Impact of Single Basepair Mismatches on Electron‐Transfer Processes at Fc‐PNA⋅DNA Modified Gold Surfaces

Gold‐surface grafted peptide nucleic acid (PNA) strands, which carry a redox‐active ferrocene tag, present unique tools to electrochemically investigate their mechanical bending elasticity based on the kinetics of electron‐transfer (ET) processes. A comparative study of the mechanical bending properties and the thermodynamic stability of a series of 12‐mer Fc‐PNA?DNA duplexes was carried out. A single basepair mismatch was integrated at all possible strand positions to provide nanoscopic insights into the physicochemical changes provoked by the presence of a single basepair mismatch with regard to its position within the strand. The ET processes at single mismatch Fc‐PNA?DNA modified surfaces were found to proceed with increasing diffusion limitation and decreasing standard ET rate constants k⁰ when the single basepair mismatch was dislocated along the strand towards its free‐dangling Fc‐modified end. The observed ET characteristics are considered to be due to a punctual increase in the strand elasticity at the mismatch position. The kinetic mismatch discrimination with respect to the fully‐complementary duplex presents a basis for an electrochemical DNA sensing strategy based on the Fc‐PNA?DNA bending dynamics for loosely packed monolayers. In a general sense, the strand elasticity presents a further physicochemical property which is affected by a single basepair mismatch which may possibly be used as a basis for future DNA sensing concepts for the specific detection of single basepair mismatches.     

Electroactive chitosan nanoparticles for the detection of single-nucleotide polymorphisms using peptide nucleic acids

Here we report an electrochemical biosensor that would allow for simple and rapid analysis of nucleic acids in combination with nuclease activity on nucleic acids and electroactive bionanoparticles. The detection of single-nucleotide polymorphisms (SNPs) using PNA probes takes advantage of the significant structural and physicochemical differences between the full hybrids and SNPs in PNA/DNA and DNA/DNA duplexes. Ferrocene-conjugated chitosan nanoparticles (Chi-Fc) were used as the electroactive indicator of hybridization. Chi-Fc had no affinity towards the neutral PNA probe immobilized on a gold electrode (AuE) surface. When the PNA probe on the electrode surface hybridized with a full-complementary target DNA, Chi-Fc electrostatically attached to the negatively-charged phosphate backbone of DNA on the surface and gave rise to a high electrochemical oxidation signal from ferrocene at ∼0.30 V. Exposing the surface to a single-stranded DNA specific nuclease, Nuclease S1, was found to be very effective for removing the nonspecifically adsorbed SNP DNA. An SNP in the target DNA to PNA made it susceptible to the enzymatic digestion. After the enzymatic digestion and subsequent exposure to Chi-Fc, the presence of SNPs was determined by monitoring the changes in the electrical current response of Chi-Fc. The method provided a detection limit of 1 fM (S/N = 3) for the target DNA oligonucleotide. Additionally, asymmetric PCR was employed to detect the presence of genetically modified organism (GMO) in standard Roundup Ready soybean samples. PNA-mediated PCR amplification of real DNA samples was performed to detect SNPs related to alcolohol dehydrogenase (ALDH). Chitosan nanoparticles are promising biometarials for various analytical and pharmaceutical applications. Figure The electrochemical method for SNP detection using PNA probes and chitosan nanoparticles takes advantage of the significant structural and physicochemical differences between PNA/DNA and DNA/DNA duplexes. Single-stranded DNA specific enzymes selectively choose these SNP sites and hydrolyze the DNA molecules on gold electrode (AuE) surface. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.     

Paramagnetic Particles and PNA Probe for Automated Separation and Electrochemical Detection of Influenza

Considerable efforts have been devoted to the development of rapid and sensitive methods allowing the detection of viral nucleic acid. We herein describe an assay for identification of a specific influenza sequence. The suggested method was based on isolation using paramagnetic particles coupled with electrochemical detection of isolated product. Peptide nucleic acid (PNA) was used as a probe for hybridization and identification of the influenza-derived specific sequence. The use of PNA can show numerous benefits: PNA probe is not degradable by enzymes and the duplex of PNA with RNA/DNA is more thermostable and more resistant to pH changes than DNA/DNA or RNA/RNA duplexes. This PNA probe assay can be applied as a magnetically guidable tool for detection of DNA/RNA samples under different conditions.     

