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Affinity proteins based on a three-helix bundle (affibodies, alphabodies, and computationally de novo designed ones) have been shown to be a general platform to discover binders with properties reminiscent of antibodies, combining high target specificity with affinities reaching well below the nanomolar. Herein, we report a strategy, coined self-assembled proteomimetic (SAP), to mimic such three-helix bundle architecture with a hybridization-enforced two-helix coiled coil that is obtained by templated native chemical ligation (T-NCL) of PNA-peptide conjugates. This SAP strategy stands out by its synthetic accessibility, reducing the length on the longest synthetic peptide to less than 30 amino acids which is readily attainable by standard SPPS methodologies. We show that the T-NCL dramatically accelerates the ligation, enabling this chemistry to proceed in a combinatorial fashion at low micromolar concentrations. We demonstrate that small combinatorial libraries of SAPs can be prepared in one operation and used directly in affinity selections against a target of interest with an LC-MS analysis of the fittest binders. Moreover, we show that the underlying design paradigm is functional for SAPs based on structurally distinct three-helix peptides aimed at different therapeutic targets, namely HER2 and spike's RBD, reaching picomolar affinities. We further illustrate that the affinity of the SAP can be allosterically regulated using a toehold displacement of the hybridizing PNAs to disrupt the coiled coil stabilization. Finally, we show that an RBD-targeting SAP effectively inhibits viral entry of SARS-CoV-2 with an IC 50 of 2.8 nM., Competing Interests: Competing interests statement:A provisional patent application is in preparation.

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 (K i  = 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., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

A label-free electrochemical DNA detection strategy based on self-assembled α-Fe 2 O 3 /Fe 3 O 4 nanosheets with PNA-DNA hybridization process was developed for ultrasensitive detection of APOE ε4 gene, one of the most robust genetic risks for Alzheimer's Disease (AD). In this work, magnetic α-Fe 2 O 3 /Fe 3 O 4 heterogeneous nanosheets were prepared by hydrothermal-calcined reduction method and loaded with Au nanoparticles (AuNPs) on their surfaces. The magnetic α-Fe 2 O 3 /Fe 3 O 4 @Au nanocomposites significantly enhanced the electrochemical response as a signal amplification matrix and were able to bind to the magnetic glassy carbon electrode (MGCE) surface by magnetic self-assembly. Moreover, owing to the high specificity and stable binding capacity of PNA with respect to the target DNA, the biosensor not only enabled accurate (the limit of detection was estimated to be 0.147 pM) and rapid detection of the APOE ε4 gene, but also exhibited excellent specificity, stability and regeneration capability. Additional, the satisfactory recoveries were also obtained in real samples of human serum, ranging from 92.83 % to 106.22 % with relative standard deviation (RSD) between 0.25 % and 1.85 %. The results possessed important reference value for exploring the application of DNA biosensor technology in the diagnosis of APOE gene mutation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Peptide nucleic acids (PNA), synthetic molecules comprising a peptide-like backbone and natural and unnatural nucleobases, have garnered significant attention for their potential applications in gene editing and other biomedical fields. The unique properties of PNA, particularly enhanced stability/specificity/affinity towards targeted DNA and RNA sequences, achieved significant attention recently for gene silencing, gene correction, antisense therapy, drug delivery, biosensing and other various diagnostic aspects. This review explores the structure, properties, and potential of PNA in transforming genetic engineering including potent biomedical challenges. In Addition, we explore future perspectives and potential limitations of PNA-based technologies, highlighting the need for further research and development to fully realize their therapeutic and biotechnological potential., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 Elsevier Inc. All rights reserved.)

Noncanonical base pairs play an important role in enabling the structural and functional complexity of RNA. Molecular recognition of such motifs is challenging because of their diversity, significant deviation from the Watson-Crick structures, and dynamic behavior, resulting in alternative conformations of similar stability. Triplex-forming peptide nucleic acids (PNAs) have emerged as excellent ligands for the recognition of Watson-Crick base-paired double helical RNA. The present study extends the recognition potential of PNA to RNA helices having noncanonical GoU, AoC, and tandem GoA/AoG base pairs. The purines of the noncanonical base pairs formed M + ·GoU, T·AoC, M + ·GoA, and T·AoG Hoogsteen triples of similar or slightly reduced stability compared to the canonical M + ·G-C and T·A-U triples. Recognition of pyrimidines was more challenging. While the P·CoA triple was only slightly less stable than P·C-G, the E nucleobase did not form a stable triple with U of the UoG wobble pair. Molecular dynamics simulations suggested the formation of expected Hoogsteen hydrogen bonds for all of the stable triples. Collectively, these results expand the scope of triple helical recognition to noncanonical structures and sequence motifs common in biologically relevant RNAs.

Cancer immunotherapy is focused on stimulating the immune system against cancer cells by exploiting immune checkpoint mechanisms. PD-1/PD-L1 is one of the most known immune checkpoints due to the widespread upregulation of the Programmed Death Ligand 1 (PD-L1) transmembrane protein in cancer tissues. Accordingly, taking advantage of the ability of oncolytic adenoviruses (OAd) to specifically infect and kill tumor cells over healthy ones, here, we developed a targeted delivery platform based on OAd to selectively deliver in cancer cells an antisense peptide nucleic acid (PNA) targeting the PD-L1 mRNA. The antisense PNA was modified with a six-lysine tail to improve water solubility and binding affinity to the polyanionic surface of the OAd carrier. Dynamic light scattering measurements confirmed the effective binding of the PNA cargo to OAd. Flow cytometry analysis evaluated the impact on PD-L1 protein expression in A549 and SK-OV3 cancer cell lines post-incubation with the OAd/PNA system. Statistically significant PD-L1 downregulation was observed in SK-OV3 cells treated with OAd-delivered PNA, surpassing the effect of free PNA. Confocal microscopy showed the cytoplasmic localization of OAd-delivered PNA, supporting the proposed antisense mechanism for PD-L1 downregulation. This targeted delivery system holds potential for enhancing the effectiveness of cancer immunotherapy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Detection of KRAS mutation in colorectal cancer (CRC) is important in the prediction of response to target therapy. The study aims to develop a novel mutation detection platform called the "PNA-LNA molecular switch" for the detection of KRAS mutation in CRC. We employed the enhanced binding specificity of peptide nucleic acid (PNA) and locked nucleic acid (LNA) in conjunction with a loop-mediated isothermal amplification (LAMP) approach to identify the mutation status of KRAS oncogene codon 12 (c.35G>T/G12V and c.35G>A/G12D) using synthetic oligonucleotides and colon cancer cell lines (Caco-2 and SW480). This method specifically blocked the amplification of the wild-type sequences while substantially amplifying the mutated ones, which was visualized by both colorimetric and fluorescence assays. We then checked the mutation profile of KRAS codon 12 in the DNA derived from tumor tissue samples (number of samples, n = 30) and circulating tumor cells (n = 24) from CRC patients. Finally, we validated the results by comparing them with the data obtained from DNA sequencing of colon tumors (n = 21) of the same CRC patients. This method showed excellent sensitivity (1 DNA copy/μl), reproducibility [relative standard deviation (%RSD) < 5%, for n = 3], and linear dynamic range (1 ag/μl-10 pg/μl, R 2  = 0.94). This platform is significantly faster, relatively cheaper, has superior sensitivity and specificity, and does not require any high-end equipment. To conclude, this method has the potential to be translated into clinical settings for the detection of mutations in diverse diseases and conditions., Competing Interests: Declaration of competing interest The authors declared no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

