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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 outermembrane 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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.

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. [ABSTRACT FROM AUTHOR]

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 cassetteclass 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 posttranscriptional 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. [ABSTRACT FROM AUTHOR]

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.

Cystic fibrosis (CF) is an autosomal recessive disease impacting ∼100,000 people worldwide. This lethal disorder is caused by mutation of the CFtransmembrane 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.

The analysis of 18S rRNA gene amplicons is an important tool to characterize the diversity of the eukaryotic soil microbiome. Here we analyzed two primer sets (TAReuk, EKeuk) and the impact of a newly designed antifungal peptide nucleic acid to enhance the detection of protists in silico and with soil DNA extracted from croplands and a forest. The in silico analyses showed for TAReuk pronounced specificities for protist SAR supergroup and Metazoa, while EKeuk was particularly specific for Ascomycota and Basidiomycota. In silico , the PNA matched with the majority of Ascomycota (81.3 %) and Basidiomycota (65.4 %), but with <6 % of protists. The contrasting primer specificities were confirmed with soil DNA, but the proportion of protist amplicons was similar. In contrast to in silico , effects of the PNA were not as clear with soil DNA, even though it completely inhibited the amplification of the targeted fungal sequences. PNA effects were more pronounced with TAReuk, and results with cropland and forest soil DNA were not consistent, e.g. , for cropland, PNA decreased the relative abundance of fungi but for forest it was the opposite, possibly because of different fungal diversity. The divergence between PNA in silico -predictions and results with soil DNA are likely an outcome of primer binding to <100 % complementary target sequences and a still limited DNA sequence databases for soil microbial eukaryotes. With TAReuk, the presence of PNA enhanced the detection of Conosa and, thus, could be a useful tool to study this group in the future. • A new PNA probe inhibited in silico PCR amplification of most fungal 18S rRNA genes. • With soil DNA, the PNA probe did not inhibit amplification of fungal 18S rRNA genes. • PNA effects on detection of protists varied between two 18S rRNA primer pairs. • PNA effects varied between DNA extracted from five different soils. • With the common TAReuk, protist primers PNA increased the detection of Conosa. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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.

Unlike conventional triplex‐forming oligonucleotide (TFO), triplex‐forming peptide nucleic acid (PNA) can tightly bind with double‐stranded RNA (dsRNA) than double‐stranded DNA (dsDNA). Here, we performed spectroscopic, thermodynamic and kinetic experiments for triplex formation by PNA to examine different binding behaviors between PNA − dsRNA and PNA − dsDNA triplexes. We found 9‐mer PNA (cytosine content of 66%) formed the thermally stable triplex with dsRNA compared to dsDNA over a wide range of pH (5.5‐8.0), salt concentration (50‐500 mM NaCl). Both the calorimetric binding constant and the association rate constant for dsRNA were larger than those for dsDNA, indicating the favorable association process for the PNA − dsRNA triplex formation. Comparison with the DNA/RNA heteroduplexes revealed that the DNA strand was detrimental to the triplex stability for PNA, a contrasting result for conventional TFO. The keys underlying the difference in the triplex formation of PNA with different duplexes appear to be the conformational adoptability and the geometric compatibility of PNA to fit the deep, narrow major groove of dsRNA and the helical rigidity difference of the duplexes. Our results emphasize the importance of both the sugar puckering of the duplex and the appropriate conformational flexibility of PNA for the triplex formation. [ABSTRACT FROM AUTHOR]

Bacterial vaginosis (BV) is the most common vaginal infection in women of reproductive age and has been associated with serious health complications, mainly in pregnant women. It is characterized by a decrease in the number of Lactobacillus species in the healthy vaginal microbiota and an overgrowth of strict and facultative anaerobic bacteria that develop a polymicrobial biofilm. Despite over 60 years of research investigating BV, its etiology is not fully understood. Gardnerella spp. is a crucial microorganism that contributes to the formation of the biofilm and the development of BV, but the role of other BV-associated bacteria is not clear. Nevertheless, Fannyhessea vaginae (previously known as Atopobium vaginae) is a highly specific species for BV, and co-colonization with Gardnerella is thought to be a very specific diagnostic marker. The diagnosis of BV still presents some limitations, since currently used methods often fail to accurately detect BV. This work aims to develop a novel peptide nucleic acid (PNA) probe targeting F. vaginae. This probe was further validated in a multiplex assay, which included a Gardnerella- specific PNA probe, as a possible method for diagnosis of BV, and was compared with quantification by qPCR. The new PNA probe showed excellent sensitivity and specificity and could discriminate F. vaginae - Gardnerella biofilms, confirming the potential to be used for the detection of BV-associated pathogens. [ABSTRACT FROM AUTHOR]

