Peptide Nucleic Acid Hybridization Research Articles

Development of supramolecular anticoagulants with on-demand reversibility.

Drugs are administered at a dosing schedule set by their therapeutic index, and termination of action is achieved by clearance and metabolism of the drug. In some cases, such as anticoagulant drugs or immunotherapeutics, it is important to be able to quickly reverse the drug's action. Here, we report a general strategy to achieve on-demand reversibility by designing a supramolecular drug (a noncovalent assembly of two cooperatively interacting drug fragments held together by transient hybridization of peptide nucleic acid (PNA)) that can be reversed with a PNA antidote that outcompetes the hybridization between the fragments. We demonstrate the approach with thrombin-inhibiting anticoagulants, creating very potent and reversible bivalent direct thrombin inhibitors (Ki = 74 pM). The supramolecular inhibitor effectively inhibited thrombus formation in mice in a needle injury thrombosis model, and this activity could be reversed by administration of the PNA antidote. This design is applicable to therapeutic targets where two binding sites can be identified.

Electrochemical sensor based on Fe3O4/α-Fe2O3@Au magnetic nanocomposites for sensitive determination of the TP53 gene

Considering the high cost and tedious process of gene sequencing, there is an urgent need to develop portable and efficient sensors for the TP53 gene. Here, we developed a novel electrochemical sensor that detected the TP53 gene using magnetic peptide nucleic acid (PNA)-modified Fe3O4/α-Fe2O3@Au nanocomposites. Cyclic voltammetry and electrochemical impedance spectroscopy confirmed the successful stepwise construction of the sensor, especially the high-affinity binding of PNA to DNA strands, which induced different electron transfer rates and resulted in current changes. Variations in the differential pulse voltammetry current observed during hybridization at different surface PNA probe densities, hybridization times, and hybridization temperatures were explored. The biosensing strategy obtained a limit of detection of 0.26 pM, a limit of quantification of 0.85 pM, and a wide linear range (1 pM–1 μM), confirming that the Fe3O4/α-Fe2O3@Au nanocomposites and the strategy based on magnetic separation and magnetically induced self-assembly improved the binding efficiency of nucleic acid molecules. The biosensor was a label-free and enzyme-free device with excellent reproducibility and stability that could identify single-base mismatched DNA without additional DNA amplification procedures, and the serum spiked experiments revealed the feasibility of the detection approach.

Using Peptide Nucleic Acid Hybridization Probes for Qualitative and Quantitative Analysis of Nucleic Acid Therapeutics by Capillary Electrophoresis.

The space of advanced therapeutic modalities is currently evolving in rapid pace necessitating continuous improvement of analytical quality control methods. In order to evaluate the identity of nucleic acid species in gene therapy products, we propose a capillary electrophoresis-based gel free hybridization assay in which fluorescently labeled peptide nucleic acids (PNAs) are applied as affinity probes. PNAs are engineered organic polymers that share the base pairing properties with DNA and RNA but have an uncharged peptide backbone. In the present study, we conduct various proof-of-concept studies to identify the potential of PNA probes for advanced analytical characterization of novel therapeutic modalities like oligonucleotides, plasmids, mRNA, and DNA released by recombinant adeno-associated virus. For single-stranded nucleic acids up to 1000 nucleotides, the method is an excellent choice that proved to be highly specific by detecting DNA traces in complex samples, while having a limit of quantification in the picomolar range when multiple probes are used. For double-stranded samples, only fragments that are similar in size to the probe could be quantified. This limitation can be circumvented when target DNA is digested and multiple probes are used opening an alternative to quantitative PCR.

Two-Helix Supramolecular Proteomimetic Binders Assembled via PNA-Assisted Disulfide Crosslinking.