PDZK1-interacting protein 1 (PDZK1IP1) has emerged as a potential therapeutic target for skin inflammatory diseases and epithelial tumors. This study investigates the modulation of PDZK1IP1 gene expression using peptide nucleic acids (PNAs), a class of oligonucleotide therapeutics known for their robust binding affinity to complementary nucleic acid sequences and their resistance to degradation by nucleases. To enhance water solubility and cellular permeability, modified PNA oligomers were synthesized by conjugating nucleobases with primary amine chains. A study using a fluorescein-labeled modified PNA oligomer demonstrated significantly enhanced cellular permeability in HaCaT cells compared to the unmodified PNA. These modified PNA oligomers effectively suppressed PDZK1IP1 gene expression and alleviated interferon γ (IFNγ)-induced inflammatory responses in normal human keratinocytes. These findings suggest the potential application of modified PNAs targeting PDZK1IP1 in the treatment of skin inflammatory diseases., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Daram Jung, Yeasel Jeon, Joonseo Kim reports financial support and equipment, drugs, or supplies were provided by OliPass Corporation. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Peptides are well known for forming nanoparticles, while DNA duplexes, triplexes and tetraplexes create rigid nanostructures. Accordingly, the covalent conjugation of peptides to DNA/RNA produces hybrid self-assembling features and may lead to interesting nano-assemblies distinct from those of their individual components. Herein, we report the preparation of a collagen mimetic peptide incorporating lysine in its backbone, with alkylamino side chains radially conjugated with G-rich PNA [collagen-(PNA-GGG) 3 ]. In the presence of complementary C-rich DNA (dCCCTTTCCC) at neutral pH, the collagen mimetic triplexes were interconnected by PNA-GGG : DNA-CCC duplexes, leading to the formation of larger assemblies of nanostructures. Upon decreasing the pH to 4.5, the dissociation of the triplex-duplex assembly released the protonated C-rich DNA, which immediately folded into an i -motif. With an increase in the pH to 7.2 (neutral), the i -motif unfolded into linear DNA, which reformed the PNA-GGG : DNA-CCC duplex interconnecting the collagen triplexes. The pH-induced switching of the assembly and disassembly was reversible over a few cycles. The hybrid collagen-(PNAGGG) 3  : DNA-C 3 T 3 C 3 triplex-duplex and the individual components of the assembly including the i -motif were characterized by UV and CD melting, fluorescence, TEM and gel electrophoresis. The pH-induced reversible switching was established by the changes in the CD and fluorescence properties. Peptide-DNA conjugates have wide applications in both biology and materials science, ranging from therapeutics and drug delivery to diagnostics and molecular switches. Thus, the prototype ensemble of the triplex peptide-PNA conjugate and its duplex with DNA described herein has potential for elaboration into rationally designed systems by varying the PNA/DNA sequences to trap functional ligands/drugs for release in pH-controlled environments.

Targeting the genes regulate the lineage commitment of human mesenchymal stem cells (hMSCs) to adipocytes provides a promising strategy for addressing obesity. In this study, we investigated the therapeutic potential of cell-penetrating anti-sense peptide nucleic acids (PNAs) designed to enhance solubility and hybridization properties, specifically targeting sulfatase 2 (SULF2), a potential reciprocal regulator of adipocyte and osteoblast differentiation in hMSCs. Cell-penetrating modified PNA oligomers effectively inhibit SULF2 gene transcription, leading to significant reductions in adiponectin protein synthesis and intracellular lipid droplet accumulation during adipogenesis in human bone marrow-derived MSCs (hBM-MSCs). Notably, PNA oligomer compound 5 exhibited the most potent anti-adipogenic activity, with an IC 50 value of 0.28 μM. These findings show the potential of SULF2-targeting cell-penetrating PNA oligomers as novel therapeutic agents for obesity-related metabolic diseases., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Daram Jung reports financial support was provided by OliPass Corporation. Yeasel Jeon reports financial support was provided by OliPass Corporation. Minhee Kim reports financial support was provided by Minhee Kim. Areum Kim reports was provided by OliPass Corporation. Minsoo Noh, Ph.D. reports a relationship with Soul National University that includes: funding grants. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Precision genetic medicine enlists antisense oligonucleotides (ASOs) to bind to nucleic acid targets important for human disease. Peptide nucleic acids (PNAs) have many desirable attributes as ASOs but lack cellular permeability. Here, we use an assay based on the corrective splicing of an mRNA to assess the ability of synthetic peptides to deliver a functional PNA into a human cell. We find that the endosomolytic peptides L17E and L17ER 4 are highly efficacious delivery vehicles. Co-treatment of a PNA with low micromolar L17E or L17ER 4 enables robust corrective splicing in nearly all treated cells. Peptide-PNA conjugates are even more effective. These results enhance the utility of PNAs as research tools and potential therapeutic agents.

We investigated a renal tubule-targeting carbohydrate (RENTAC) that can selectively deliver small-molecule and nucleic acid analogs to the proximal convoluted tubules of the kidney following systemic delivery in mice. We comprehensively evaluated anti-miR-21-peptide nucleic acid-RENTAC, and fluorophore-RENTAC conjugates in cell culture and in vivo. We established that RENTAC conjugates showed megalin- and cubilin-dependent endocytic uptake in the immortalized kidney cell line. In vivo biodistribution studies confirmed the retention of RENTAC conjugates in the kidneys for several days compared with other organs. Immunofluorescence staining confirmed the selective distribution of the RENTAC conjugates in proximal convoluted tubules. We further demonstrated proximal convoluted tubule targeting features of RENTAC conjugates in a folic acid-induced kidney fibrosis mouse model. As a biological readout, we targeted miR-33 using antisense peptide nucleic acid (PNA) 33-RENTAC conjugates in the fibrotic kidney disease model. The targeted delivery of PNA 33-RENTAC resulted in slower fibrosis progression and decreased collagen deposition. We also confirmed that the RENTAC ligand did not exert any adverse reactions. Thus, we established that the RENTAC ligand can be used for broad clinical applications targeting the kidneys selectively., Competing Interests: Declaration of interests R.B., V.K., and A.W. are inventors of patents related to kidney delivery assigned to the University of Connecticut., (Copyright © 2024 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Biosensors are capable of diagnosing tumors through imaging in vivo or liquid biopsy, but they face the challenges of inefficient delivery into tumor sites and the lack of reliable tumor-associated biomarkers. Herein, we constructed a dual-mode biosensor based on a multifunctional peptide nucleic acid (PNA)/peptide copolymer and DNA tetrahedron for tumor imaging and urinalysis. The biosensor could enter the cancer cells to initiate a microRNA-21-specific catalytic hairpin assembly reaction after cleavage by matrix-metalloprotease (MMP) in the tumor microenvironment, and the MMP cleavage product was released into the bloodstream and then was filtered out by the kidney. As PNA was a synthetic DNA analogue that could not be degraded by nucleases and proteases, it could serve as a reliable synthetic biomarker and be easily detected by high-performance liquid chromatography in urine. Importantly, the biosensor was hitchhiked on the macrophage membrane to realize efficient delivery in the depth of tumor utilizing the macrophage ability of actively homing to the tumor site and infiltrating into the tumor. The results indicated that the signal output of the biosensor was improved remarkably and mice with a tumor volume as little as 30-40 mm 3 could be reliably discriminated through urine assay. This innovative macrophage-hitchhiking dual-mode biosensor holds a great potential as a non-invasive and convenient tool for tumor diagnosis and tumor progression evaluation.