Bacterial vaginosis (BV) is the most common vaginal infection in women of reproductive age and has been associated with serious health complications, mainly in pregnant women. It is characterized by a decrease in the number of Lactobacillus species in the healthy vaginal microbiota and an overgrowth of strict and facultative anaerobic bacteria that develop a polymicrobial biofilm. Despite over 60 years of research investigating BV, its etiology is not fully understood. Gardnerella spp. is a crucial microorganism that contributes to the formation of the biofilm and the development of BV, but the role of other BV-associated bacteria is not clear. Nevertheless, Fannyhessea vaginae (previously known as Atopobium vaginae) is a highly specific species for BV, and co-colonization with Gardnerella is thought to be a very specific diagnostic marker. The diagnosis of BV still presents some limitations, since currently used methods often fail to accurately detect BV. This work aims to develop a novel peptide nucleic acid (PNA) probe targeting F. vaginae. This probe was further validated in a multiplex assay, which included a Gardnerella-specific PNA probe, as a possible method for diagnosis of BV, and was compared with quantification by qPCR. The new PNA probe showed excellent sensitivity and specificity and could discriminate F. vaginae-Gardnerella biofilms, confirming the potential to be used for the detection of BV-associated pathogens.

• PNAs exhibited antibacterial efficiency at a micromolar dose. • Efficiency of PNAs is influenced by several design factors. • Optimisation of the delivery system and infection testing models is required. • PNAs have potential as target-based antibacterial agents. 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. [Display omitted] [ABSTRACT FROM AUTHOR]

Photoswitchable oligonucleotides can determine specific biological outcomes by light-induced conformational changes. In particular, artificial probes activated by visible-light irradiation are highly desired in biological applications. Here, we report two novel types of visible-light photoswitchable peptide nucleic acids (PNAs) based on the molecular transducers: hemithioindigo and tetra-ortho-fluoroazobenzene. Our study reveals that the tetra-ortho-fluoroazobenzene–PNA conjugates have promising properties (fast reversible isomerization, exceptional thermal stability, high isomer conversions and sensitivity to visible-light irradiation) as reversible modulators to control oligonucleotide hybridization in biological contexts. Furthermore, we verified that this switchable modification delivers a slightly different hybridization behavior in the PNA. Thus, both melting experiments and strand-displacement assays showed that in all the cases the trans-isomer is the one with superior binding affinities. Alternative versions, inspired by our first compounds here reported, may find applications in different fields such as chemical biology, nanotechnology and materials science.

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.

Peptide Nucleic Acid (PNA) serves as an artificial functional analog of DNA. Being immune to enzymatic degradation and possessing strong affinity towards DNA and RNA, it is an ideal candidate for many medical and biotechnological applications that are of antisense and antigene in nature. PNAs are anticipated to have its application in DNA and RNA detection as well as quantification, to serve as antibacterial and antiviral agents, and silencing gene for developing anticancer strategies. Although, their restricted entry in both eukaryotic and prokaryotic cells limit their applications. In addition, aggregation of PNA in storage containers reduces the quality and quantity of functional PNA that makes it inadequate for their mass production and storage. To overcome these limitations, researchers have modified PNA either by the addition of diverse functional groups at various loci on its backbone, or by synthesizing chimeras with other moieties associated with various delivery agents that aids their entry into the cell. Here, this review article summarizes few of the structural modifications that are performed with PNA, methods used to improve their cellular uptake and shedding light on the applications of PNA in various prospects in biological sciences. [ABSTRACT FROM AUTHOR]

