In vivo interference of pea aphid endosymbiont Buchnera groEL gene by synthetic peptide nucleic acids.

The unculturable nature of intracellular obligate symbionts presents a significant challenge for elucidating gene functionality, necessitating the development of gene manipulation techniques. One of the best-studied obligate symbioses is that between aphids and the bacterial endosymbiont Buchnera aphidicola. Given the extensive genome reduction observed in Buchnera, the remaining genes are crucial for understanding the host-symbiont relationship, but a lack of tools for manipulating gene function in the endosymbiont has significantly impeded the exploration of the molecular mechanisms underlying this mutualism. In this study, we introduced a novel gene manipulation technique employing synthetic single-stranded peptide nucleic acids (PNAs). We targeted the critical Buchnera groEL using specially designed antisense PNAs conjugated to an arginine-rich cell-penetrating peptide (CPP). Within 24 h of PNA administration via microinjection, we observed a significant reduction in groEL expression and Buchnera cell count. Notably, the interference of groEL led to profound morphological malformations in Buchnera, indicative of impaired cellular integrity. The gene knockdown technique developed in this study, involving the microinjection of CPP-conjugated antisense PNAs, provides a potent approach for in vivo gene manipulation of unculturable intracellular symbionts, offering valuable insights into their biology and interactions with hosts.

Investigating the Effectiveness of mqsR-Peptide Nucleic Acid as a Novel Solution for the Eradication of Persister Cells in Clinical Isolates of Escherichia coli.

BACKGROUND: Bacterial persisters are non- or slow-growing phenotypic variants that may be responsible for recalcitrance and relapse of persistent infections and antibiotic failure. In Escherichia coli, mqsRA is a well-known type II toxin-antitoxin system associated with persister cell formation. This study aimed to investigate the efficiency of an antisense peptide nucleic acid (PNA) targeting mqsRA in eliminating E. coli persisters. METHODS: The study included 600 non-duplicated urine samples from adult patients with suspected urinary tract infections. The isolates were subjected to antimicrobial susceptibility testing and bacterial persister cells assay. The presence of mqsRA in the isolates was evaluated by polymerase chain reaction. Finally, expression of the mqsR and mqsA genes was assessed after exposure to normal conditions, stress, and different concentrations of mqsR-PNA (1 - 35 µM). RESULTS: The mqsR gene was significantly overexpressed under stress conditions, which was compensated by the PNA treatment. Complete inhibition of E. coli persister cells was achieved after overnight treatment with the anti-mqsR-PNA at concentrations ≥ 15 µM. CONCLUSIONS: The growth of E. coli persister cells can be inhibited by the anti-mqsR-PNA. Further studies are required to evaluate the effectiveness of this antisense PNA in both preclinical and clinical settings.

Peptide Nucleic Acid Clamp-Assisted Photothermal Multiplexed Digital PCR for Identifying SARS-CoV-2 Variants of Concern.

The unprecedented demand for variants diagnosis in response to the COVID-19 epidemic has brought the spotlight onto rapid and accurate detection assays for single nucleotide polymorphisms (SNPs) at multiple locations. However, it is still challenging to ensure simplicity, affordability, and compatibility with multiplexing. Here, a novel technique is presented that combines peptide nucleic acid (PNA) clamps and near-infrared (NIR)-driven digital polymerase chain reaction (dPCR) to identify the Omicron and Delta variants. This is achieved by simultaneously identifying highly conserved mutated signatures at codons 19, 614, and 655 of the spike protein gene. By microfluidically introducing graphene-oxide-nanocomposite into the assembled gelatin microcarriers, they achieved a rapid temperature ramping-up rate and switchable gel-to-sol phase transformation synchronized with PCR activation under NIR irradiation. Two sets of duplex PCR reactions, each classifying respective PNA probes, are emulsified in parallel and illuminated together using a homemade vacuum-based droplet generation device and a programmable NIR control module. This allowed for selective amplification of mutant sequences due to single-base-pair mismatch with PNA blockers. Sequence-recognized bioreactions and fluorescent-color scoring enabled quick identification of variants. This technique achieved a detection limit of 5,100 copies and a 5-fold quantitative resolution, which is promising to unfold minor differences and dynamic changes.