Antisense oligonucleotides have been developed as therapeutic compounds, with peptide nucleic acid (PNA) emerging as a promising nucleic acid mimic for antimicrobial applications. To be effective, PNAs must be internalized into bacterial cells, as they are not naturally absorbed. A strategy to improve PNA membrane penetration and cellular uptake involves covalently conjugating them to cell-penetrating peptides. However, these membrane-active peptides can exhibit cytotoxicity, and their efficiency as PNA carriers needs to be enhanced. Therefore, we explored new peptide-PNA conjugates and their linkers to understand how they affect PNA uptake into bacteria. We conjugated PNA to two peptides, anoplin and (KFF) 3 K, along with their structurally stabilized hydrocarbon-stapled derivatives, and evaluated their transport into various bacterial strains. The PNA sequence targeted bacterial mRNA encoding the essential acyl carrier protein. As linkages, we used either a non-cleavable 8-amino-2,6-dioxaoctanoyl (ethylene glycol, eg1) linker or a reducible disulfide bridge. We found that the hydrocarbon-stapled peptides did not enhance PNA delivery, despite the strong inner- and outer-membrane-penetrating capabilities of the standalone peptides. Additionally, the disulfide bridge linkage, which is cleavable in the bacterial cytoplasm, decreased the antimicrobial activity of the peptide-PNA conjugates. Notably, we identified anoplin as a new potent PNA carrier peptide, with the anoplin-eg1-PNA conjugate demonstrating antibacterial activity against E. coli and S. Typhimurium strains in the 2-4 µM range., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Nitrobiphenyl photosensitizer (NBP)-peptide nucleic acids (PNA) conjugates were synthesized to develop a tool for photo-knockdown of target DNAs. The presence of NBP hardly hindered duplex formation with the complementary single strand DNA as demonstrated by the comparison of T m values and CD spectra with those for standard PNA/DNA duplexes. However, the photooxidation of guanines in NBP-PNA/DNAs was significantly less effective than those of corresponding NBP-DNA/DNA. Production of singlet oxygen ( 1 O 2 ) during the photooxidation was confirmed by consumption of furfuryl alcohol, a 1 O 2 detector. The poor photooxidation efficiency was ameliorated with 1 O 2 generated from an externally added NBP derivative. It was found that, when complexed with the sticky end of a double strand DNA, NBP-PNA was able to photooxidize G in the DNA/DNA duplex region, whereas G in the PNA/DNA duplex region was considerably unreactive. These results suggest that 1 O 2 produced from NBP-PNA tends to quench during diffusion along the PNA/DNA backbone, whereas quenching is less likely during diffusion along DNA/DNA region., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

We have explored the reaction of a three-components mixture of aminomalononitrile, urea and α-amino acid methyl esters for the multicomponent synthesis substituted purines resembling PNA's building blocks. 2,6-diamino-purines, 6-amino-3,9-dihydro-2H-purin-2-one (iso-guanines), and 3,9-dihydro-6H-purin-6-one derivatives, selectively decorated at C(8)-position of the purine ring with different amino acid residues, were obtained from acceptable to good yields. The regio-selectivity of the transformation was controlled by the use of urea in the ternary mixture and by the annulation agent involved in the ring-closure of amino-imidazole carbonitrile intermediates. Solvent free conditions, microwave irradiation and simple one-carbon containing reagents further satisfied the major requirement of atom economy and sustainable chemistry. Due to the prebiotic nature of the three-components mixture and of annulation agents, it also embodies the possibility for the synthesis of novel PNAs bearing purine nucleobases decorated at C(8)-position of the imidazole ring as alternative RNA analogues in molecular evolution., (© 2024 The Authors. ChemistryOpen published by Wiley-VCH GmbH.)

Spherical nucleic acids (SNAs) with functional peptide cores are an emerging nanoplatform for synergistic cancer therapy but have been rarely reported. We construct herein the first SNA nanoplatform based on a biodegradable binary peptide backbone of methionine (Met) and cysteine (Cys) for codelivery of a photosensitizer, Chlorin e6 (Ce6) and human liver-specific miR122 for synergistic photodynamic-gene therapy of hepatic cell carcinoma (HCC). Met supplementation by the peptide core improves the infiltration of T cells and enhances the effector function of T cells for turning a "cold" tumor into a "hot" one. The resulting SNA(+) shows the most significant inhibitory effect in a Hepa1-6 HCC primary/distal tumor model, with tumor growth inhibition (TGI) values of 98.5 ± 0.5 % and 99.1 ± 0.4 % for the primary and distant tumors, respectively. This SNA nanoplatform achieves superior high TGI values reported thus far to our knowledge with almost complete eradication for both tumors due to the simultaneous adaptive and innate immunity activation via photodynamic therapy (PDT) induced immunogenic cell death (ICD) and Met supplementation-promoted adaptive immunity, and miR122-enhanced innate immunity. Overall, this study not only develops a reliable synthetic strategy toward peptide-backboned multifunctional SNA nanoplatform, but also reports the modulation of amino acid metabolism for enhanced innate immunity for highly efficient HCC immunotherapy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Fast and accurate identification of pathogenic microbes in patient samples is crucial for the timely treatment of acute infectious diseases such as sepsis. The fluorescence in situ hybridization (FISH) technique allows the rapid detection and identification of microbes based on their variation in genomic sequence without time-consuming culturing or sequencing. However, the recent explosion of microbial genomic data has made it challenging to design an appropriate set of probes for microbial mixtures. We developed a novel set of peptide nucleic acid (PNA)-based FISH probes with optimal target specificity by analyzing the variations in 16S ribosomal RNA sequence across all bacterial species. Owing to their superior penetration into bacteria and higher mismatch sensitivity, the PNA probes distinguished seven bacterial species commonly observed in bacteremia with 96-99.9% accuracy using our optimized FISH procedure. Detection based on Förster resonance energy transfer (FRET) between pairs of adjacent binding PNA probes eliminated crosstalk between species. Rapid sequential species identification was implemented, using chemically cleavable fluorophores, without compromising detection accuracy. Owing to their outstanding accuracy and enhanced speed, this set of techniques shows great potential for clinical use., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

We present a novel activity-based detection strategy for matrix metalloproteinase 2 (MMP2), a critical cancer protease biomarker, leveraging a mechanism responsive to the proteolytic activity of MMP2 and its integration with CRISPR-Cas12a-assisted signal amplification. We designed a chemical translator comprising two functional units─a peptide and a peptide nucleic acid (PNA), fused together. The peptide presents the substrate of MMP2, while the PNA serves as a nucleic acid output for subsequent processing. This chemical translator was immobilized on micrometer magnetic beads as a physical support for an activity-based assay. We incorporated into our design a single-stranded DNA partially hybridized with the PNA sequence and bearing a region complementary to the RNA guide of CRISPR-Cas12a. The target-induced nuclease activity of Cas12a results in the degradation of FRET-labeled DNA reporters and amplified fluorescence signal, enabling the detection of MMP2 in the low picomolar range, showing a limit of detection of 72 pg/mL. This study provides new design principles for a broader applicability of CRISPR-Cas-based biosensing.