It has been reported that dysregulation of microRNA-155 expression and function is associated with tumorigenesis, growth, tumor subtypes, invasion, and poor survival rates. Peptide nucleic acid (PNA), an artificially synthesized nucleic acid mimic, has been applied for molecular diagnosis. In this study, a PNA sequence that undergoes complementary binding to miR-155 was labeled with 99mTc to evaluate whether the tracer could visualize the expression of miR-155 in breast cancer. Both antisense PNA (anti-PNA, fully complementary bound to human mature miR-155, referred to as "anti-PNA-155") and mismatched PNA (referred to as "mis-PNA") single strands containing 23-mer were synthesized. The relative expression of miR-155 in MCF-7 cells and tumors was higher than that in MDA-MB-231 cells and tumors. Single-photon emission computed tomography (SPECT) scan showed that radioactivity mainly accumulated in kidney. MCF-7 tumors, but not MDA-MB-231 tumors, were clearly visualized after [99mTc]anti-PNA-155 injection. MCF-7 tumors were less visible when coinjected with 100-fold excess of anti-PNA-155 or injected with [99mTc]mis-PNA, which suggested specific binding. Biodistribution study results were consistent with SPECT imaging. We successfully demonstrated that [99mTc]anti-PNA-155 could visualize miR-155 expression in vivo, suggesting it may be a promising probe applied in breast cancer. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • An electrochemical biosensor for miRNA-21 based on PNA-DNA H3WJ formation and CHA amplification. • PNA-DNA H3WJs exhibits superior thermal stability compared to their DNA counterparts. • It has a low LOD of 0.15 fM and a wide linear dynamic range spanning from 0.5 fM to 5 nM. • It exhibits outstanding stability and exceptional selectivity towards the target miRNAs. [ABSTRACT FROM AUTHOR]

In the field of anti-aging research, a groundbreaking study introduces a novel isothermal amplification strategy for the rapid and sensitive detection of Matrix Metalloproteinase 2 (MMP-2) utilizing a bipedal DNA walker. MMP-2 plays a crucial role in skin aging due to its involvement in the degradation of collagen and elastin, key components of the extracellular matrix. The dysregulation of MMP-2 activity contributes to the breakdown of the extracellular matrix, leading to manifestations of skin aging such as wrinkles, sagging, and loss of elasticity. This research presents a novel method for detecting MMP-2 using a bipedal DNA walker. The method uses peptide nucleic acid (PNA) as a substrate for MMP-2, which can release a bandage strand that forms the bipedal walker "leg" with two DNA strands. The bipedal walker "leg" initiates the bipedal walker isothermal amplification strategy that involves entropy-driven DNA walking and catalytic hairpin assembly on a PEI@Ru(bpy) 3 2+-Ti 3 C 2 @AuNPs-modified electrode. The walker's motion produces an electrochemiluminescence (ECL) signal that can be rapidly analyzed by a biosensor. The detection limit of this method is 80.6 fM, which is significantly lower than other methods. Overall, this study represents an important step towards the development of effective anti-aging research methods. [Display omitted] • Novel isothermal amplification strategy for rapid and sensitive detection of MMP2. • Utilizes a bipedal DNA walker and peptide nucleic acid (PNA) as a substrate for MMP2. • High sensitivity, specificity, and versatility for MMP2 detection. • Detection limit of 80.6 fM, significantly lower than other methods for MMP2 detection. • Potential applications in biotechnology, medicine, and environmental science. [ABSTRACT FROM AUTHOR]

Peptide-oligonucleotide conjugates (POCs) represent one of the increasingly successful albeit costly approaches to increasing the cellular uptake, tissue delivery, bioavailability, and, thus, overall efficiency of therapeutic nucleic acids, such as, antisense oligonucleotides and small interfering RNAs. This review puts the subject of chemical synthesis of POCs into the wider context of therapeutic oligonucleotides and the problem of nucleic acid drug delivery, cell-penetrating peptide structural types, the mechanisms of their intracellular transport, and the ways of application, which include the formation of non-covalent complexes with oligonucleotides (peptide additives) or covalent conjugation. The main strategies for the synthesis of POCs are viewed in detail, which are conceptually divided into (a) the stepwise solid-phase synthesis approach and (b) post-synthetic conjugation either in solution or on the solid phase, especially by means of various click chemistries. The relative advantages and disadvantages of both strategies are discussed and compared.