Peptidic motifs folded in a defined conformation are able to inhibit protein-protein interactions (PPIs) covering large interfaces and as such they are biomedical molecules of interest. Mimicry of such natural structures with synthetically tractable constructs often requires complex scaffolding and extensive optimization to preserve the fidelity of binding to the target. Here, we present a novel proteomimetic strategy based on a 2-helix binding motif that is brought together by hybridization of peptide nucleic acids (PNA) and stabilized by a rationally positioned intermolecular disulfide crosslink. Using a solid phase synthesis approach (SPPS), the building blocks are easily accessible and such supramolecular peptide-PNA helical hybrids could be further coiled using precise templated chemistry. The elaboration of the structural design afforded high affinity SARS CoV-2 RBD (receptor binding domain) binders without interference with the underlying peptide sequence, creating a basis for a new architecture of supramolecular proteomimetics.

A Single-Molecule Insight into the Ionic Strength-dependent, Cationic Peptide Nucleic Acids-Oligonucleotides Interactions.

To alleviate solubility-related shortcomings associated with the use of neutral peptide nucleic acids (PNA), a powerful strategy is incorporate various charged sidechains onto the PNA structure. Here we employ a single-molecule technique and prove that the ionic current blockade signature of free poly(Arg)-PNAs and their corresponding duplexes with target ssDNAs interacting with a single α-hemolysin (α-HL) nanopore is highly ionic strength dependent, with high salt-containing electrolytes facilitating both capture and isolation of such complexes. Our data illustrate the effect of low ionic strength in reducing the effective volume of free poly(Arg)-PNAs and augmentation of their electrophoretic mobility while traversing the nanopore. We found that unlike in high salt electrolytes, the specific hybridization of cationic moiety-containing PNAs with complementary negatively charged ssDNAs in a salt concentration as low as 0.5 M is dramatically impeded. We suggest a scenario in which reduced charge screening by counterions in low salt electrolytes enables non-specific, electrostatic interactions with the anionic backbone of polynucleotides, thus reducing the ability of PNA-DNA complementary association via hydrogen bonding patterns. We applied an experimental strategy with spatially-separated poly(Arg)-PNAs and ssDNAs, and present evidence at the single-molecule level suggestive of the real-time, long-range interactions-driven formation of poly(Arg)-PNA-DNA complexes, as individual strands entering the nanopore from opposite directions collide inside a nanocavity.

Site-specific conjugation of recognition tags to trastuzumab for peptide nucleic acid-mediated radionuclide HER2 pretargeting

Pretargeting is a promising strategy to reach high imaging contrast in a shorter time than by targeting with directly radiolabeled monoclonal antibodies (mAbs). One of problems in pretargeting is a site-specific, reproducible and uniform conjugation of recognition tags to mAbs. To solve this issue we propose a photoconjugation to covalently couple a recognition tag to a mAb via a photoactivatable Z domain. The Z-domain, a 58-amino acid protein derived from the IgG-binding B-domain of Staphylococcus aureus protein A, has a well-characterized binding site in the Fc portion of IgG. We tested the feasibility of this approach using pretargeting based on hybridization between peptide nucleic acids (PNAs). We have used photoconjugation to couple trastuzumab with the PNA-based hybridization probe, HP1. A complementary [57Co]Co-labeled PNA hybridization probe ([57Co]Co-HP2) was used as the secondary targeting probe. In vitro studies demonstrated that trastuzumab-ZHP1 bound specifically to human epidermal growth factor receptor 2 (HER2)-expressing cells with nanomolar affinity. The binding of the secondary [57Co]Co-HP2 probe to trastuzumab-PNA-pretreated cells was in the picomolar affinity range. A two-fold increase in SKOV-3 tumor targeting was achieved when [57Co]Co-HP2 (0.7 nmol) was injected 48 h after injection of trastuzumab-ZHP1 (0.5 nmol) compared with trastuzumab-ZHP1 alone (0.8 ± 0.2 vs. 0.33 ± 0.06 %ID/g). Tumor accumulation of [57Co]Co-HP2 was significantly reduced by pre-saturation with trastuzumab or when no trastuzumab-ZHP1 was preinjected. A tumor-to-blood uptake ratio of 1.5 ± 0.3 was achieved resulting in a clear visualization of HER2-expressing xenografts as confirmed by SPECT imaging. In conclusion, the feasibility of stable site-specific coupling of a PNA-based recognition tag to trastuzumab and successful pretargeting has been demonstrated. This approach can hopefully be used for a broad range of mAbs and recognition tags.

