been examined. Well-defined triplex to duplex melting transitions of PNA₂ DNA complexes is clearly observed by variable temperature UV absorbance with the cyclopropane-constrained PNA.     

Synthesis of pyrrolidine-based oxy-peptide nucleic acids carrying four types of nucleobases and their transport into cytoplasm

We synthesized 16 pyrrolidine-based oxy-peptide nucleic acid (POPNA) monomers carrying four different nucleobases onto four different stereoisomers of pyrrolidine rings. Using these monomers, we prepared POPNA oligomers, which formed sequence-specific hybrids with DNAs. The oligomer configurations influenced the hybrid stability. The oligomers were not taken into CHO cells. However, they could enter the cell cytoplasm when mixed with the influenza virus hemagglutinin peptide-arginine heptamer conjugate.     

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.     

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.     

PNA for one-base differentiating protection of DNA from nuclease and its use for SNPs detection

By the combination of peptide nucleic acid (PNA) with single-stranded DNA specific nucleases, alteration of a single base to another in DNA has been detected with high accuracy. Only the DNAs in DNA/PNA duplexes involving a mismatch are efficiently hydrolyzed by these enzymes, whereas fully matching sequences are kept intact. This difference is visually scored by adding 3,3'-diethylthiadicarbocyanine, which changes its color from blue to purple upon binding to DNA/PNA duplexes. These findings are applied to the convenient and straightforward detection of single nucleotide polymorphisms (SNPs). When the target site in the sample DNA is completely complementary with the PNA, a notable amount of DNA/PNA duplex remains and thus the solution exhibits purple color. In the presence of even one mismatch between PNA and DNA, however, the DNA is completely digested by the enzyme and therefore the dye shows its intrinsic blue color. The SNPs in the apolipoprotein E gene of human DNA have been successfully genotyped by this method.     

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.     

Monofunctionally trans-diammine platinum(II)-modified peptide nucleic acid oligomers: a new generation of potential antisense drugs

A solid-phase approach is described that provides facile access to monofunctionally trans-PtII-modified PNA oligomers of arbitrary sequence for potential use both in antigene and antisense strategies. The approach includes the synthesis of a platinated building block 1 and its subsequent incorporation into three different PNA oligomers 5-7 by solid-phase synthesis. In a model cross-linking reaction one of the latter is found to recognize sequence-specifically a target oligonucleotide 8 and to cross-link to it. The resulting structure is the trans-PtII-cross-linked PNA/DNA duplex 9 as revealed by mass spectrometry in combination with a Maxam-Gilbert sequencing experiment.     

(2′‐O‐Methyl‐RNA)‐3′‐PNA Chimeras: A New Class of Mixed Backbone Oligonucleotide Analogues with High Binding Affinity to RNA

The automated on‐line synthesis of DNA‐3′‐PNA chimeras 1 – 4 and (2′‐O‐methyl‐RNA)‐3′‐PNA chimeras 5 – 8 is described, in which the 3′‐terminal part of the oligonucleotide is linked to the N‐terminal part of the PNA via N‐(ω‐hydroxyalkyl)‐N‐[(thymin‐1‐yl)acetyl]glycine units (alkyl=Et, Ph, Bu, and pentyl). By means of UV thermal denaturation, the binding affinities of all chimeras were directly compared by determining their T_m values in the duplex with complementary DNA and RNA. All investigated DNA‐3′‐PNA chimeras and (2′‐O‐methyl‐RNA)‐3′‐PNA chimeras form more‐stable duplexes with complementary DNA and RNA than the corresponding unmodified DNA. Interestingly, a N‐(3‐hydroxypropyl)glycine linker resulted in the highest binding affinity for DNA‐3′‐PNA chimeras, whereas the (2′‐O‐methyl‐RNA)‐3′‐PNA chimeras showed optimal binding with the homologous N‐(4‐hydroxybutyl)glycine linker. The duplexes of (2′‐O‐methyl‐RNA)‐3′‐PNA chimeras and RNA were significantly more stable than those containing the corresponding DNA‐3′‐PNA chimeras. Surprisingly, we found that the charged (2′‐O‐methyl‐RNA)‐3′‐PNA chimera with a N‐(4‐hydroxybutyl)glycine‐based unit at the junction to the PNA part shows the same binding affinity to RNA as uncharged PNA. Potential applications of (2′‐O‐methyl‐RNA)‐3′‐PNA chimeras include their use as antisense agents acting by a RNase‐independent mechanism of action, a prerequisite for antisense‐oligonucleotide‐mediated correction of aberrant splicing of pre‐mRNA.     