Colorectal cancer (CRC) is the third most common cancer in the world and its disease-specific mortality is estimated to be approximately 33% in the developed world. KRAS mutations have been shown to predict response to anti-EGFR (epidermal growth factor receptor) targeted monoclonal antibody therapy. Therefore, KRAS mutation testing of metastatic CRC patients is mandatory in the clinical setting to aid in the choice of appropriate therapy. Currently, the most common strategy for KRAS mutation detection consists of conventional polymerase chain reaction (PCR) and direct sequencing. However, it is a time-consuming and complicated procedure, not suitable for routine clinical test. The objective of this study is to develop and evaluate a highly sensitive and rapid method using peptide nucleic acid (PNA) oligomers mediated real-time PCR clamping for detection of KRAS mutations. The PNA-mediated PCR clamping assay can real-time detect a mutation in a sample containing 1% of the mutant allele in a mixture of wild-type genomic DNA, which also enables the accurate and rapid detection of all KRAS codon 12 and 13 mutations in a single reaction. The total assay time is short as it requires only 1.5 hours after the samples preparation. Thus, the present method offers a potential alternative to be applied in clinical samples of CRC for detection of DNA carrying KRAS mutations.

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.

The search for new, non-standard targets is currently a high priority in the design of new antibacterial compounds. Bacterial toxin–antitoxin systems (TAs) are genetic modules that encode a toxin protein that causes growth arrest by interfering with essential cellular processes, and a cognate antitoxin, which neutralizes the toxin activity. TAs have no human analogs, are highly abundant in bacterial genomes, and therefore represent attractive alternative targets for antimicrobial drugs. This study demonstrates how artificial activation of Escherichia coli mazEF and hipBA toxin–antitoxin systems using sequence-specific antisense peptide nucleic acid oligomers is an innovative antibacterial strategy. The growth arrest observed in E. coli resulted from the inhibition of translation of the antitoxins by the antisense oligomers. Furthermore, two other targets, related to the activities of mazEF and hipBA, were identified as promising sites of action for antibacterials. These results show that TAs are susceptible to sequence-specific antisense agents and provide a proof-of-concept for their further exploitation in antimicrobial strategies.

Infections by carbapenem-resistant A. baumannii (CRAB), a widespread nosocomial pathogen, are becoming increasingly difficult to prevent and treat. Therefore, there is an urgent need for discovery of novel antibiotics against CRAB. Programmable, precision antisense antibiotics, e.g., based on the nucleic acid mimic PNA (peptide nucleic acid) have shown promise in this respect in the form of PNA-BPP (bacteria penetrating peptide) conjugates targeting essential bacterial genes. In the present study, we designed and synthesized a series of PNA-BPPs targeting the translation initiation region of the ftsZ, acpP, or rne gene of CRAB strains. The antimicrobial activity of the compounds and effects on gene expression level was compared to that of analogous mismatch PNA controls. Three antisense conjugates (KFF)3K-eg1-(acpP)PNA (5639), (KFF)3K-eg1-(ftsZ)PNA (5612), and (KFF)3-K-eg1-(rne)PNA (5656) exhibited complete growth inhibition against several CRAB strains at 1–2, 2–8, and 2 µM, respectively, and the compounds were bactericidal at 1–2× MIC. The bactericidal effect was correlated to reduction of target gene mRNA level using RT-qPCR, and the compounds showed no bacterial membrane disruption activity at 1–2× MIC. PNA5612 was tested against a series of 12 CRAB isolates and all were sensitive at 2–8 µM. In addition, the conjugates exhibited no cellular toxicity in the HepG2 cell line (up to 20 μM) and did not shown significant antibacterial activity against other Gram negatives (E. coli, P. aeruginosa). These results provide a starting point for discovery of antisense precision designer antibiotics for specific treatment of CRAB infections.

miRNAs are well known for being implicated in a myriad of biological situations, including those related to serious diseases. Amongst miRNAs, miRNA-21 has the spotlight as it is reported to be up-regulated in multiple severe pathological conditions, being its quantification a key point in medicine. To date, most of the techniques for miRNA quantification have shown to be less effective than expected; thus, we herein present a novel, rapid, cost-effective, robust and PCR-free approach, based on dynamic chemistry, for the identification and quantification of miRNA directly from tumour cells using both FACS and a fluorescent microplate. This dynamic chemistry novel application involves bead based reagents and allows quantifying the number of miR-21 molecules presented in MDA-MB-468 and H1975 tumour cells. fx1 • Chem-NAT based protocol to count miRNA molecules per cell with single base resolution directly from cell lysates. • Chem-NAT implementation for miRNA quantification with standard FACS and fluorescence microplate readers. • Three and half hours protocol from cell pellet to result which do not require nucleic acid amplification steps. • Quantification of miR-21 molecules presented in tumour cells MDA-MB-468 and H1975. [ABSTRACT FROM AUTHOR]