Direct Quantitative Analysis of Multiple microRNAs (DQAMmiR) with Peptide Nucleic Acid Hybridization Probes.

Direct quantitative analysis of multiple miRNAs (DQAMmiR) is a hybridization-based assay, in which the excess of the DNA hybridization probes is separated from the miRNA-probe hybrids, and the hybrids are separated from each other in gel-free capillary electrophoresis (CE) using two types of mobility shifters: single-strand DNA binding protein (SSB) added to the CE running buffer and peptide drag tags conjugated with the probes. Here we introduce the second-generation DQAMmiR, which utilizes peptide nucleic acid (PNA) rather than DNA hybridization probes and requires no SSB in the CE running buffer. PNA probes are electrically neutral, while PNA-miRNA hybrids are negatively charged, and this difference in charge can be a basis for separation of the hybrids from the probes. In this proof-of-principle work, we first experimentally confirmed that the PNA-RNA hybrid was separable from the excess of the PNA probe without SSB in the running buffer, resulting in a near 10 min time window, which would allow, theoretically, separation of up to 30 hybrids. Then, we adapted to PNA-RNA hybrids our previously developed theoretical model for predicting hybrid mobilities. The calculation performed with the modified theoretical model indicated that PNA-RNA hybrids of slightly different lengths could be separated from each other without drag tags. Accordingly, we designed a simple experimental model capable of confirming: (i) separation of tag-free hybrids of different lengths and (ii) separation of same-length hybrids due to a drag tag on the PNA probe. The experimental model included three miRNAs: 20-nt miR-147a, 20-nt miR-378g, and 22-nt miR-21. The three complementary PNA probes had lengths matching those of the corresponding target miRNAs. The probe for miR-147a had a short five-amino-acid drag tag; the other two had no drag tags. We were able to achieve baseline separation of the three hybrids from each other. The LOQ of 14 pM along with the high accuracy (recovery >90%) and precision (RSD ≈ 10%) of the assay at picomolar target concentrations suggest that PNA-facilitated DQAMmiR could potentially support practical miRNA analysis of clinical samples.

Clickable PNA Probes for Imaging Human Telomeres and Poly(A) RNAs.

The ability to bind strongly to complementary nucleic acid sequences, invade complex nucleic acid structures, and resist degradation by cellular enzymes has made peptide nucleic acid (PNA) oligomers as very useful hybridization probes in molecular diagnosis. For such applications, the PNA oligomers have to be labeled with appropriate reporters as they lack intrinsic labels that can be used in biophysical assays. Although solid-phase synthesis is commonly used to attach reporters onto PNA, development of milder and modular labeling methods will provide access to PNA oligomers labeled with a wider range of biophysical tags. Here, we describe the establishment of a postsynthetic modification strategy based on bioorthogonal chemical reactions in functionalizing PNA oligomers in solution with a variety of tags. A toolbox composed of alkyne- and azide-modified monomers were site-specifically incorporated into PNA oligomers and postsynthetically click-functionalized with various tags, ranging from sugar, amino acid, biotin, to fluorophores, by using copper(I)-catalyzed azide–alkyne cycloaddition, strain-promoted azide–alkyne cycloaddition, and Staudinger ligation reactions. As a proof of utility of this method, fluorescent PNA hybridization probes were developed and used in imaging human telomeres in chromosomes and poly(A) RNAs in cells. Taken together, this simple approach of generating a wide range of functional PNA oligomers will expand the use of PNA in molecular diagnosis.

Radionuclide Therapy of HER2-Expressing Human Xenografts Using Affibody-Based Peptide Nucleic Acid–Mediated Pretargeting: In Vivo Proof of Principle