Antitumor efficacy of a sequence-specific DNA-targeted γPNA-based c-Myc inhibitor.

Targeting oncogenes at the genomic DNA level can open new avenues for precision medicine. Significant efforts are ongoing to target oncogenes using RNA-targeted and protein-targeted platforms, but no progress has been made to target genomic DNA for cancer therapy. Here, we introduce a gamma peptide nucleic acid (γPNA)-based genomic DNA-targeted platform to silence oncogenes in vivo. γPNAs efficiently invade the mixed sequences of genomic DNA with high affinity and specificity. As a proof of concept, we establish that γPNA can inhibit c-Myc transcription in multiple cell lines. We evaluate the in vivo efficacy and safety of genomic DNA targeting in three pre-clinical models. We also establish that anti-transcription γPNA in combination with histone deacetylase inhibitors and chemotherapeutic drugs results in robust antitumor activity in cell-line- and patient-derived xenografts. Overall, this strategy offers a unique therapeutic platform to target genomic DNA to inhibit oncogenes for cancer therapy.

PNA-Pdx: Versatile Peptide Nucleic Acid-Based Detection of Nucleic Acids and SNPs.

Monitoring diseases caused by pathogens or by mutations in DNA sequences requires accurate, rapid, and sensitive tools to detect specific nucleic acid sequences. Here, we describe a new peptide nucleic acid (PNA)-based nucleic acid detection toolkit, termed PNA-powered diagnostics (PNA-Pdx). PNA-Pdx employs PNA probes that bind specifically to a target and are then detected in lateral flow assays. This can precisely detect a specific pathogen or genotype genomic sequence. PNA probes can also be designed to invade double-stranded DNAs (dsDNAs) to produce single-stranded DNAs for precise CRISPR-Cas12b-based detection of genomic SNPs without requiring the protospacer-adjacent motif (PAM), as Cas12b requires PAM sequences only for dsDNA targets. PNA-Pdx identified target nucleic acid sequences at concentrations as low as 2 copies/µL and precisely detected the SARS-CoV-2 genome in clinical samples in 40 min. Furthermore, the specific dsDNA invasion by the PNA coupled with CRISPR-Cas12b precisely detected genomic SNPs without PAM restriction. Overall, PNA-Pdx provides a novel toolkit for nucleic acid and SNP detection as well as highlights the benefits of engineering PNA probes for detecting nucleic acids.

Combined mutations of the penA with ftsX genes contribute to ceftriaxone resistance in Neisseria gonorrhoeae and peptide nucleic acids targeting these genes reverse ceftriaxone resistance.

OBJECTIVES: To investigate the gene mutations associated with ceftriaxone (CRO) resistance among gonococcal isolates, and to determine the effects of the mutated genes on CRO minimum inhibitory concentrations (MICs) with transformation assays and antisense peptide nucleic acids (asPNAs). METHODS: Ceftriaxone-resistant (CROR) and ceftriaxone-susceptible (CROS) isolates were identified using EUCAST and paired according to similarity in their MICs to other antimicrobials. The two groups of gonococci were sequenced and analysed. Mutated genes that showed a statistical difference between the two groups were transformed into gonococcal reference strains to determine their functions. AsPNAs were designed and transformed into the former transformant to further confirm the effects of the mutated genes. RESULTS: Twenty-two paired CROR and CROS isolates were obtained. The incidence of the penA-A501T and penA-G542S mutations individually, as well as combined mutations (penA-A501T and ftsX-R251H, penA-G542S and ftsX R251H), was statistically different between the two groups. The MIC of ATCC43069 (A43) increased 2 times following transformation with penA-A501T, and the MICs of A43 and ATCC49226 (A49) increased 32 times and 2 times following transformation with penA-A501T and ftsX-R251H, respectively. Antisense PNA-P3 reduced the MIC of the A43 transformant most significantly when transformed individually. PNA-P3 and PNA-F1 (asPNAs of the penA and ftsX) restored CRO susceptibility. CONCLUSIONS: PenA-A501T and penA-G542S mutations are important in CRO resistance among gonococci isolates. The ftsX-R251H mutation is also related to CRO resistance, and combined mutations of ftsX-R251H and penA-A501T comediate a significant reduction in CRO susceptibility. The combined application of PNA-P3 and PNA-F1 could effectively reverse the resistance to CRO in N. gonorrhoeae.