Obtaining new materials by exploiting the self-assembly of biomolecules is a very challenging field. In recent years, short peptides and nucleic acids have been used as scaffolds to produce supramolecular structures for different applications in the biomedical and technological fields. In this review, we will focus on the self-assembly of peptide nucleic acids (PNAs), their conjugates with peptides, or other molecules. We will describe the physical properties of the assembled systems and, where described, the application they were designed for.

Nucleic acid sensors based on a peptide nucleic acid (PNA) probe have seen a surge in interest since their discovery in the 1990s, and after the patent protecting them expired in 2013. The appeal of PNA as capture and/or sensing probes as an alternative to standard DNA or RNA oligonucleotides originates from their superior chemical stability and affinity for complementary oligonucleotides, as well as their increased responsiveness to single base mismatches. The implementation of PNA probes onto optical and electrochemical sensors has showed great promise although progress has been hampered by issues mostly associated with surface chemistry, probe accessibility and non-specific binding. Herein, we report on a systematic comparison between various PNA immobilisation strategies on carbon substrates based on both covalent and non-covalent chemistries. Besides the use of standard electrochemical techniques to characterise the extent of surface modification, the ability of immobilised PNAs to engage in chemical interactions with freely diffusing molecules was also investigated. Using original chemical tags, this study provides a unique insight into the impact of immobilisation chemistries on PNA's (bio)availability. Rapid immobilisation of biotinylated PNA oligomers on screen-printed carbon electrode (SPCE) coated with adsorbed polystreptavidin (pSA) demonstrated highest efficiency and ease in the preparation process. An original nucleic acid sensor using this immobilisation chemistry is reported that is based on a sandwich assay between a surface bound PNA capture probe and a freely diffusing electrochemically active PNA sensing probe., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Peptide nucleic acids (PNAs) combine the programmability of native nucleic acids with the robustness and ease of synthesis of a peptide backbone. These designer biomolecules have demonstrated tremendous utility across a broad range of applications, from the formation of bespoke biosupramolecular architectures to biosensing and gene regulation. Herein, we explore some of the key developments in the application of PNA in chemical biology and biotechnology in the last 5 years and present anticipated key areas of future development., (© 2024 The Authors. ChemPlusChem published by Wiley-VCH GmbH.)

The similar transmission patterns and early symptoms of respiratory viral infections, particularly severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza (H1N1), and respiratory syncytial virus (RSV), pose substantial challenges in the diagnosis, therapeutic management, and handling of these infectious diseases. Multiplexed point-of-care testing for detection is urgently needed for prompt and efficient disease management. Here, we introduce an electrochemical paper-based analytical device (ePAD) platform for multiplexed and label-free detection of SARS-CoV-2, H1N1, and RSV infection using immobilized pyrrolidinyl peptide nucleic acid probes. Hybridization between the probes and viral nucleic acid targets causes changes in the electrochemical response. The resulting sensor offers high sensitivity and low detection limits of 0.12, 0.35, and 0.36 pM for SARS-CoV-2 (N gene), H1N1, and RSV, respectively, without showing any cross-reactivities. The amplification-free detection of extracted RNA from 42 nasopharyngeal swab samples was successfully demonstrated and validated against reverse-transcription polymerase chain reaction (range of cycle threshold values: 17.43-25.89). The proposed platform showed excellent clinical sensitivity (100 %) and specificity (≥97 %) to achieve excellent agreement (κ ≥ 0.914) with the standard assay, thereby demonstrating its applicability for the screening and diagnosis of these respiratory diseases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Alpha-synuclein, encoded by the SNCA gene, is a pivotal protein implicated in the pathogenesis of synucleinopathies, including Parkinson's disease. Current approaches for modulating alpha-synuclein levels involve antisense nucleotides, siRNAs, and small molecules targeting SNCA's 5'-UTR mRNA. Here, we propose a groundbreaking strategy targeting G-quadruplex structures to effectively modulate SNCA gene expression and lowering alpha-synuclein amount. Novel G-quadruplex sequences, identified on the SNCA gene's transcription starting site and 5'-UTR of SNCA mRNAs, were experimentally confirmed for their stability through biophysical assays and in vitro experiments on human genomic DNA. Biological validation in differentiated SH-SY5Y cells revealed that well-known G-quadruplex ligands remarkably stabilized these structures, inducing the modulation of SNCA mRNAs expression, and the effective decrease in alpha-synuclein amount. Besides, a novel peptide nucleic acid conjugate, designed to selectively disrupt of G-quadruplex within the SNCA gene promoter, caused a promising lowering of both SNCA mRNA and alpha-synuclein protein. Altogether our findings highlight G-quadruplexes' key role as intriguing biological targets in achieving a notable and successful reduction in alpha-synuclein expression, pointing to a novel approach against synucleinopathies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Although CRISPR-Cas9 gene therapies have proven to be a powerful tool across many applications, improvements are necessary to increase the specificity of this technology. Cas9 cutting in off-target sites remains an issue that limits CRISPR's application in human-based therapies. Treatment of autosomal dominant diseases also remains a challenge when mutant alleles differ from the wild-type sequence by only one base pair. Here, we utilize synthetic peptide nucleic acids (PNAs) that bind selected spacer sequences in the guide RNA (gRNA) to increase Cas9 specificity up to 10-fold. We interrogate variations in PNA length, binding position, and degree of homology with the gRNA. Our findings reveal that PNAs bound in the region distal to the protospacer adjacent motif (PAM) site effectively enhance specificity in both on-target/off-target and allele-specific scenarios. In addition, we demonstrate that introducing deliberate mismatches between PNAs bound in the PAM-proximal region of the gRNA can modulate Cas9 activity in an allele-specific manner. These advancements hold promise for addressing current limitations and expanding the therapeutic potential of CRISPR technology.