Affibody molecules are small proteins engineered using a nonantibody scaffold. Radiolabeled Affibody molecules are excellent imaging probes, but their application to radionuclide therapy has been prevented by high renal reabsorption. The aim of this study was to test the hypothesis that Affibody-based peptide nucleic acid (PNA)-mediated pretargeted therapy of human epidermal growth factor receptor 2 (HER2)-expressing cancer extends survival without accompanying renal toxicity. Methods: A HER2-targeting Affibody molecule ligated with an AGTCGTGATGTAGTC PNA hybridization probe (ZHER2:342-SR-HP1) was used as the primary pretargeting agent. A complementary AGTCGTGATGTAGTC PNA conjugated to the chelator DOTA and labeled with the radionuclide 177Lu (177Lu-HP2) was used as the secondary agent. The influence of different factors on pretargeting was investigated. Experimental radionuclide therapy in mice bearing SKOV-3 xenografts was performed in 6 cycles separated by 7 d. Results: Optimal tumor targeting was achieved when 16 MBq/3.5 μg (0.65 nmol) of 177Lu-HP2 was injected 16 h after injection of 100 μg (7.7 nmol) of ZHER2:342-SR-HP1. The calculated absorbed dose to tumors was 1,075 mGy/MBq, whereas the absorbed dose to kidneys was 206 mGy/MBq and the absorbed dose to blood (surrogate of bone marrow) was 4 mGy/MBq. Survival of mice was significantly longer (P < 0.05) in the treatment group (66 d) than in the control groups treated with the same amount of ZHER2:342-SR-HP1 only (37 d), the same amount and activity of 177Lu-HP2 only (32 d), or phosphate-buffered saline (37 d). Conclusion: The studied pretargeting system can deliver an absorbed dose to tumors appreciably exceeding absorbed doses to critical organs, making Affibody-based PNA-mediated pretargeted radionuclide therapy highly attractive.

A microfluidic streaming potential analyzer for label-free DNA detection

In this work, a streaming potential analyzer was constructed based on a composite PDMS-on-glass microchannel and was applied to the label-free DNA detection. Peptide nucleic acid (PNA) probes were covalently attached to the glass substrate to facilitate hybridization with the target DNA at low ionic strength and provide minimized baseline signal for streaming potential analysis. The hybridization of the neutral PNA in the microchannel with the target DNA produced a negatively charged complex, which caused the change of zeta potential and in turn the measured streaming potential. The change of zeta potential was dependent on the hybridization time as well as the concentration and length of the target DNA, through which the complementary target DNA was distinguished from non-complementary DNA with a detection limit of 10 nM. The PNA probes could be recovered in situ and reused without significant capacity loss. The change of the zeta potential was decreased by only 4% after three cycles of PNA-DNA hybridization and PNA recovery. The microfluidic streaming potential analyzer functionalized with PNA probes provides a simple, inexpensive and reusable platform for nucleic acid detection.

Pharmacokinetic Profiling of Conjugated Therapeutic Oligonucleotides: A High-Throughput Method Based Upon Serial Blood Microsampling Coupled to Peptide Nucleic Acid Hybridization Assay.

Therapeutic oligonucleotides, such as small interfering RNAs (siRNAs), hold great promise for the treatment of incurable genetically defined disorders by targeting cognate toxic gene products for degradation. To achieve meaningful tissue distribution and efficacy in vivo, siRNAs must be conjugated or formulated. Clear understanding of the pharmacokinetic (PK)/pharmacodynamic behavior of these compounds is necessary to optimize and characterize the performance of therapeutic oligonucleotides in vivo. In this study, we describe a simple and reproducible methodology for the evaluation of in vivo blood/plasma PK profiles and tissue distribution of oligonucleotides. The method is based on serial blood microsampling from the saphenous vein, coupled to peptide nucleic acid hybridization assay for quantification of guide strands. Performed with minimal number of animals, this method allowed unequivocal detection and sensitive quantification without the need for amplification, or further modification of the oligonucleotides. Using this methodology, we compared plasma clearances and tissue distribution profiles of two different hydrophobically modified siRNAs (hsiRNAs). Notably, cholesterol-hsiRNA presented slow plasma clearances and mainly accumulated in the liver, whereas, phosphocholine-docosahexaenoic acid-hsiRNA was rapidly cleared from the plasma and preferably accumulated in the kidney. These data suggest that the PK/biodistribution profiles of modified hsiRNAs are determined by the chemical nature of the conjugate. Importantly, the method described in this study constitutes a simple platform to conduct pilot assessments of the basic clearance and tissue distribution profiles, which can be broadly applied for evaluation of new chemical variants of siRNAs and micro-RNAs.

