Bacteria secrete various iron-chelators (siderophores), which scavenge Fe3+ from the environment, bind it with high affinity, and retrieve it inside the cell. After the Fe3+ uptake, bacteria extract the soluble iron(II) from the siderophore. Ferric siderophores are transported inside the cell via the TonB-dependent receptor system. Importantly, siderophore uptake paths have been also used by sideromycins, natural antibiotics. Our goal is to hijack the transport system for hydroxamate-type siderophores to deliver peptide nucleic acid oligomers into Escherichia coli cells. As siderophore mimics we designed and synthesized linear and cyclic Nδ-acetyl-Nδ-hydroxy-l-ornithine based peptides. Using circular dichroism spectroscopy, we found that iron(III) is coordinated by the linear trimer with hydroxamate groups but not by the cyclic peptide. The internal flexibility of the linear siderophore oxygen atoms and their interactions with Fe3+ were confirmed by all-atom molecular dynamics simulations. Using flow cytometry we found that the designed hydroxamate trimer transports PNA oligomers inside the E. coli cells. Growth recovery assays on various E. coli mutants suggest the pathway of this transport through the FhuE outer-membrane receptor, which is responsible for the uptake of the natural iron chelator, ferric-coprogen. This pathway also involves the FhuD periplasmic binding protein. Docking of the siderophores to the FhuE and FhuD receptor structures showed that binding of the hydroxamate trimer is energetically favorable corroborating the experimentally suggested uptake path. Therefore, this siderophore mimic, as well as its conjugate with PNA, is most probably internalized through the hydroxamate pathway.

In recent years, nucleic acids have emerged as powerful biomaterials, revolutionizing the field of biomedicine. This review explores the multifaceted applications of nucleic acids, focusing on their pivotal role in various biomedical applications. Nucleic acids, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), possess unique properties such as molecular recognition ability, programmability, and ease of synthesis, making them versatile tools in biosensing and for gene regulation, drug delivery, and targeted therapy. Their compatibility with chemical modifications enhances their binding affinity and resistance to degradation, elevating their effectiveness in targeted applications. Additionally, nucleic acids have found utility as self-assembling building blocks, leading to the creation of nanostructures whose high order underpins their enhanced biological stability and affects the cellular uptake efficiency. Furthermore, this review delves into the significant role of oligonucleotides (ODNs) as indispensable tools for biological studies and biomarker discovery. ODNs, short sequences of nucleic acids, have been instrumental in unraveling complex biological mechanisms. They serve as probes for studying gene expression, protein interactions, and cellular pathways, providing invaluable insights into fundamental biological processes. By examining the synergistic interplay between nucleic acids as powerful biomaterials and ODNs as indispensable tools for biological studies and biomarkers, this review highlights the transformative impact of these molecules on biomedical research. Their versatile applications not only deepen our understanding of biological systems but also are the driving force for innovation in diagnostics and therapeutics, ultimately advancing the field of biomedicine.

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.

Peptide nucleic acid (PNA) is a nucleic acid mimic with high specificity and binding affinity to natural DNA or RNA, as well as resistance to enzymatic degradation. PNA sequences can be designed to selectively silence gene expression, which makes PNA a promising tool for antimicrobial applications. However, the poor membrane permeability of PNA remains the main limiting factor for its applications in cells. To overcome this obstacle, PNA conjugates with different molecules have been developed. This mini-review focuses on covalently linked conjugates of PNA with cell-penetrating peptides, aminosugars, aminoglycoside antibiotics, and non-peptidic molecules that were tested, primarily as PNA carriers, in antibacterial and antiviral applications. The chemistries of the conjugation and the applied linkers are also discussed.

Robust and sensitive methods for the detection of microRNAs (miRNAs) are crucial in the clinical diagnosis of cancers. In this study, a novel electrochemical biosensor with high sensitivity for miRNA-21 detection is developed, which relies on the formation of a peptide nucleic acid (PNA)-DNA hetero-three-way junction (H3WJ) and target-recycling catalytic hairpin assembly (CHA) amplification. The electroneutral PNA probes are initially immobilized onto a gold electrode to construct the sensor. Upon introduction of miRNA-21, target-recycling CHA is initiated, resulting in abundant double-stranded CHA products. Subsequently, association between the PNA probes and these products leads to the formation of PNA-DNA H3WJs. Consequently, the electrode surface is densely populated with numerous electroactive Ferrocene (Fc) groups, resulting in a significantly amplified current response for highly sensitive detection of miRNA-21 at concentrations as low as 0.15 fM. This approach demonstrates remarkable specificity towards target miRNAs and can be utilized for quantitative monitoring of miRNA-21 expression in human cancer cells. More importantly, the sensor exhibits exceptional stability and shows a significant reduction in background noise during miRNA detection, making this method a highly promising sensing platform for monitoring various miRNA biomarkers to facilitate the diagnosis of diverse cancers.