The analogs of DNA are unique biomedical tools that are broadly utilized to develop different types of biosensors. Peptide nucleic acids (PNA) are an individual and notable class of nucleic acid analogs due to their unique, novel physicochemical and biochemical characteristics, stability and resistance to nuclease and protease enzymes, significant interactions with complementary strands and remarkable hybridization attributes. Therefore, they are employed in the preparation and fabrication of various types of functional biosensors. In other words, immobilization of PNA as an appropriate diagnostic probe on the surface of electrochemical and optical converters lead to the fabrication of PNA-based biosensors which could be applied for sensitive and selective determination of diverse analytes. In this article, we are purposed to demonstrate an overview of physicochemical and biochemical features, modifications of peptide nucleic acid backbone and its usages as a bio-receptor in the PNA-based biosensors in recognition of viruses, RNA, bacteria, DNA, SNP (single-nucleotide polymorphism) and etc. Application of Peptide Nucleic Acid as bioreceptor in biosensors development. Image 1 • Physicochemical and biochemical characteristics of peptide nucleic acid was explained. • The plus points of PNA over DNA-based biosensors were described. • Application of PNA in different biosensors such as electrochemical, optical, and microarrays was explained. • PNA as a bio-receptor in biosensors was utilized for viruses, RNA, bacteria, DNA, SNP, point mutations detection. [ABSTRACT FROM AUTHOR]

The development of new RNA‐binding ligands is attracting increasing interest in fundamental science and the pharmaceutical industry. The goal of this study was to improve the RNA binding properties of triplex‐forming peptide nucleic acids (PNAs) by further increasing the pKa of 2‐aminopyridine (M). Protonation of M was the key for enabling triplex formation at physiological pH in earlier studies. Substitution on M by an electron‐donating 4‐methoxy substituent resulted in slight destabilization of the PNA–dsRNA triplex, contrary to the expected stabilization due to more favorable protonation. To explain this unexpected result, the first NMR structural studies were performed on an M‐modified PNA–dsRNA triplex which, combined with computational modeling identified unfavorable steric and electrostatic repulsion between the 4‐methoxy group of M and the oxygen of the carbonyl group connecting the adjacent nucleobase to PNA backbone. The structural studies also provided insights into hydrogen‐bonding interactions that might be responsible for the high affinity and unusual RNA‐binding preference of PNAs. RNA binding improved: A 2‐aminopyridine (M) nucleobase enables strong and selective triple helical binding of peptide nucleic acid (PNA) to dsRNA. However, further modification of M with the electron‐donating 4‐methoxy group did not enhance the binding affinity. NMR structural studies showed that this is likely due to steric and electronic repulsion (denoted by the red spheres) between the oxygen of 4‐methoxy group and the oxygen of the carbonyl connecting the adjacent PNA nucleobase to the PNA backbone. [ABSTRACT FROM AUTHOR]

We aim to understand the structure and stability of the backbone tailored Watson-Crick base pairs, Guanine-Cytosine (GC), Adenine-Thymine (AT) and Adenine-Uracil (AU) by incorporating N-(2-aminoethyl) glycine units (linked by amide bonds) at the purine and pyrimidine sites of the nucleobases. Density functional theory (DFT) is employed in which B3LYP/6-311++G∗∗ level of theory has been used to optimize all the structures. The peptide attached base pairs are compared with the natural deoxyribose nucleic acid (DNA)/ribonucleic acid (RNA) base pairs and the calculations are carried out in both the gas and solution phases. The structural propensities of the optimized base pairs are analyzed using base pair geometries, hydrogen bond distances and stabilization energies and, compared with the standard reference data. The structural parameters were found to correlate well with the available data. The addition of peptide chain at the back bone of the DNA/RNA base pairs results only with a minimal distortion and hence does not alter the structural configuration of the base pairs. Also enhanced stability of the base pairs is spotted while adding peptidic chain at the purine site rather than the pyrimidine site of the nucleobases. The stability of the complexes is further interpreted by considering the hydrogen bonded N–H stretching frequencies of the respective base pairs. The discrimination in the interaction energies observed in both gas and solution phases are resulted due to the existence of distinct lowest unoccupied molecular orbitals (LUMO) in the solution phase. The reactivity of the base pairs is also analyzed through the in-depth examinations on the highest occupied molecular orbital (HOMO)-LUMO orbitals. [ABSTRACT FROM AUTHOR]