Role of Cell-Penetrating Peptides in Intracellular Delivery of Peptide Nucleic Acids Targeting Hepadnaviral Replication.

Peptide nucleic acids (PNAs) are potentially attractive antisense agents against hepatitis B virus (HBV), although poor cellular uptake limits their therapeutic application. In the duck HBV (DHBV) model, we evaluated different cell-penetrating peptides (CPPs) for delivery to hepatocytes of a PNA-targeting hepadnaviral encapsidation signal (ε). This anti-ε PNA exhibited sequence-specific inhibition of DHBV RT in a cell-free system. Investigation of the best in vivo route of delivery of PNA conjugated to (D-Arg)8 (P1) showed that intraperitoneal injection to ducklings was ineffective, whereas intravenously (i.v.) injected fluorescein-P1-PNA reached the hepatocytes. Treatment of virus carriers with i.v.-administered P1-PNA resulted in a decrease in viral DNA compared to untreated controls. Surprisingly, a similar inhibition of viral replication was observed in vivo as well as in vitro in primary hepatocyte cultures for a control 2 nt mismatched PNA conjugated to P1. By contrast, the same PNA coupled to (D-Lys)4 (P2) inhibited DHBV replication in a sequence-specific manner. Interestingly, only P1, but not P2, displayed anti-DHBV activity in the absence of PNA cargo. Hence, we provide new evidence that CPP-PNA conjugates inhibit DHBV replication following low-dose administration. Importantly, our results demonstrate the key role of CPPs used as vehicles in antiviral specificity of CPP-PNA conjugates.

Fischer carbene mediated covalent grafting of a peptide nucleic acid on gold surfaces and IR optical detection of DNA hybridization with a transition metalcarbonyl label

Amine-reactive surfaces comprising N-hydroxysuccinimide ester groups as well as much more unusual Fischer alkoxymetallocarbene groups were generated on gold-coated surfaces via self-assembled monolayers of carboxy- and azido-terminated thiolates, respectively. These functions were further used to immobilize homothymine peptide nucleic acid (PNA) decamer in a covalent fashion involving the primary amine located at its N-terminus. These stepwise processes were monitored by polarization modulation reflection – absorption infrared spectroscopy (PM-RAIRS) that gave useful information on the molecular composition of the organic layers. PNA grafting and hybridization with complementary DNA strand were successfully transduced by quartz crystal microbalance (QCM) measurements. Unfortunately, attempts to transduce the hybridization optically by IR in a label-free fashion were inconclusive. Therefore we undertook to introduce an IR reporter group, namely a transition metalcarbonyl (TMC) entity at the 5′ terminus of complementary DNA. Evidence for the formation of PNA-DNA heteroduplex was brought by the presence of ν(CO) bands in the 2000cm−1 region of the IR spectrum of the gold surface owing to the metalcarbonyl label.

Rapid Colorimetric Detection of the Fungal Phytopathogen Synchytrium endobioticum Using Cyanine dye-Indicated PNA Hybridization

Synchytrium endobioticum is a parasitic fungus that is considered to be the most important potato pathogen worldwide. Spread of the fungus can result in catastrophic impacts to agriculture and economy, as it is very infective and can survive in spore form for over 70 years. To date, the gold standard for S. endobioticum identification relies on visual classification by highly trained personnel. This paper presents the colorimetric detection of S. endobioticum from potato wart samples using a peptide nucleic acid (PNA) probe and a cyanine dye. A segment of the 18S ribosomal RNA gene was sequenced for several S. endobioticum pathotypes and used to design a species-specific PNA probe. The probe was used in a rapid colorimetric hybridization assay that detected RNA from infected wart tissue down to 10−17 mol within 15 min. This technique demonstrates potential for rapid identification of potato wart in non-laboratory settings with minimally trained staff.