Cystic fibrosis (CF) is an autosomal recessive disease impacting ∼100,000 people worldwide. This lethal disorder is caused by mutation of the CF transmembrane conductance regulator (CFTR) gene, which encodes an ATP-binding cassette-class C protein. More than 2,100 variants have been identified throughout the length of CFTR. These defects confer differing levels of severity in mRNA and/or protein synthesis, folding, gating, and turnover. Drug discovery efforts have resulted in recent development of modulator therapies that improve clinical outcomes for people living with CF. However, a significant portion of the CF population has demonstrated either no response and/or adverse reactions to small molecules. Additional therapeutic options are needed to restore underlying genetic defects for all patients, particularly individuals carrying rare or refractory CFTR variants. Concerted focus has been placed on rescuing variants that encode truncated CFTR protein, which also harbor abnormalities in mRNA synthesis and stability. The current mini-review provides an overview of CFTR mRNA features known to elicit functional consequences on final protein conformation and function, including considerations for RNA-directed therapies under investigation. Alternative exon usage in the 5\'-untranslated region, polypyrimidine tracts, and other sequence elements that influence splicing are discussed. Additionally, we describe mechanisms of CFTR mRNA decay and post-transcriptional regulation mediated through interactions with the 3\'-untranslated region (e.g. poly-uracil sequences, microRNAs). Contributions of synonymous single nucleotide polymorphisms to CFTR transcript utilization are also examined. Comprehensive understanding of CFTR RNA biology will be imperative for optimizing future therapeutic endeavors intended to address presently untreatable forms of CF.

Exon skipping is an efficient technique to inhibit specific gene expression induced by a short-sequence peptide nucleic acid (PNA). To date, there has been no study on the effects of PNA on skin pigmentation. In melanocytes, the tripartite complex is responsible for the transport of mature melanosomes from the nucleus to the dendrites. The tripartite complex is composed of Rab27a, Mlph (Melanophilin), and Myosin Va. Defects in the protein Mlph, a melanosome transport-related protein, are known to cause hypopigmentation. Our study shows that Olipass peptide nucleic acid (OPNA), a cell membrane-permeable PNA, targets exon skipping in the Mlph SHD domain, which is involved in Rab27a binding. Our findings demonstrate that OPNA induced exon skipping in melan-a cells, resulting in shortened Mlph mRNA, reduced Mlph protein levels, and melanosome aggregation, as observed by microscopy. Therefore, OPNA inhibits the expression of Mlph by inducing exon skipping within the gene. These results suggest that OPNA, which targets Mlph, may be a potential new whitening agent to inhibit melanosome movement.

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.

Legionella are opportunistic intracellular pathogens that are found throughout the environment. The Legionella contamination of water systems represents a serious social problem that can lead to severe diseases, which can manifest as both Pontiac fever and Legionnaires\' disease (LD) infections. Fluorescence in situ hybridization using nucleic acid mimic probes (NAM-FISH) is a powerful and versatile technique for bacterial detection. By optimizing a peptide nucleic acid (PNA) sequence based on fluorescently selective binding to specific bacterial rRNA sequences, we established a new PNA-FISH method that has been successfully designed for the specific detection of the genus Legionella. The LEG22 PNA probe has shown great theoretical performance, presenting 99.9% specificity and 96.9% sensitivity. We also demonstrated that the PNA-FISH approach presents a good signal-to-noise ratio when applied in artificially contaminated water samples directly on filtration membranes or after cells elution. For water samples with higher turbidity (from cooling tower water systems), there is still the need for further method optimization in order to detect cellular contents and to overcome interferents\' autofluorescence, which hinders probe signal visualization. Nevertheless, this work shows that the PNA-FISH approach could be a promising alternative for the rapid (3-4 h) and accurate detection of Legionella.

The physical and chemical properties of the outer membrane of Gram-negative bacteria including Escherichia coli have a significant impact on the antibacterial activity and uptake of antibiotics, including antimicrobial peptides and antisense peptide-peptide nucleic acid (PNA) conjugates. Using a defined subset of E. coli lipopolysaccharide (LPS) and envelope mutants, components of the LPS-core, which provide differential susceptibility toward a panel of bacterial penetrating peptide (BPP)-PNA conjugates, were identified. Deleting the outer core of the LPS and perturbing the inner core only sensitized the bacteria toward (KFF)3K-PNA conjugates, but not toward conjugates carrying arginine-based BPPs. Interestingly, the chemical composition of the outer LPS core as such, rather than overall hydrophobicity or surface charge, appears to determine the susceptibility to different BPP-PNA conjugates thereby clearly demonstrating the complexity and specificity of the interaction with the LPS/outer membrane. Notably, mutants with outer membrane changes conferring polymyxin resistance did not show resistance toward the BPP-PNA conjugates, thereby eliminating one possible route of resistance for these molecules. Finally, envelope weakening, through deletion of membrane proteins such as OmpA as well as some proteins previously identified as involved in cationic antimicrobial peptide uptake, did not significantly influence BPP-PNA conjugate activity.