Abstract Graphene-oxide (GO) quenching-based molecular beacon was developed for rapid and sensitive detection of RNAs in living cells and tissues. Here, we applied GO quenching-based molecular beacon sensor to visualize neurogenic miR-193a levels delivered via exosomes during cell-non-autonomous neurogenesis in neural progenitor cells on a microfluidic platform. Exosomal transport was visualized using CD63-RFP plasmid vector, and FAM-labeled peptide nucleic acid (PNA) probe for the miR-193 sequence was designed to detect endogenous miR-193 expression. Fluorescence signals of FAM-PNA193a-GO were recovered in dibutyryl-cAMP-induced F11 cells, resulting from increased expression of miR-193a after neuronal differentiation. We observed delivery of miR-193a-containing exosomes released from differentiated donor F11 cells to recipient undifferentiated F11 cells. Fluorescence recovery was evident in exosome-stimulated recipient individual F11 cells in the microfluidic system. We propose molecular beacon imaging using PNA-GO complex for visualization of individual cellular expression of mature microRNAs. This system reveals the precise spatial localization and temporal sequences of mature miRNAs by intercellular exosomal delivery of messages for processes such as cell-non-autonomous neurogenesis. Highlights • PNA-GO probe has high selectivity for target miR-193a. • It provides more sensitive imaging compared to other reported methods. • PNA-GO probe has also little or no cytotoxicity. [ABSTRACT FROM AUTHOR]

The search for new, non-standard targets is currently a high priority in the design of new antibacterial compounds. Bacterial toxin–antitoxin systems (TAs) are genetic modules that encode a toxin protein that causes growth arrest by interfering with essential cellular processes, and a cognate antitoxin, which neutralizes the toxin activity. TAs have no human analogs, are highly abundant in bacterial genomes, and therefore represent attractive alternative targets for antimicrobial drugs. This study demonstrates how artificial activation of Escherichia coli mazEF and hipBA toxin–antitoxin systems using sequence-specific antisense peptide nucleic acid oligomers is an innovative antibacterial strategy. The growth arrest observed in E. coli resulted from the inhibition of translation of the antitoxins by the antisense oligomers. Furthermore, two other targets, related to the activities of mazEF and hipBA , were identified as promising sites of action for antibacterials. These results show that TAs are susceptible to sequence-specific antisense agents and provide a proof-of-concept for their further exploitation in antimicrobial strategies. [ABSTRACT FROM AUTHOR]

Atopic dermatitis (AD) is a chronic inflammatory skin disease whose prevalence is increasing worldwide. Filaggrin (FLG) is essential for the development of the skin barrier, and its genetic mutations are major predisposing factors for AD. In this study, we developed a convenient and practical method to detect FLG mutations in AD patients using peptide nucleic acid (PNA) probes labelled with fluorescent markers for rapid analysis. Fluorescence melting curve analysis (FMCA) precisely identified FLG mutations based on the distinct difference in the melting temperatures of the wild‐type and mutant allele. Moreover, PNA probe‐based FMCA easily and accurately verified patient samples with both heterozygote and homozygote FLG mutations, providing a high‐throughput method to reliable screen AD patients. Our method provides a convenient, rapid and accurate diagnostic tool to identify potential AD patients allowing for early preventive treatment, leading to lower incidence rates of AD, and reducing total healthcare expenses. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs or miRs) are small noncoding RNAs involved in the fine regulation of post-transcriptional processes in the cell. The physiological levels of these short (20−22-mer) oligonucleotides are important for the homeostasis of the organism, and therefore dysregulation can lead to the onset of cancer and other pathologies. Their importance as biomarkers is constantly growing and, in this context, detection methods based on the hybridization to peptide nucleic acids (PNAs) are gaining their place in the spotlight. After a brief overview of their biogenesis, this review will discuss the significance of targeting miR, providing a wide range of PNA-based approaches to detect them at biologically significant concentrations, based on electrochemical, fluorescence and colorimetric assays.