Paramagnetic Particles and PNA Probe for Automated Separation and Electrochemical Detection of Influenza

Considerable efforts have been devoted to the development of rapid and sensitive methods allowing the detection of viral nucleic acid. We herein describe an assay for identification of a specific influenza sequence. The suggested method was based on isolation using paramagnetic particles coupled with electrochemical detection of isolated product. Peptide nucleic acid (PNA) was used as a probe for hybridization and identification of the influenza-derived specific sequence. The use of PNA can show numerous benefits: PNA probe is not degradable by enzymes and the duplex of PNA with RNA/DNA is more thermostable and more resistant to pH changes than DNA/DNA or RNA/RNA duplexes. This PNA probe assay can be applied as a magnetically guidable tool for detection of DNA/RNA samples under different conditions.

     

(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.     

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.     

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.     

In situ scanning tunneling microscopy of DNA-modified gold surfaces: bias and mismatch dependence

In situ scanning tunneling microscopy has been performed on DNA-modified gold surfaces under physiological conditions. The STM images of DNA-modified gold surfaces are strongly dependent on the applied potential and percentage of DNA duplexes containing a single base mismatch. At negative surface potentials we observe reproducible features that are attributed to DNA agglomerates where the DNA duplexes are in the upright orientation; at positive potentials, when DNA molecules lie down on the surface, the film is transparent, and only the gold surface is distinguishable. These observations indicate that DNA possesses a non-negligible local density of states which can be probed when the DNA duplex is in the upright orientation. By varying the percentage of DNA duplexes containing a single base mismatch, we have observed a dramatic change in the image contrast as a result of the perturbation induced by the mismatch on the electronic pathway inside the DNA. These results emphasize the central role of the integrity of the pi-stack for DNA charge transport. Duplex DNA is a promising candidate in molecular electronics, but only in arrangements where the orbitals can efficiently overlap with the electronic states of the electrodes and the environment does not constrain the DNA in non-native, poorly stacked conformations.     

Effect of secondary structure on the thermodynamics and kinetics of PNA hybridization to DNA hairpins.

The binding of a series of PNA and DNA probes to a group of unusually stable DNA hairpins of the tetraloop motif has been observed using absorbance hypochromicity (ABS), circular dichroism (CD), and a colorimetric assay for PNA/DNA duplex detection. These results indicate that both stable PNA-DNA and DNA-DNA duplexes can be formed with these target hairpins, even when the melting temperatures for the resulting duplexes are up to 50 degrees C lower than that of the hairpin target. Both hairpin/single-stranded and hairpin/hairpin interactions are considered in the scope of these studies. Secondary structures in both target and probe molecules are shown to depress the melting temperatures and free energies of the probe-target duplexes. Kinetic analysis of hybridization yields reaction rates that are up to 160-fold slower than hybridization between two unstructured strands. The thermodynamic and kinetic obstacles to hybridization imposed by both target and probe secondary structure are significant concerns for the continued development of antisense agents and especially diagnostic probes.     

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.     

Mechanistic studies of Fc-PNA(⋅DNA) surface dynamics based on the kinetics of electron-transfer processes

N‐Terminally ferrocenylated and C‐terminally gold‐surface‐grafted peptide nucleic acid (PNA) strands were exploited as unique tools for the electrochemical investigation of the strand dynamics of short PNA(?DNA) duplexes. On the basis of the quantitative analysis of the kinetics and the diffusional characteristics of the electron‐transfer process, a nanoscopic view of the Fc‐PNA(?DNA) surface dynamics was obtained. Loosely packed, surface‐confined Fc‐PNA single strands were found to render the charge‐transfer process of the tethered Fc moiety diffusion‐limited, whereas surfaces modified with Fc‐PNA?DNA duplexes exhibited a charge‐transfer process with characteristics between the two extremes of diffusion and surface limitation. The interplay between the inherent strand elasticity and effects exerted by the electric field are supposed to dictate the probability of a sufficient approach of the Fc head group to the electrode surface, as reflected in the measured values of the electron‐transfer rate constant, k⁰. An in‐depth understanding of the dynamics of surface‐bound PNA and PNA?DNA strands is of utmost importance for the development of DNA biosensors using (Fc‐)PNA recognition layers.     