Rapid detection of Mycobacterium bovis in bovine cytological smears and tissue sections by peptide nucleic acid fluorescence in-situ hybridization.

BACKGROUND: Bovine tuberculosis is the leading cause of death in cattle and other species worldwide. Quick and precise identification of mycobacteria is critical to control the occurrence of tuberculosis in cattle. METHODS: We developed a fluorescent peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) approach to detect Mycobacterium bovis and Mycobacterium avium in cytological smears and tissue sections of bovines suspected of having tuberculosis. PNA-FISH was conducted on smears of lung and lymph node tissues. Standard bovine mycobacterial cultures were used to standardize the probes using 50 % formamide for M. bovis and 30 % formamide for M. avium. M. bovis probe (MTBCcy3), which was standardized at hybridization conditions of (55 °C and 40 % formamide) concentrations, was positive in all cytological smears. RESULTS: Four out of twenty five samples tested positive in tissue sections observed as a bright red fluorescence with a cy3 filter (MTBC probe). No results were observed with (MAVTAMRA) probe for M. avium which was standardized at hybridization conditions of (55 °C and 30 % formamide). No fluorescence was observed in the control tissue sections. Additionally, the results were juxtaposed with those of other commonly used detection methods such as immunohistochemistry and Polymerase Chain Reaction (PCR) by targeting the esxA gene. None of the samples tested positive for M. avium infection. CONCLUSION: PNA-FISH can be used to obtain cytological impression smears and tissue sections. When compared to PCR it consumes less time in the diagnosis of bovine tuberculosis in post mortem cases.

Localization of EGFR Mutations in Non-small-cell Lung Cancer Tissues Using Mutation-specific PNA-DNA Probes.

BACKGROUND/AIM: Epidermal growth factor receptor (EGFR) signaling inhibitors are potent therapeutic agents for EGFR-mutant non-small-cell lung cancer, but the effects of such inhibitors on the localization of EGFR mutations in tumor tissues remain to be elucidated. Thus, a simple and efficient technology for the detection of mutations in tumor tissue specimens needs to be developed. MATERIALS AND METHODS: Using an EGFR mutation-specific peptide nucleic acid (PNA)-DNA probe, the EGFR mutation-positive part of whole NSCLC tissues was visualized by immunofluorescence. Formalin-fixed paraffin-embedded sections obtained from A549, NCI-H1975, HCC827 and PC-9 tumors transplanted into nude mice were subjected to staining using PNA-DNA probes specific for the mRNA sequences producing the L858R, del E746-A750 and T790M mutations. RESULTS: The probes for the L858R mutation showed intense positive staining in H1975 cells, and the probe for the del E746-A750 mutation exhibited positive staining specifically in HCC827 and PC-9 tumors. On the other hand, A549 tumors without EGFR mutation did not show any significant staining for any PNA-DNA probe. In combination staining, the addition of cytokeratin stain increased the positive staining rate of each PNA-DNA probe. In addition, the positive staining rate of the probes for the L858R mutation was comparable to that of the antibody to EGFR L858R mutated protein. CONCLUSION: PNA-DNA probes specific for EGFR mutations might be useful tools to detect heterogeneous mutant EGFR expression in cancer tissues and efficiently evaluate the effect of EGFR signaling inhibitors on tissues of EGFR-mutant cancer.

Ultrasound-assisted Peptide Nucleic Acids synthesis (US-PNAS).

Herein, we developed an innovative and easily accessible solid-phase synthetic protocol for Peptide Nucleic Acid (PNA) oligomers by systematically investigating the ultrasonication effects in all steps of the PNA synthesis (US-PNAS). When compared with standard protocols, the application of the so-obtained US-PNAS approach succeeded in improving the crude product purities and the isolated yields of different PNA, including small or medium-sized oligomers (5-mer and 9-mer), complex purine-rich sequences (like a 5-mer Guanine homoligomer and the telomeric sequence TEL-13) and longer oligomers (such as the 18-mer anti-IVS2-654 PNA and the 23-mer anti-mRNA 155 PNA). Noteworthy, our ultrasound-assisted strategy is compatible with the commercially available PNA monomers and well-established coupling reagents and only requires the use of an ultrasonic bath, which is a simple equipment generally available in most synthetic laboratories.