Antibiotic resistance is an escalating, worldwide problem. Due to excessive use of antibiotics, multidrug-resistant bacteria have become a serious threat and a major global healthcare problem of the 21st century. This fact creates an urgent need for new and effective antimicrobials. The common strategies for antibiotic discovery are based on either modifying existing antibiotics or screening compound libraries, but these strategies have not been successful in recent decades. An alternative approach could be to use gene-specific oligonucleotides, such as peptide nucleic acid (PNA) oligomers, that can specifically target any single pathogen. This approach broadens the range of potential targets to any gene with a known sequence in any bacterium, and could significantly reduce the time required to discover new antimicrobials or their redesign, if resistance arises. We review the potential of PNA as an antibacterial molecule. First, we describe the physicochemical properties of PNA and modifications of the PNA backbone and nucleobases. Second, we review the carriers used to transport PNA to bacterial cells. Furthermore, we discuss the PNA targets in antibacterial studies focusing on antisense PNA targeting bacterial mRNA and rRNA.

Peptide nucleic acids (PNAs) are structural mimics of nucleic acids that form stable hybrids with DNA and RNA. In addition, PNAs can invade double-stranded DNA. Due to these characteristics, PNAs are widely used as biochemical tools, for example, in antisense/antigene therapy. Interstrand crosslink formation in nucleic acids is one of the strategies for preparing a stable duplex by covalent bond formation. In this study, we have synthesized PNAs incorporating 4-amino-6-oxo-2-vinylpyrimidine (AOVP) as a crosslinking agent and evaluated their reactivities for targeting DNA and RNA.

Bleeding and thrombosis represent common complications in myeloproliferative neoplasms (MPN) and significantly contribute to morbidity and mortality. Molecular markers, including CALR mutations, were considered not only as diagnostic markers, but also as risk factors for bleeding and thrombosis associated with MPN, especially for patients in remote primary hospitals. We sought to develop an easy-to-use assay for the rapid detection of CALR type 1 (CALR-1) and type 2 (CALR-2) mutations in Philadelphia chromosome-negative MPN patients. Peptide nucleic acid-locked nucleic acid (PNA-LNA) clamping loop-mediated isothermal amplification (LAMP) assays were established, which were integrated into a centrifugal compact disc (CD) microfluidic platform. A total of 158 clinical blood samples were tested simultaneously by this microfluidic platform and an in-house real time PCR assay. The detection performance of the LAMP arrays was validated and conflicting results were identified by Sanger sequencing. The results suggested that the LAMP methods we developed exhibited good sensitivity, specificity, and precision. By real time fluorescence assay the detection limit for CALR-1 and CALR-2 mutations could reach as low as 1% and 0.5% respectively, and 10% and 5% respectively by visual method. There were no nonspecific background amplifications among different detection systems. For the CALR-1 and CALR-2 LAMP detection systems, intra-batch CV values of 1% mutated plasmid were 10.56% and 10.51% respectively, and the inter-batch CV values were 19.55% and 18.39%, respectively. The products were all analyzed by melting curve analysis and electrophoresis followed by Sanger sequencing analysis, which were consistent with the database sequences. The microfluidic platform could complete rapid detection of CALR-1/2 mutations within 60 min. The results of clinical samples detected by our CD-like microfluidic chipLAMP assay and rtPCR assay suggested that 133 samples were CALR wild type, 15 were CALR-1 mutation type, and 9 were CALR-2 mutation type. The correlation coefficient value (Kendall's tau_b) of the two assays was 0.99. Interestingly, by the newly established detection platform, we were surprised to find that one patient of Chinese origin harbored both CALR-1 and CALR-2 mutations. This result was verified by Sanger sequencing analysis. The LAMP detection systems developed herein displayed good sensitivity, specificity, and stability. Additionally, the detection results could be directly judged by color changes of the reaction systems without any auxiliary equipment. Thus, the platform we developed has the potential of being widely used in remote and economically undeveloped areas in the future. [ABSTRACT FROM AUTHOR]