Enzyme-amplified electrochemical hybridization assay based on PNA, LNA and DNA probe-modified micro-magnetic beads

In the present study, we investigated the properties of PNA and LNA capture probes in the development of an electrochemical hybridization assay. Streptavidin-coated paramagnetic micro-beads were used as a solid phase to immobilize biotinylated DNA, PNA and LNA capture probes, respectively. The target sequence was then recognized via hybridization with the capture probe. After labeling the biotinylated hybrid with a streptavidin–enzyme conjugate, the electrochemical detection of the enzymatic product was performed onto the surface of a disposable electrode. The assay was applied to the analytical detection of biotinylated DNA as well as RNA sequences. Detection limits, calculated considering the slope of the linear portion of the calibration curve in the range 0–2 nM were found to be 152, 118 and 91 pM, coupled with a reproducibility of the analysis equal to 5, 9 and 6%, calculated as RSD%, for DNA, PNA and LNA probes respectively, using the DNA target. In the case of the RNA target, the detection limits were found to be 51, 60 and 78 pM for DNA, PNA and LNA probes respectively.     

Label‐Free Bioelectronic Detection of Point Mutation by Using Peptide Nucleic Acid Probes

An electrochemical hybridization biosensor based on peptide nucleic acid (PNA) probes with a label‐free protocol is described. The detection of PNA‐DNA and DNA‐DNA hybridizations were accomplished based on the oxidation signal of guanine by using differential pulse voltammetry (DPV) at carbon paste electrode (CPE). It was observed that the oxidation signals of guanine obtained from the PNA and DNA probe modified CPEs were higher than those obtained from the PNA‐DNA and DNA‐DNA hybrid modified CPEs due to the accessible unbound guanine bases. The detection of hybridization between PNA probe and point mutation containing DNA target sequences was clearly observed due to the difference of the oxidation signals of guanine bases, because the point mutation was guanine nearly at the middle of the sequence. The effect of the DNA target concentration on the hybridization signal was also observed. The PNA probe was also challenged with excessive and equal amount of noncomplementary DNA and also mixtures of point mutation and target DNA.     

Pyrrolidine nucleic acids: DNA/PNA oligomers with 2-hydroxy/aminomethyl- 4-(thymin-1-yl)pyrrolidine-N-acetic acid

Synthesis of pyrrolidine-based chiral positively charged DNA analogues is reported. The synthesis of (2S,4S) and (2R,4R) thymin-1-ylpyrrolidine-N-acetic acid, its site specific incorporation in PNA:DNA chimera and PNA, and the study of their binding properties with complementary DNA/RNA sequences is presented.     

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.     

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.     

Synthesis and evaluation of (1S,2R/1R,2S)-aminocyclohexylglycyl PNAs as conformationally preorganized PNA analogues for DNA/RNA recognition

Conformationally constrained cis-aminocyclohexylglycyl PNAs have been designed on the basis of stereospecific imposition of 1,2-cis-cyclohexyl moieties on the aminoethyl segment of aminoethylglycyl PNA (aegPNA). The introduction of the cis-cyclohexyl ring may allow the restriction of the torsion angle beta in the ethylenediamine segment to 60-70 degrees that is prevalent in PNA(2):DNA and PNA:RNA complexes. The synthesis of the optically pure monomers (10a and 10b) is achieved by stereoselective enzymatic hydrolysis of an intermediate ester 2. The chiral PNA oligomers were synthesized with (1S,2R/1R,2S)-aminocyclohexylglycyl thymine monomers in the center and N-terminus of aegPNA. Differential gel shift retardation with one or more units of modified monomer units was observed as a result of hybridization of PNA sequences with complementary DNA sequences. Hybridization studies with complementary DNA and RNA sequences using UV-T(m) measurements indicate that PNA with (1S,2R)-cyclohexyl stereochemistry enhances selective binding with RNA over DNA as compared to control aegPNA and PNA with the other (1R,2S) isomer.     

Formation of a PNA2-DNA2 hybrid quadruplex

DNA guanine (G) quadruplexes are stabilized by an interesting variation of the hydrogen-bonding schemes encountered in nucleic acid duplexes and triplexes. In an attempt to use this mode of molecular recognition, we target a dimeric G-quadruplex formed by the Oxytricha nova telomeric sequence d(G(4)T(4)G(4)) with a peptide nucleic acid (PNA) probe having a homologous rather than complementary sequence. UV-vis and CD spectroscopy reveal that a stable hybrid possessing G-quartets is formed between the PNA and DNA. The four-stranded character of the hybrid and the relative orientation of the strands is determined by fluorescence resonance energy transfer (FRET) experiments. FRET results indicate that (i) the two PNA strands are parallel to each other, (ii) the two DNA strands are parallel to each other, and (iii) the 5'-termini of the DNA strands align with the N-termini of the PNA strands. The resulting PNA(2)-DNA(2) quadruplex shows a preference of Na(+) over Li(+) and displays thermodynamic behavior consistent with alternating PNA and DNA strands in the hybrid. The formation of this novel supramolecular structure demonstrates a new high-affinity DNA recognition mechanism and expands the scope of molecular recognition by PNA.     

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.     

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.