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.     

Nucleic acid sensor for M. tuberculosis detection based on surface plasmon resonance

Cysteine modified NH(2)-end peptide nucleic acid (PNA) (24-mer) probe and 5'-thiol end labeled deoxyribonucleic acid (DNA) probes specific to Mycobacterium tuberculosis have been immobilized onto BK-7 gold coated glass plates for the detection of complementary, one-base mismatch, non-complementary targets and complementary target sequence in genomic DNA of Mycobacterium tuberculosis using a surface plasmon resonance (SPR) technique. The DNA/Au and PNA/Au bio-electrodes have been characterized using contact angle, atomic force microscopy (AFM), electrochemical impedance spectroscopy (EIS) and cyclic voltammetric (CV) techniques, respectively. It is revealed that there is a 252 millidegrees SPR angle change in the case of PNA immobilization and 205 millidegrees for DNA immobilization, indicating increased amount of immobilized PNA molecules. Hybridization studies reveal that there is no binding of the non-complementary target to DNA/Au and PNA/Au electrode. Compared to the DNA/Au bioelectrode, PNA/Au electrode has been found to be more efficient for detection of one-base mismatch sequence. The PNA/Au bioelectrode shows better detection limit (1.0 ng ml(-1)) over the DNA-Au bioelectrode (3.0 ng ml(-1)). The values of the association (k(a)) and dissociation rate constant (k(d)) for the complementary sequence in case of the PNA/Au bioelectrode have been estimated as 8.5 x 10(4) m(-1) s(-1) and 3.6 x 10(-3) s(-1), respectively.     

Highly Stable Duplex Formation by Artificial Nucleic Acids Acyclic Threoninol Nucleic Acid (aTNA) and Serinol Nucleic Acid (SNA) with Acyclic Scaffolds

The stabilities of duplexes formed by strands of novel artificial nucleic acids composed of acyclic threoninol nucleic acid (aTNA) and serinol nucleic acid (SNA) building blocks were compared with duplexes formed by the acyclic glycol nucleic acid (GNA), peptide nucleic acid (PNA), and native DNA and RNA. All acyclic nucleic acid homoduplexes examined in this study had significantly higher thermal stability than DNA and RNA duplexes. Melting temperatures of homoduplexes were in the order of aTNA>PNA≈GNA≥SNA?RNA>DNA. Thermodynamic analyses revealed that high stabilities of duplexes formed by aTNA and SNA were due to large enthalpy changes upon formation of duplexes compared with DNA and RNA duplexes. The higher stability of the aTNA homoduplex than the SNA duplex was attributed to the less flexible backbone due to the methyl group of D ‐threoninol on aTNA, which induced clockwise winding. Unlike aTNA, the more flexible SNA was able to cross‐hybridize with RNA and DNA. Similarly, the SNA/PNA heteroduplex was more stable than the aTNA/PNA duplex. A 15‐mer SNA/RNA was more stable than an RNA/DNA duplex of the same sequence.     

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.     