RNA-binding proteins (RBPs) are a major class of proteins that interact with RNAs to change their fate or function. RBPs and the ribonucleoprotein complexes they constitute are involved in many essential cellular processes. In many cases, the molecular details of RBP:RNA interactions differ between viruses, prokaryotes and eukaryotes, making prokaryotic and viral RBPs good potential drug targets. However, targeting RBPs with small molecules has so far been met with limited success as RNA-binding sites tend to be extended, shallow and dynamic with a mixture of charged, polar and hydrophobic interactions. Here, we show that peptide nucleic acids (PNAs) with nucleic acid-like binding properties and a highly stable peptide-like backbone can be used to target some RBPs. We have designed PNAs to mimic the short RNA stem-loop sequence required for the initiation of prokaryotic signal recognition particle (SRP) assembly, a target for antibiotics development. Using a range of biophysical and biochemical assays, the designed PNAs were demonstrated to fold into a hairpin structure, bind the targeted protein and compete with the native RNA hairpin to inhibit SRP formation. To show the applicability of PNAs against other RBPs, a PNA was also shown to bind Nsp9 from SARS-CoV-2, a protein that exhibits non-sequence-specific RNA binding but preferentially binds hairpin structures. Taken together, our results support that PNAs can be a promising class of compounds for targeting RNA-binding activities in RBPs., Competing Interests: NO authors have competing interests., (Copyright: © 2024 Zhong et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Site-specific nucleases are crucial for genome engineering applications in medicine and agriculture. The ideal site-specific nucleases are easily reprogrammable, highly specific in target site recognition, and robust in nuclease activities. Prokaryotic Argonaute (pAgo) proteins have received much attention as biotechnological tools due to their ability to recognize specific target sequences without a protospacer adjacent motif, but their lack of intrinsic dsDNA unwinding activity limits their utility in key applications such as gene editing. Recently, we developed a pAgo-based system for site-specific DNA cleavage at physiological temperatures independently of the DNA form, using peptide nucleic acids (PNAs) to facilitate unwinding dsDNA targets. Here, we fused catalytically dead pAgos with the nuclease domain of the restriction endonuclease FokI and named this modified platform PNA-assisted FokI-(d)pAgo (PNFP) editors. In the PNFP system, catalytically inactive pAgo recognizes and binds to a specific target DNA sequence based on a programmable guide DNA sequence; upon binding to the target site, the FokI domains dimerize and introduce precise dsDNA breaks. We explored key parameters of the PNFP system including the requirements of PNA and guide DNAs, the specificity of PNA and guide DNA on target cleavage, the optimal concentration of different components, reaction time for invasion and cleavage, and ideal temperature and reaction buffer, to ensure efficient DNA editing in vitro. The results demonstrated robust site-specific target cleavage by PNFP system at optimal conditions in vitro. We envision that the PNFP system will provide higher editing efficiency and specificity with fewer off-target effects in vivo., Competing Interests: Conflicts of interest Authors have a pending patent application on the PNFP editors and their diverse uses and applications in diverse organisms., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Forced intercalation peptide nucleic acids (FIT-PNAs) are DNA mimics that act as RNA sensors. The sensing event occurs due to sequence-specific RNA hybridization, leading to a substantial increase in fluorescence. The fluorophore in the FIT-PNA is termed a surrogate base. This molecule typically replaces a purine in the PNA sequence. BisQ is a surrogate base that connects two quinolines via a monomethine bond. BisQ-based FIT-PNAs have excellent biophysical features that include high brightness and red-shifted emission (λ em, max = 613 nm). In this report, we detail two chemical approaches that allow for the facile synthesis of the BisQ PNA monomer. In both cases, the key compound used for the synthesis of BisQ-CH 2 COOH is the tBu-ester-modified quinoline synthon (compound 5 ). Subsequently, one method uses the Alloc acid-protected PNA backbone, whereas the other uses the tBu ester-protected PNA backbone. In the latter case, the overall yield for BisQ acid (compound 7 ) and BisQ PNA monomer syntheses was 61% in six synthetic steps. This is a substantial improvement to the published procedures to date (7% total yield). Lastly, we have prepared an 11-mer FIT-PNA with either BisQ or thiazole orange (TO) and studied their photophysical properties. We find superior photophysical properties for the BisQ FIT-PNA in terms of the brightness and selectivity, highlighting the added value of using this surrogate base for RNA sensing.

The presence of non-essential metals in the environment as contaminants is prone to cause hazardous health problems following accumulation in the human body and the ensuing toxic effects. This calls for continuous discovery and innovation in the realm of developing easy-to-operate, cheap and sensitive sensors. Herein, we describe the proof of concept approach for designing a molecular receptor-like, chimeric sensor based on the pore-forming peptide alamethicin (Alm), tethered via a linker with an ultrashort peptide nucleic acid (PNA) moiety, capable of generating functional ion channel oligomers in planar lipid membranes. The working principle of the sensor exploits the ability of Hg 2+ ions to complex mismatching thymine-thymine sequences between the PNA receptor moiety on Alm oligomers and free, thymine-based, single-stranded DNAs (ssDNAs) in solution, thus creating a stable base pair at the oligomer entrance. This generates a transducing mechanism which converts the metal ion complexation into a specific electrical signature of the self-assembled Alm oligomers, enabling selective Hg 2+ ion detection. The platform is programmable, whereby the simple exchange of the PNA sequence and its ssDNA counterpart in solution rendered the system selective for Cu 2+ ion detection. With further optimization, the presented solution has the potential to translate into miniaturized, cost-effective biosensors suitable for the real-time, label-free and continuous detection of metal ions or other biomolecules.

Nucleic acids (DNA and RNA) can form diverse secondary structures ranging from hairpins to duplex, triplex, G4-tetraplex and C4-i-motifs. Many of the DNA analogues designed as antisense oligonucleotides (ASO) are also adept at embracing such folded structures, although to different extents with altered stabilities. One such analogue, peptide nucleic acid (PNA), which is uncharged and achiral, forms hybrids with complementary DNA/RNA with greater stability and specificity than DNA:DNA/RNA hybrids. Like DNAs, these single-stranded PNAs can form PNA:DNA/RNA duplexes, PNA:DNA:PNA triplexes, PNA-G4 tetraplexes and PNA-C4-i-motifs. We have recently designed Janus-like bimodal PNAs endowed with two different nucleobase sequences on either side of a single aminoethylglycyl ( aeg ) PNA backbone and shown that these can simultaneously bind to two complementary DNA sequences from both faces of PNA. This leads to the formation of supramolecular polyplexes such as double duplexes, triple duplexes and triplexes of double duplexes with appropriate complementary DNA/RNA. Herein, we demonstrate that Janus/bimodal PNA with a poly G-sequence on the triazole side of the PNA backbone and mixed bases on the t-amide side, templates the initial formation of a (PNA-G5) 4 tetraplex (triazole side), followed by the formation of a PNA:DNA duplex (t-amide side). Such a polyplex shows synergistic overall stabilisation compared to the isolated duplexes/quadruplex. The assembly of polyplexes with a shared backbone for duplexes and tetraplexes is programmable and may have potential applications in the self-assembly of nucleic acid nano- and origami structures. It is also shown that Janus PNAs enter the cells better than the standard aeg -PNA oligomers, and hence have implications for in vivo applications as well.

Tailored healthcare, an approach focused on individual patients, requires integrating emerging interdisciplinary technologies to develop accurate and user-friendly diagnostic tools. KRAS mutations, prevalent in various common cancers, are crucial determinants in selecting patients for novel KRAS inhibitor therapies. This study presents a novel state-of-the-art Lab-on-a-Disc system utilizing peptide nucleic acids-loop backward (PNA-LB) mediated allele-specific loop-mediated isothermal amplification (LAMP) for detecting the frequent G12D KRAS mutation, signifying its superiority over alternative mutation detection approaches. The designed Lab-on-a-Disc system demonstrated exceptional preclinical and technical precision, accuracy, and versatility. By applying varying cutoff values to PNA- LB LAMP reactions, the assay's sensitivity and specificity were increased by 80 % and 90 %, respectively. The device's key advantages include a robust microfluidic Lab-on-a-Disc design, precise rotary control, and a cutting-edge induction heating module. These features enable multiplexing of LAMP reactions with high reproducibility and repeatability, with CV% values less than 3.5 % and 5.5 %, respectively. The device offers several methods for accurate endpoint result detection, including naked-eye observation, RGB image analysis using Python code, and time of fluorescence (T f ) values. Preclinical specificity and sensitivity, assessed using different cutoffs for Eva-Green fluorescence T f values and pH-sensitive dyes, demonstrated comparable performance to the best standard methods. Overall, this study represents a significant step towards tailoring treatment strategies for cancer patients through precise and efficient mutation detection technologies., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Siamak Salami reports financial support was provided by Iran National Science Foundation. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The small molecule epiberberine (EPI) is a natural alkaloid with versatile bioactivities against several diseases including cancer and bacterial infection. EPI can induce the formation of a unique binding pocket at the 5' side of a human telomeric G-quadruplex (HTG) sequence with four telomeric repeats (Q4), resulting in a nanomolar binding affinity ( K D approximately 26 nM) with significant fluorescence enhancement upon binding. It is important to understand (1) how EPI binding affects HTG structural stability and (2) how enhanced EPI binding may be achieved through the engineering of the DNA binding pocket. In this work, the EPI-binding-induced HTG structure stabilization effect was probed by a peptide nucleic acid (PNA) invasion assay in combination with a series of biophysical techniques. We show that the PNA invasion-based method may be useful for the characterization of compounds binding to DNA (and RNA) structures under physiological conditions without the need to vary the solution temperature or buffer components, which are typically needed for structural stability characterization. Importantly, the combination of theoretical modeling and experimental quantification allows us to successfully engineer Q4 derivative Q4-ds-A by a simple extension of a duplex structure to Q4 at the 5' end. Q4-ds-A is an excellent EPI binder with a K D of 8 nM, with the binding enhancement achieved through the preformation of a binding pocket and a reduced dissociation rate. The tight binding of Q4 and Q4-ds-A with EPI allows us to develop a novel magnetic bead-based affinity purification system to effectively extract EPI from Rhizoma coptidis (Huang Lian) extracts.