Design and off-target prediction for antisense oligomers targeting bacterial mRNAs with the MASON web server.

Antisense oligomers (ASOs), such as peptide nucleic acids (PNAs), designed to inhibit the translation of essential bacterial genes, have emerged as attractive sequence- and species-specific programmable RNA antibiotics. Yet, potential drawbacks include unwanted side effects caused by their binding to transcripts other than the intended target. To facilitate the design of PNAs with minimal off-target effects, we developed MASON (make antisense oligomers now), a web server for the design of PNAs that target bacterial mRNAs. MASON generates PNA sequences complementary to the translational start site of a bacterial gene of interest and reports critical sequence attributes and potential off-target sites. We based MASON's off-target predictions on experiments in which we treated Salmonella enterica serovar Typhimurium with a series of 10-mer PNAs derived from a PNA targeting the essential gene acpP but carrying two serial mismatches. Growth inhibition and RNA-sequencing (RNA-seq) data revealed that PNAs with terminal mismatches are still able to target acpP, suggesting wider off-target effects than anticipated. Comparison of these results to an RNA-seq data set from uropathogenic Escherichia coli (UPEC) treated with eleven different PNAs confirmed that our findings are not unique to Salmonella We believe that MASON's off-target assessment will improve the design of specific PNAs and other ASOs.

Identification of single nucleotide polymorphisms by a peptide nucleic acid-based sandwich hybridization assay coupled with toehold-mediated strand displacement reactions.

In this work, we developed a simple and accurate peptide nucleic acid (PNA)-based sandwich hybridization assay for single nucleotide polymorphisms (SNPs) in the p53 gene. Our approach combines the enzyme-free toehold-mediated strand displacement reaction (SDR) with real-time enzyme-linked immunosorbent assay (ELISA) to detect SNPs with high sensitivity and specificity. A PNA-DNA heteroduplex with an external toehold is designed and fixed on well surface of a 96-well plate. The strand displacement from PNA-DNA heteroduplexes is initiated by the hybridization of target sequence with the toehold domain and ends with the fully displacing of the incumbent DNA. Finally, the as formed PNA-target DNA duplex with overhang at its 5'-end hybridizes with a biotin-labeled reporter PNA to form a sandwich structure on surface for signal amplification. The proposed PNA-based sandwich biosensor displays high sensitivity and greatly enhanced discriminability to target p53 gene segments against single-base mutant sequences compared to its all-DNA counterpart. Furthermore, the probe design is elegantly simple and the sensing procedure is easy to operate. We believe that this strategy may provide a simple and universal strategy for SNPs detection through easily altering the sequences of probes according to the sequences around target SNPs.

Genital mycobacteriosis caused by Mycobacterium marinum detected in two captive sharks by peptide nucleic acid-fluorescence in situ hybridization.

Mycobacterium marinum is a prevalent nontuberculous mycobacterium (NTM)-infecting teleosts. Conversely, little is known about mycobacteriosis in elasmobranchs, and M. marinum infection has never been reported from the subclass. This study investigated the histopathological characteristics and localization of this mycobacterium through molecular analysis of two captive sharks, a scalloped hammerhead Sphyrna lewini and a Japanese bullhead shark Heterodontus japonicus, exhibited in the same aquarium tank. We detected genital mycobacteriosis caused by M. marinum infection using molecular analyses, including polymerase chain reaction (PCR) and DNA sequencing targeting the 60 kDa heat-shock protein gene (hsp65), and peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) targeting the 16S rRNA gene. Both sharks showed granulomas in connective tissues of the gonads without central necrosis or surrounding fibrous capsules, which is unlike the typical mycobacterial granulomas seen in teleosts. This study reveals that elasmobranchs can be aquatic hosts of M. marinum. Because M. marinum is a representative waterborne NTM and a potential zoonotic agent, cautious and intensive research is needed to overcome a lack of data on the relationship between NTM and the aquatic environment in association with this subclass of Chondrichthyes.