Effect of PNA backbone modifications on cyanine dye binding to PNA-DNA duplexes investigated by optical spectroscopy and molecular dynamics simulations

Optical spectroscopy and molecular dynamics simulations have been used to study the interaction between a cationic cyanine dye and peptide nucleic acid (PNA)-DNA duplexes. This recognition event is important because it leads to a visible color change, signaling successful hybridization of PNA with a complementary DNA strand. We previously proposed that the dye recognized the minor groove of the duplex, using it as a template for the assembly of a helical aggregate. Consistent with this, we now report that addition of isobutyl groups to the PNA backbone hinders aggregation of the dye when the substituents project into the minor groove but have a weaker effect if directed out of the groove. UV-Visible and circular dichroic spectroscopy were used to compare aggregation on the different PNA-DNA duplexes, while molecular dynamics simulations were used to confirm that the substituents block the minor groove to varying degrees, depending on the configuration of the starting amino acid. In addition to a simple steric blockage effect of the substituent, the simulations suggest that directing the isobutyl group into the minor groove causes the groove to narrow and the duplex to become more rigid, structural perturbations that are relevant to the growing interest in backbone-modified PNA for applications in the biological and materials sciences.     

A new peptide nucleotide acid biosensor for electrochemical detection of single nucleotide polymorphism in duplex DNA via triplex structure formation

In this paper, we report a new PNA biosensor for electrochemical detection of point mutation or single nucleotide polymorphism (SNP) in p53 gene corresponding oligonucleotide based on PNA/ds-DNA triplex formation following hybridization of PNA probe with double-stranded DNA (ds-DNA) sample without denaturing the ds-DNA into single-stranded DNA (ss-DNA). As p53 gene is mutated in many human tumors, this research is useful for cancer therapy and genomic study. In this approach, methylene blue (MB) is used for electrochemical signal generation and the interaction between MB and oligonucleotides is studied by differential pulse voltammety (DPV). Probe-modified electrode is prepared by self-assembled monolayer (SAM) formation of thiolated PNA molecules on the surface of Au electrode. A significant increase in the reduction signal of MB following hybridization of the probe with the complementary double-stranded oligonucleotide (ds-oligonucleotide) confirms the function of the biosensor. The selectivity of the PNA sensor is investigated by non-complementary ds-oligonucleotides and the results support the ability of the sensor to detect single-base mismatch directly on ds-oligonucleotide. The influence of probe and ds-DNA concentrations on the effective discrimination against complementary sequence and point mutation is studied and the concentration of 10^?6 M is selected as appropriate concentration. Diagnostic performance of the biosensor is described and the detection limit is found to be 4.15 × 10^?12 M.     

Fluorescence and visual detection of single nucleotide polymorphism using cationic conjugated polyelectrolyte

We report a simple assay for visual detection of single nucleotide polymorphisms (SNPs) with good sensitivity and selectivity. The selectivity is determined by Escherichia coli (E. coli) DNA ligase mediated circular formation upon recognition of the point mutation on DNA targets. Rolling cycle amplification (RCA) of the perfect-matched DNA target is then initiated using the in situ formed circular template in the presence of Phi29 enzyme. Due to amplification of the DNA target, the RCA product has a tandem-repeated sequence, which is significantly longer than that for the SNP strand. Direct addition of a cationic conjugated polymer of poly[9,9'-bis(6'-(N,N,N-trimethylammonium)hexyl)fluorene-co-9,9'-bis(2-(2-(2-(N,N,N-trimethylammonium)ethoxyl)-ethoxy)-ethyl)fluorene tetrabromide] containing 20 mol% 2,1,3-benzothiadiazole (PFBT(20)) into the RCA solution leads to blue-whitish fluorescent color for SNP strand and yellowish fluorescent color for amplified DNA, due to PFBT(20)/DNA complexation induced intrachain/interchain energy transfer. To further improve the contrast for visual detection, FAM-labeled peptide nucleic acid (PNA) was hybridized to each amplified sequence, which is followed by the addition of poly{2,7-[9,9-bis(6'-N,N,N-trimethylammoniumhexyl)]fluorene-co-2,5-difluoro-1,4-phenylene dibromide} (PFP). The PNA/DNA hybridization brings PFP and FAM-PNA into close proximity for energy transfer, and the solution fluorescent color appears green in the presence of target DNA with a detection limit of 1 nM, which is significantly improved as compared to that for most reported visual SNP assay.     

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.