We demonstrate that the attachment of 30-170 bp dsDNA oligomers to ssDNA viral genomes gives a significant additional mobility shift in micelle-tagging electrophoresis (MTE). In MTE, a modified peptide nucleic acid amphiphile is attached to the viral genome to bind drag-inducing micelles present in capillary electrophoresis running buffers. Further attachment of 30-170 bp dsDNA oligomers drastically shifts the mobility of the 5.1 kB ssDNA genome of mouse minute virus (MMV), providing a new mechanism to improve resolution in CE-based analysis of kilobase nucleic acids. A model based on biased-reptation electrophoresis, end-labeled free-solution electrophoresis, and Ferguson gel-filtration theory is presented to describe the observed mobility shifts.

Enzymatic peptide synthesis is a powerful alternative to solid-phase methods, as enzymes can have high regio- and stereoselectivity and high yield and require mild reaction conditions. This is beneficial in formulation research due to the rise of nucleic acid therapies. Peptide nucleic acids (PNAs) have a high affinity toward DNA and RNA, and their solubility and cellular delivery can be improved via conjugation to peptides. Here, we designed and assessed the viability of the papain enzyme to conjugate four PNA-peptide models in water and an organic solvent using QM/MM metadynamics. We found that the reactions in water yield better results, where three conjugates could potentially be synthesized by the enzyme, with the first transition state as the rate-limiting step, with an associated energy of 14.53 kcal mol -1 , although with a slight endergonic profile. The results highlight the importance of considering the enzyme pockets and different substrate acceptivities and contribute to developing greener, direct, and precise synthetic routes for nucleic acid-based therapies. By exploring the enzyme's potential in conjunction with chemical synthesis, current protocols can be simplified for the synthesis of longer nucleic acids and peptide sequences (and, by extension, proteins) from smaller oligo or peptide blocks.

Although MYCN has been considered an undruggable target, MYCN alterations confer poor prognosis in many pediatric and adult cancers. The novel MYCN -specific inhibitor BGA002 is an antigene peptide nucleic acid oligonucleotide covalently bound to a nuclear localization signal peptide. In the present study, we characterized the pharmacokinetics (PK) of BGA002 after single and repeated administration to mice using a novel specific enzyme-linked immunosorbent assay. BGA002 concentrations in plasma showed linear PK, with dose proportional increase across the tested dose levels and similar exposure between male and female and between intravenous and subcutaneous route of administration. Repeated dosing resulted in no accumulation in plasma. Biodistribution up to 7 days after single subcutaneous administration of [ 14 C]-radiolabeled BGA002 showed broad tissues and organ distribution (suggesting a potential capability to reach primary tumor and metastasis in several body sites), with high concentrations in kidney, liver, spleen, lymph nodes, adrenals, and bone marrow. Remarkably, we demonstrated that BGA002 concentrates in tumors after repeated systemic administrations in three mouse models with MYCN amplification (neuroblastoma, rhabdomyosarcoma, and small-cell lung cancer), leading to a significant reduction in tumor weight. Taking into account the available safety profile of BGA002, these data support further evaluation of BGA002 in patients with MYCN -positive tumors.

Peptide nucleic acid (PNA) is a prominent artificial nucleic acid mimetic and modifications at the γ-position of the peptidic backbone are known to further enhance the desirable properties of PNA in terms of duplex stability. Here, we leveraged a propargyl ether modification at this position for late stage functionalization of PNA to obtain positively charged (cationic amino and guanidinium groups), negatively charged (anionic carboxylate and alkyl phosphonate groups) and neutral (PEG) PNAs to assess the impact of these charges on DNA : PNA and PNA : PNA duplex formation. Thermal stability analysis findings concurred with prior studies showing PNA : DNA duplexes are moderately more stable with cationic PNAs than anionic PNAs at physiological salt concentrations. We show that this effect is derived predominantly from differences in the association kinetics. For PNA : PNA duplexes, anionic PNAs were found to form the most stable duplexes, more stable than neutral PNA : PNA duplexes.

Peptide nucleic acids (PNAs) are charge-neutral synthetic DNA/RNA analogues. In many aspects of biology and biotechnology, the details of DNA and PNA melting reaction coordinates are crucial, and their associative/dissociative details remain inadequately understood. In the current study, we have attempted to gain insights into comparative melting pathways and binding affinity of iso-sequences of an 18-mer PNA-DNA-PNA triplex and the analogous DNA-DNA-DNA triplex, and DNA-DNA and PNA-DNA duplexes. It is intriguing that while the DNA-DNA-DNA triplex melts in two sequential steps, the PNA-DNA-PNA triplex melts in a single step and the mechanistic aspects for this difference are still not clear. We report an all-atom molecular dynamics simulation of both complexes in the temperature range of 300 to 500 K with 20 K intervals. Based on the trajectory analysis, we provide evidence that the association and dissociation are dictated by the differences in fraying-peeling effects from either terminus to the center in a zipper pattern among the PNA-DNA-PNA triplex and DNA-DNA-DNA triplexes. These are shown to be governed by the different characteristics of H-bonding, RMSD, and Free Energy Landscape (FEL) as analyzed by PCA, leading to the DNA-DNA-DNA triplex exhibiting sequential melting, while the PNA-DNA-PNA triplex shows cooperative melting of the whole fragment in a single-step. The PNA-DNA-PNA triplex base pairs are thermodynamically more stable than the DNA-DNA-DNA triplex, with the binding affinity of PNA-TFO to the PNA : DNA duplex being higher than that of DNA-TFO to the DNA : DNA duplex. The investigation of the association/dissociation of PNA-TFO to the PNA-DNA duplex has relevance and importance in the emerging effective applications of oligonucleotide therapy.

The self-assembly of peptides and peptide analogues may be exploited to develop platforms for different biomedical applications, among which CEST-MRI (chemical exchange saturation transfer magnetic resonance imaging) represents one of the most attractive techniques to be explored as a novel metal-free contrast approach in imaging acquisitions. A lysine-containing peptide sequence (LIVAGK-NH 2 , named K2) was thus modified by insertion, at the N-terminus, of a peptide nucleic acid (PNA) base, leading to a primary amine suitable for the signal generation. a-K2, c-K2, g-K2 and t-K2 peptides were synthesized and characterized. The c-K2 sequence displayed gelling properties and the Watson and Crick pairing, arising from its combination with g-K2, allowed a significant increase in the mechanical responsivity of the hydrogel. These matrices were able to generate a CEST signal around 2.5 ppm from water and, after assessing their cytocompatibility on GL261 (murine glioma), TS/a (murine breast carcinoma), and 3T3-NIH (murine fibroblasts) cell lines, their capability to work as implants for in vivo detection, was proved by intratumor injection in Balb/c mice inoculated with TS/a murine breast cancer cells.