Development of a PNA-DiSc2 based portable absorbance platform for the detection of pathogen nucleic acids.

We report the development of a portable absorption (PortAbs)-based pathogen nucleic acid detection system using peptide nucleic acid (PNA) and a cyanine dye, DiSc2(5). When the dye binds to the PNA-DNA hybrid, it results in a characteristic ∼110 nm shift in the dye absorbance, which we measure using PortAbs. The protocol involves amplification of the target DNA, PNA-DNA hybridization and dye complexing steps followed by absorption measurement. The system is built using a broad-spectrum photodiode whose output is amplified and then measured by a high resolution (24 or 32 bit) analog-to-digital converter. The excitation pulses of light are delivered by a color-changing LED. The sequence of excitation, measurement and display of results are all controlled by an embedded Raspberry-Pi board (or alternatively a laptop). At higher concentrations of the target amplicon (∼200 ng), the color change can be detected visually. At lower concentrations, PortAbs outperforms a plate reader and can detect target DNA as low as 30 ng or approximately 10 nM which is at least 10 fold better than previously reported studies. We validate the methodology using SARS-CoV-2 clinical samples containing about 1000 copies of the viral RNA and show that the entire workflow takes about 90 min. The cost of the complete standalone system is less than INR 40 000 (approx. 500 USD).

Ultrahigh sensitive and selective detection of single nucleotide polymorphism using peptide nucleic acid and ribonuclease H assembled DNA amplification (PRADA).

Early diagnosis and monitoring of cancer is the best way to increase the survival rate among patients with cancer. However, the current cancer screening technology is expensive and time-consuming; hence, cancer screening remains challenging. Therefore, developing a relatively inexpensive and high-performance analytical method is necessary. Especially, mutations in KRAS can be characterized as single nucleotide polymorphism mutations. Therefore, discrimination of single nucleotide polymorphism is essential to detect cancer mutations. This study introduces a method with high sensitivity and selectivity of real-time PCR using peptide nucleic acid (PNA) and RNase H II to detect KRAS single nucleotide polymorphism. This method was developed via the fusion of the existing PNA clamping PCR technique and the RNase H-dependent PCR technique. A synergistic effect was realized by mitigating the shortcomings of each method. Our method had a detection limit of 1 aM and selectivity of 0.01%. This study demonstrated completed validation tests with DNA-spiked plasma sample analysis, cell culture, and analysis of blood samples collected from patients with cancer. Furthermore, we demonstrated the applicability of this method for breath biopsy.

Machine Learning Guides Peptide Nucleic Acid Flow Synthesis and Sequence Design.

Peptide nucleic acids (PNAs) are potential antisense therapies for genetic, acquired, and viral diseases. Efficiently selecting candidate PNA sequences for synthesis and evaluation from a genome containing hundreds to thousands of options can be challenging. To facilitate this process, this work leverages machine learning (ML) algorithms and automated synthesis technology to predict PNA synthesis efficiency and guide rational PNA sequence design. The training data is collected from individual fluorenylmethyloxycarbonyl (Fmoc) deprotection reactions performed on a fully automated PNA synthesizer. The optimized ML model allows for 93% prediction accuracy and 0.97 Pearson's r. The predicted synthesis scores are validated to be correlated with the experimental high-performance liquid chromatography (HPLC) crude purities (correlation coefficient R2 = 0.95). Furthermore, a general applicability of ML is demonstrated through designing synthetically accessible antisense PNA sequences from 102 315 predicted candidates targeting exon 44 of the human dystrophin gene, SARS-CoV-2, HIV, as well as selected genes associated with cardiovascular diseases, type II diabetes, and various cancers. Collectively, ML provides an accurate prediction of PNA synthesis quality and serves as a useful computational tool for informing PNA sequence design.

Electrochemical Detection of Femtomolar DNA via Boronate Affinity-Mediated Decoration of Polysaccharides with Electroactive Tags.