A Review of Peptide Nucleic Acid

Peptide Nucleic Acid (PNA) is a nucleobase oligomer in which the whole backbone is mainly replaced by N-(2- aminoethyl) glycine units. PNA is considered as DNA with a neutral peptide backbone due to negative charged sugar–phosphate backbone. It is chemically stable and resistant to hydrolytic cleavage. PNA can be categorized specific sequences of DNA and RNA according to Watson–Crick hydrogen bonding structure. Hybridization process showed high thermal stability and unique ionic strength effects. It is formed a stable PNA/DNA/PNA triplex with a looped-out DNA strand. PNA hybridization technology is promptly developed within in situ hybridization. In our review paper was elaborated the PNA superior hybridization characteristics, importance’s of PNA and major applications of PNA in the diagnostic and pharmaceutical fields. And also PNA could be replaced DNA in uses as a probe for many investigation purposes. PNAs antisense activities have found in nerve cells and even in rats upon injection into the brain, and in Escherichia coli.

Abstract B55: Hypoxanthine wobble base multimutant KRAS2 mRNA PET imaging agent in G12D mice

Abstract Activating mutations in KRAS2 oncogene lead to constitutive K-Ras-GTP signals regardless of EGFR signaling, enabling resistance to EGFR-targeted therapies. Some cancers are activated by mutation of the KRAS2 12th codon from the wild type GGU that encodes glycine (G) to a mutant GAU that encodes aspartate (D) or a mutant GUU that encodes valine (V). For this reason, we hypothesize that determining multiple KRAS2 mutations in vivo by external mRNA PET imaging will enable physicians to decide on alternatives to EGFR-directed therapy. We previously pioneered tumor mRNA PET imaging in pancreatic cancer and breast cancer xenografts [Paudyal, et al. (2013) Nuclear Medicine and Biology 40(8):994-9] with peptide nucleic acid (PNA) 12-mers coupled to receptor-targeting peptides and imaging reporter moieties, showing single mismatch specificity. Thus, we designed and synthesized a 12-mer PNA hybridization agent with a hypoxanthine wobble base opposite the middle base of the KRAS2 mRNA 12th codon. At the C-terminus, we included a cyclized tetrapeptide based on insulin-like growth factor 1 (IGF1) to enable IGF1R-mediated cellular uptake. We included an N-terminal N2S2 chelator in order to bind 64Cu. Molecular dynamics and circular dichroism analysis of the wobble base PNA H-bonded to KRAS2 RNA showed preference for mutant A or U over wild type G [Sanders, et al. (2013) Journal of Physical Chemistry B 117(39):11584-95]. We then administered 200 μCi of the [64Cu]PNA wobble base agent, or the complementary G12D agent, or the wild type agent, by tail vein into 3-month-old G12D transgenic mice that developed spontaneous lung tumors. 4 hr and 24 hr later, mice were imaged in a Siemens Inveon microPET/CT scanner. The PET standard uptake value (SUV) with the G12D agent was 1.4, while SUV was 1.3 with the G12DVA wobble base agent, vs. 0.04 with the G12 wild type sequence. The wobble base approach enables detection of three different KRAS2 mutations, G12D, G12V, or G12A, with a single agent. mRNA PET imaging also enables external monitoring of therapeutic efficacy. Supported by NIH CA148565; IP owned by EW/MLT, licensed to MTTI. Citation Format: Eric Wickstrom, Chang-Po Chen, Matthew E. Wampole, Kaijun Zhang, Bishnuhari Paudyal, Antican Wang, Sushil Tripathi, Pardeep Kumar, Edith P. Mitchell, Bo Lu, Mathew L. Thakur, Brian D. Gray, Jeffrey A. Mattis. Hypoxanthine wobble base multimutant KRAS2 mRNA PET imaging agent in G12D mice. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B55. doi: 10.1158/1557-3125.RASONC14-B55

Human leukocyte antigen haplotype phasing by allele-specific enrichment with peptide nucleic acid probes.