Advantages like biocompatibility, biodegradability and tunability allowed the exploitation of peptides and peptidomimetics as versatile therapeutic or diagnostic agents. Because of their selectivity towards transmembrane receptors or cell membranes, peptides have also been identified as suitable molecules able to deliver in vivo macromolecules, proteins or nucleic acids. However, after the identification of the homodimer diphenylalanine (FF) as an aggregative motif inside the Aβ 1-42 polypeptide, short and ultrashort peptides have been studied as building blocks for the fabrication of supramolecular, ordered nanostructures for applications in biotechnological, biomedical and industrial fields. In this perspective, many hybrid molecules that combine FF with other chemical entities have been synthesized and characterized. Two novel hybrid derivatives (tFaF and cFgF), in which the FF homodimer is alternated with the peptide-nucleic acid (PNA) heterodimer "g-c" (guanine-cytosine) or "a-t" (adenine-thymine) and their dimeric forms (tFaF) 2 and (cFgF) 2 were synthesized. The structural characterization performed by circular dichroism (CD), Fourier transform infrared (FTIR) and fluorescence spectroscopies highlighted the capability of all the FF-PNA derivatives to self-assemble into β-sheet structures. As a consequence of this supramolecular organization, the resulting aggregates also exhibit optoelectronic properties already reported for other similar nanostructures. This photoemissive behavior is promising for their potential applications in bioimaging., (© 2024 European Peptide Society and John Wiley & Sons Ltd.)

Peptide nucleic acid (PNA) based antisense strategy is a promising therapeutic approach to specifically inhibit target gene expression. However, unlike protein coding genes, identification of an ideal PNA binding site for non-coding RNA is not straightforward. Here, we compare the inhibitory activities of PNA molecules that bind a non-coding 4.5S RNA called SRP RNA, a key component of the bacterial signal recognition particle (SRP). A 9-mer PNA (PNA 9 ) complementary to the tetraloop region of the RNA was more potent in inhibiting its interaction with the SRP protein, compared to an 8-mer PNA (PNA 8 ) targeting a stem-loop. PNA 9 , which contained a homo-pyrimidine sequence could form a triplex with the complementary stretch of RNA in vitro as confirmed using a fluorescent derivative of PNA 9 (F-PNA 13 ). The RNA-PNA complex formation resulted in inhibition of SRP function with PNA 9 and F-PNA 13 , but not PNA 8 highlighting the importance of target site selection. Surprisingly, F-PNA 13 which was more potent in inhibiting SRP function in vitro, showed weaker antibacterial activity compared to PNA 9 likely due to poor cell penetration of the longer PNA. Our results underscore the importance of suitable target site selection and optimum PNA length to develop better antisense molecules against non-coding RNA., (© 2024 Wiley-VCH GmbH.)

Ovarian cancer (OC) is one of the most lethal gynecologic cancers that is typically diagnosed at the very late stage of disease progression. Thus, there is an unmet need to develop diagnostic probes for early detection of OC. One approach may rely on RNA as a molecular biomarker. In this regard, FLJ22447 lncRNA is an RNA biomarker that is over-expressed in ovarian cancer (OC) and in cancer-associated fibroblasts (CAFs). CAFs appear early on in OC as they provide a metastatic niche for OC progression. FIT-PNAs (forced intercalation-peptide nucleic acids) are DNA analogs that are designed to fluoresce upon hybridization to their complementary RNA target sequence. In recent studies, we have shown that the introduction of cyclopentane PNAs into FIT-PNAs (cpFIT-PNA) results in superior RNA sensors. Herein, we report the design and synthesis of cpFIT-PNAs for the detection of this RNA biomarker in living OC cells (OVCAR8) and in CAFs. cpFIT-PNA was compared to FIT-PNA and the cell-penetrating peptide (CPP) of choice was either a simple one (four L-lysines) or a CPP with enhanced cellular uptake (CLIP6). The combination of CLIP6 with cpFIT-PNA resulted in a superior sensing of FLJ22447 lncRNA in OVCAR8 cells as well as in CAFs. Moreover, incubation of CLIP6-cpFIT-PNA in OVCAR8 cells leads to a significant decrease (ca. 60%) in FLJ22447 lncRNA levels and in cell viability, highlighting the potential theranostic use of such molecules.

Antisense oligomer (ASO)-based antibiotics that target mRNAs of essential bacterial genes have great potential for counteracting antimicrobial resistance and for precision microbiome editing. To date, the development of such antisense antibiotics has primarily focused on using phosphorodiamidate morpholino (PMO) and peptide nucleic acid (PNA) backbones, largely ignoring the growing number of chemical modalities that have spurred the success of ASO-based human therapy. Here, we directly compare the activities of seven chemically distinct 10mer ASOs, all designed to target the essential gene acpP upon delivery with a KFF-peptide carrier into Salmonella. Our systematic analysis of PNA, PMO, phosphorothioate (PTO)-modified DNA, 2'-methylated RNA (RNA-OMe), 2'-methoxyethylated RNA (RNA-MOE), 2'-fluorinated RNA (RNA-F), and 2'-4'-locked RNA (LNA) is based on a variety of in vitro and in vivo methods to evaluate ASO uptake, target pairing and inhibition of bacterial growth. Our data show that only PNA and PMO are efficiently delivered by the KFF peptide into Salmonella to inhibit bacterial growth. Nevertheless, the strong target binding affinity and in vitro translational repression activity of LNA and RNA-MOE make them promising modalities for antisense antibiotics that will require the identification of an effective carrier., (© 2024 Ghosh et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

MicroRNAs (miRNAs) are endogenous and noncoding single-stranded RNA molecules with a length of approximately 18-25 nucleotides, which play an undeniable role in early cancer screening. Therefore, it is very important to develop an ultrasensitive and highly specific method for detecting miRNAs. Here, we present a bottom-up assembly approach for modifying glass microtubes with silica nanowires (SiNWs) and develop a label-free sensing platform for miRNA-21 detection. The three-dimensional (3D) networks formed by SiNWs make them abundant and highly accessible sites for binding with peptide nucleic acid (PNA). As a receptor, PNA has no phosphate groups and exhibits an overall electrically neutral state, resulting in a relatively small repulsion between PNA and RNA, which can improve the hybridization efficiency. The SiNWs-filled glass microtube (SiNWs@GMT) sensor enables ultrasensitive, label-free detection of miRNA-21 with a detection limit as low as 1 aM at a detection range of 1 aM-100 nM. Noteworthy, the sensor can still detect miRNA-21 in the range of 10 2 -10 8 fM in complex solutions containing 1000-fold homologous interference of miRNAs. The high anti-interference performance of the sensor enables it to specifically recognize target miRNA-21 in the presence of other miRNAs and distinguish 1-, 3-mismatch nucleotide sequences. Significantly, the sensor platform is able to detect miRNA-21 in the lysate of breast cancer cell lines ( e.g ., MCF-7 cells and MDA-MB-231 cells), indicating that it has good potential in the screening of early breast cancers.