In view of their high efficiency and cost-effectiveness, polymers are of great promise as carriers for signal tags in amplified detection. Herein, we present a polysaccharide-amplified method for the electrochemical detection of a BRCA1 breast cancer gene-derived DNA target at the femtomolar levels. Briefly, peptide nucleic acid (PNA) with a complementary sequence was tethered as the capture probe for the DNA target, to which carboxyl group-containing polysaccharides were then attached via facile phosphate-Zr(IV)-carboxylate crosslinking, followed by the decoration of polysaccharide chains with electroactive ferrocene (Fc) signal tags via affinity coupling between a cis-diol site and phenylboronic acid (PBA) group. As the polysaccharide chain contains hundreds of cis-diol sites, boronate affinity can enable the site-specific decoration of each polysaccharide chain with hundreds of Fc signal tags, efficiently transducing each target capture event into the decoration of many Fc signal tags. As polysaccharides are cheap, renewable, ubiquitous, and biodegradable natural biopolymers, the use of polysaccharides for signal amplification offers the benefits of high efficiency, cost-effectiveness, excellent biocompatibility, and environmental friendliness. The linear range of the polysaccharide-amplified method for DNA detection was demonstrated to be from 10 fM to 10 nM (R2 = 0.996), with the detection limit as low as 2.9 fM. The results show that this method can also discriminate single base mismatch with satisfactory selectivity and can be applied to DNA detection in serum samples. In view of these merits, the polysaccharide-amplified PNA-based electrochemical method holds great promise in DNA detection with satisfactory sensitivity and selectivity.

The role of Nucleic Acid Mimics (NAMs) on FISH-based techniques and applications for microbial detection.

Fluorescent in situ hybridization (FISH) is a powerful tool that for more than 30 years has allowed to detect and quantify microorganisms as well as to study their spatial distribution in three-dimensional structured environments such as biofilms. Throughout these years, FISH has been improved in order to face some of its earlier limitations and to adapt to new research objectives. One of these improvements is related to the emergence of Nucleic Acid Mimics (NAMs), which are now employed as alternatives to the DNA and RNA probes that have been classically used in FISH. NAMs such as peptide and locked nucleic acids (PNA and LNA) have provided enhanced sensitivity and specificity to the FISH technique, as well as higher flexibility in terms of applications. In this review, we aim to cover the state-of-the-art of the different NAMs and explore their possible applications in FISH, providing a general overview of the technique advancement in the last decades.

Quantifying telomeric lncRNAs using PNA-labelled RNA-Flow FISH (RNA-Flow).

Here we present a method to detect and quantify long non-coding RNAs, in particular those related to telomeres. By coupling the specificity of a peptide nucleic acid (PNA) probe with flow cytometry we have quantified cellular levels of TERRA and TERC lncRNAs in culture cell lines and PBMCs. This easy-to-use method appointed RNA-Flow allows reliable lncRNA quantification with broad applications in basic research and clinical diagnostics. In addition, the staining protocol presented here was proven useful for the detection and quantification of such lncRNAs on unfixed cells using confocal microscopy.

Effectiveness of blocking primers and a peptide nucleic acid (PNA) clamp for 18S metabarcoding dietary analysis of herbivorous fish.

The structure of food webs and carbon flow in aquatic ecosystems can be better understood by studying contributing factors such as the diets of herbivorous fish. Metabarcoding using a high-throughput sequencer has recently been used to clarify prey organisms of various fish except herbivorous fish. Since sequences of predator fish have dominated in sequences obtained by metabarcoding, we investigated a method for suppressing the amplification of fish DNA by using a blocking primer or peptide nucleic acid (PNA) clamp to determine the prey organisms of herbivorous fish. We designed three blocking primers and one PNA clamp that anneal to fish-specific sequences and examined how efficient they were in suppressing DNA amplification in various herbivorous fish. The results showed that the PNA clamp completely suppressed fish DNA amplification, and one of the blocking primers suppressed fish DNA amplification but less efficiently than the PNA clamp. Finally, we conducted metabarcoding using mock community samples as templates to determine whether the blocking primer or the PNA clamp was effective in suppressing fish DNA amplification. The results showed that the PNA clamp suppressed 99.3%-99.9% of fish DNA amplification, whereas the blocking primer suppressed 3.3%-32.9%. Therefore, we propose the application of the PNA clamp for clarifying the prey organisms and food preferences of various herbivorous fish.

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.