Targeted capture of large fragments of genomic DNA that enrich for human leukocyte antigen (HLA) system haplotypes has utility in haematopoietic stem cell transplantation. Current methods of HLA matching are based on inference or familial studies of inheritance; and each approach has its own inherent limitations. We have designed and tested a probe–target-extraction method for capturing specific HLA haplotypes by hybridization of peptide nucleic acid (PNA) probes to alleles of the HLA-DRB1 gene. Short target fragments contained in plasmids were initially used to optimize the method followed by testing samples of genomic DNA from human subjects with preselected HLA haplotypes and obtained approximately 10% enrichment for the specific haplotype. When performed with high-molecular-weight genomic DNA, 99.0% versus 84.0% alignment match was obtained for the specific haplotype probed. The allele-specific target enrichment that we obtained can facilitate the elucidation of haplotypes between the 65 kb separating the HLA-DRB1 and the HLA-DQA1 genes, potentially spanning a total distance of at least 130 kb. Allele-specific target enrichment with PNA probes is a straightforward technique that has the capability to improve the resolution of DNA and whole genome sequencing technologies by allowing haplotyping of enriched DNA and crucially, retaining the DNA methylation profile.

Immobilization of Silver Nanoparticles by Peptide Nucleic Acids in Surface Plasmon Enhanced Dye-Sensitized Solar Cells

Light absorption of dye molecules can be increased by the localized surface plasmons of silver nanoparticles (AgNPs), which can be used to enhance the photocurrent generation of dye-sensitized solar cells (DSSCs). Simultaneously, however, reverse electron transfer from the TiO2 working electrode to the I3−/I− electrolyte is also increased. In this work, peptide nucleic acid (PNA) was successfully used to immobilize AgNPs at different distances from the TiO2 layer and dye molecules. Together with results from previous studies, results here reveal that the optimal distance is between the lengths of possibly bent 2-nm alkane-thiols and 3.2- to 4.2-nm PNA. AgNPs immobilized by hybridization of PNA of this length lead to an increase in energy conversion efficiency from 3.1% to 4.0%. Furthermore, PNA forms π-electron stacks upon hybridization, which can reduce reverse electron transfer. With double-stranded PNA, the internal quantum efficiency of DSSCs could be increased more than 10%, from 73% to 88%, with 5.8- to 6.8-nm long double-stranded PNA.

Detection and discrimination of recombinant plasmid encoding hepatitis C virus core/E1 gene based on PNA and double-stranded DNA hybridization

Development of an electrochemical DNA biosensor for direct detection and discrimination of double-stranded plasmid (ds-Pl) without the need for denaturation of the target plasmid sample using a peptide nucleic acid (PNA) oligomer as the probe is described. This goal was achieved by modification of gold electrode with 6-mercapto-1-hexanol following monolayer self-assembly of cysteine conjugated 20-mer PNA oligomer probe, complementary to the HCV core/E1 region, which binds to ds-Pl and forms PNA/ds-Pl structure. The significant variation in differential pulse voltammetric response of methylene blue on the probe modified electrode upon contacting with complementary double-strand plasmid to form PNA/ds-Pl triplex structure is the principle of target plasmid detection. The results indicated that the reduction peak current was linear with the concentration of complementary strand in the range of 10–300pg/μl with a detecion limit of 9.5pg/μl.

Label-free potentiometry for detecting DNA hybridization using peptide nucleic acid and DNA probes.

Peptide nucleic acid (PNA) has outstanding affinity over DNA for complementary nucleic acid sequences by forming a PNA-DNA heterodimer upon hybridization via Watson-Crick base-pairing. To verify whether PNA probes on an electrode surface enhance sensitivity for potentiometric DNA detection or not, we conducted a comparative study on the hybridization of PNA and DNA probes on the surface of a 10-channel gold electrodes microarray. Changes in the charge density as a result of hybridization at the solution/electrode interface on the self-assembled monolayer (SAM)-formed microelectrodes were directly transformed into potentiometric signals using a high input impedance electrometer. The charge readout allows label-free, reagent-less, and multi-parallel detection of target oligonucleotides without any optical assistance. The differences in the probe lengths between 15- to 22-mer dramatically influenced on the sensitivity of the PNA and DNA sensors. Molecular type of the capturing probe did not affect the degree of potential shift. Theoretical model for charged rod-like duplex using the Gouy-Chapman equation indicates the dominant effect of electrostatic attractive forces between anionic DNA and underlying electrode at the electrolyte/electrode interface in the potentiometry.