PNAzymes are a group of artificial enzymes which show promising results in selective and efficient cleavage of RNA targets. In the present study, we introduce a series of metal chelating groups based on N , N -bis(2-picolyl) groups (parent, 6-methyl and 6-amino substituted) as the active sites of novel PNAzymes. An improved synthetic route for the 6-amino analogues is described. The catalytic activity of the chelating groups for cleaving phosphodiesters were assessed with the model substrate 2-hydroxypropyl p-nitrophenyl phosphate (HPNPP), confirming that the zinc complexes have the reactivity order of parent < 2-methyl < 2-amino. The three ligands were conjugated to a PNA oligomer to form three PNAzymes which showed the same order of reactivity and some sensitivity to the size of the RNA bulge designed into the catalyst-substrate complex. This work demonstrates that the kinetic activity observed for the model substrate HPNPP could be translated onto the PNAzymes, but that more reactive Zn complexes are required for such PNAzymes to be viable therapeutic agents.

The international peptide community rejoiced when one of its most distinguished members, Morten Meldal of Denmark, shared the 2022 Nobel Prize in Chemistry. In fact, the regiospecific solid-phase "copper(I)-catalyzed 1,3-dipolar cycloaddition of terminal alkynes to azides" (CuACC) reaction-that formed the specific basis for Meldal's recognition-was reported first at the 17 th American Peptide Symposium held in San Diego in June 2001. The present perspective outlines intertwining conceptual and experimental threads pursued concurrently in Copenhagen and Minneapolis, sometimes by the same individuals, that provided context for Meldal's breakthrough discovery. Major topics covered include orthogonality in chemistry; the dithiasuccinoyl (Dts) protecting group for amino groups in α-amino acids, carbohydrates, and monomers for peptide nucleic acids (PNA); and poly(ethylene glycol) (PEG)-based solid supports such as PEG-PS, PEGA, and CLEAR [and variations inspired by them] for solid-phase peptide synthesis (SPPS), solid-phase organic synthesis (SPOS), and combinatorial chemistry that can support biological assays in aqueous media.

Peptide nucleic acids (PNAs) are synthetic molecules that are like DNA/RNA, but with different building blocks. PNAs target and bind to mRNAs and disrupt the function of a targeted gene, hence they have been studied as potential antibacterials. The aim of this systematic review was to provide an in-depth analysis of the current status of PNAs as antibacterial agents, define the characteristics of the effective PNA constructs, and address the gap in advancing PNAs to become clinically competent agents. Following the PRISMA model, four electronic databases were searched: Web of Science, PubMed, SciFinder and Scopus. A total of 627 articles published between 1994 and 2023 were found. After screening and a rigorous selection process using explicit inclusion and exclusion criteria, 65 scientific articles were selected, containing 656 minimum inhibitory concentration (MIC) data. The antibacterial activity of PNAs was assessed against 20 bacterial species. The most studied Gram-negative and Gram-positive bacteria were Escherichia coli (n=266) and Staphylococcus aureus (n=53), respectively. In addition, the effect of PNA design, including construct length, binding location, and carrier agents, on antibacterial activity was shown. Finally, antibacterial test models to assess the inhibitory effects of PNAs were examined, emphasising gaps and prospects. This systematic review provides a comprehensive assessment of the potential of PNAs as antibacterial agents and offers valuable insights for researchers and clinicians seeking novel therapeutic strategies in the context of increasing rates of antibiotic-resistant bacteria., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Peptide nucleic acid (PNA) is a unique combination of peptides and nucleic acids. PNA can exhibit hydrogen bonding interactions with complementary nucleobases like DNA/RNA. Also, its polyamide backbone allows easy incorporation of biomolecules like peptides and proteins to build hybrid molecular constructs. Because of chimeric structural properties, PNA has lots of potential to build diverse nanostructures. However, progress in the PNA material field is still immature compared with its massive applications in antisense oligonucleotide research. Examples of well-defined molecular assemblies have been reported with PNA amphiphiles, self-assembling guanine-PNA monomers/dimers, and PNA-decorated nucleic acids/ polymers/ peptides. All these works indicate the great potential of PNA to be used as bionanomaterials. The review summarizes the recent reports on PNA-based nanostructures and their versatile applications. Additionally, this review shares a perspective to promote a better understanding of controlling molecular assembly by the systematic structural modifications of PNA monomers., (© 2023 Wiley Periodicals LLC.)

Over the last three decades, our view of RNA has changed from a simple intermediate supporting protein synthesis to a major regulator of biological processes. In the expanding area of RNA research, peptide nucleic acid (PNA) is emerging as a promising ligand for triple-helical recognition of complex RNAs. As discussed in this feature article, the key advantages of PNAs are high sequence specificity and affinity for RNA (>10 fold higher than for DNA) that are difficult to achieve with small molecule ligands. Emerging studies demonstrate that triple-helical binding of PNAs can modulate biological function and control dynamic conformational equilibria of complex folded RNAs. These results suggest that PNA has a unique potential as a research tool and therapeutic compound targeting RNA. The remaining problems hampering advances in these directions are limitations of sequences that can be recognized by Hoogsteen triplexes (typically purine rich tracts), poor cellular uptake and bioavailability of PNA, and potential off-target effects in biological systems. Recent exciting studies are discussed that illustrate how synthetic nucleic acid chemistry provides innovative solutions for these problems.

Programmable site-specific nucleases (SSNs) hold extraordinary promise to unlock myriad gene editing applications in medicine and agriculture. However, developing small and specific SSNs is needed to overcome the delivery and specificity translational challenges of current genome engineering technologies. Structure-guided nucleases have been harnessed to generate double-strand DNA breaks but with limited success and translational potential. Here, we harnessed the power of peptide nucleic acids (PNAs) for site-specific DNA invasion and the generation of localized DNA structures that are recognized and cleaved by the eukaryotic resolvase AtMOC1 from Arabidopsis thaliana . We named this technology PN A-assisted R esolvase-mediated (PNR) editing. We tested the PNR editing concept in vitro and demonstrated its precise target specificity, examined the nucleotide requirement around the PNA invasion for the AtMOC1-mediated cleavage, mapped the AtMOC1-mediated cleavage sites, tested the role of different types and lengths of PNA molecules invasion into dsDNA for the AtMOC1-mediated cleavage, optimized the in vitro PNA invasion and AtMOC1 cleavage conditions such as temperature, buffer conditions, and cleavage time points, and demonstrated the multiplex cleavage for precise fragment release. We discuss the best design parameters for efficient, site-specific in vitro cleavage using PNR editors.

Herein, peptide nucleic acids (PNAs) are employed in the design of a participatory duplex PNA-peptide crosslinking agent. Biophysical and mechanical studies show that crosslinkers present during peptide assembly leading to hydrogelation participate in the formation of fibrils while simultaneously installing crosslinks into the higher-order network that constitutes the peptide gel. The addition of 2 mol % crosslinker into the assembling system results in a ~100 % increase in mechanical stiffness without affecting the rate of peptide assembly or the local morphology of fibrils within the gel network. Stiffness enhancement is realized by only affecting change in the elastic component of the viscoelastic gel. A synthesis of the PNA-peptide duplex crosslinkers is provided that allows facile variation in peptide composition and addresses the notorious hydrophobic content of PNAs. This crosslinking system represents a new tool for modulating the mechanical properties of peptide-based hydrogels., (© 2023 Wiley-VCH GmbH.)