Herein, we report that peptide nucleic acid sequences (PNAs) have been used as the probe species for detection of RNA and that a microfluidic microarray (MMA) chip is used as the platform for detection of hybridizations between immobilized PNA probes and RNA targets. The RNA targets used are derived from influenza A sequences. This paper discusses the optimization of two probe technologies used for RNA detection and investigates how the composition of the probe buffer and the content of the hybridization solution can influence the overall results. Our data show that the PNA probe is a better choice than the DNA probe when there is low salt in the probe buffer composition. Furthermore, we show that the absence of salt (NaCl) in the hybridization buffer does not hinder the detection of RNA sequences. The results provide evidence that PNA probes are superior to DNA probes in term of sensitivity and adaptability, as PNA immobilization and PNA-RNA hybridization are less affected by salt content in the reaction buffers unlike DNA probes. [ABSTRACT FROM AUTHOR]

In the presented assay, we elaborated a method for distinguishing sequences that are genetically closely related to each other. This is particularly important in a situation where a fine balance of the allele abundance is a point of research interest. We developed a peptide nucleic acid (PNA) strand invasion technique for the differentiation between multiple sclerosis-associated retrovirus (MSRV) and ERVWE1 sequences, both molecularly similar, belonging to the human endogenous retrovirus HERV-W family. We have found that this method may support the PCR technique in screening for minor alleles which, in certain conditions, may be undetected by the standard PCR technique. We performed the analysis of different ERVWE1 and MSRV template mixtures ranging from 0 to 100% of ERVWE1 in the studied samples, finding the linear correlation between template composition and signal intensity of final reaction products. Using the PNA strand invasion assay, we were able to estimate the relative ERVWE1 expression level in human specimens such as U-87 MG, normal human astrocytes cell lines and placental tissue. The results remained in concordance with those obtained by semi-quantitative or quantitative PCR. [ABSTRACT FROM AUTHOR]

Protozoan parasites of the Trypanosomatidae family can cause devastating diseases in humans and animals, such as Human African Trypanosomiasis or Sleeping Sickness, Chagas disease and Leishmaniasis. Currently, there are molecular assays for detecting parasitic infections and their post-treatment monitoring based on nucleic acid amplification, but there are still certain limitations which limit the development of assays that can detect and discriminate between parasite infections with a single test. Here, we present the development of a novel molecular assay for the rapid identification of Trypanosomatids, integrating DNA analysis by dynamic chemistry in conjunction with Matrix-Assisted Laser Desorption Ionization – Time-of-Flight Mass Spectrometry (MALDI-ToF). Differentiation of Trypanosoma cruzi , Trypanosoma brucei and Leishmania spp . is now possible using a single reaction tube, and enables rapid identification of Trypanosomatids. The test is based on a singleplex PCR, using a specific primer pair that amplifies a 155 base pair segment of the 28S ribosomal RNA gene, within a conserved homology region of Trypanosomatidae species. Amplified fragments are analysed by dynamic chemistry using two abasic PNA probes and the four reactive nucleobases – containing an aldehyde functional group - with MALDI-ToF to identify unique molecular patterns created by each specie due to their single base differences (Single Nucleotide Fingerprint ‘SNF’) in this highly homologous region. This novel assay offers the possibility to expand routine diagnostic testing for Trypanosomatids, and monitoring of therapeutic responses to these infectious diseases. [ABSTRACT FROM AUTHOR]

Accurate taxonomic identification of plant materials in herbal medicines is important for product quality control. The genus Paeonia (Saxifragales) is the source of the herbal preparations Paeoniae Radix (Paeoniae Radix Alba and Paeoniae Radix Rubra) and Moutan Radicis Cotex. However, confusion has arisen regarding their contents due to linguistic and taxonomic ambiguities, similar morphologies and different definitions of Paeoniae Radix in the Korean and Chinese national pharmacopoeias, leading to the distribution of adulterated products. To develop a method for identifying the four Paeonia species used in these medicines, three fluorescently-labeled peptide nucleic acid (PNA) probes were designed against ITS2 sequences containing single nucleotide polymorphisms (SNPs) and used in a real-time PCR melting curve assay. Each of the four Paeonia species was accurately identified using this analysis. The accuracy and analytical stability of the PNA melting curve assay was confirmed using commercially available samples of the four Paeonia species. This assay is a reliable genetic tool to distinguish between different Paeonia-derived herbal medicines and identify the botanical origins of Paeoniae Radix and Moutan Radicis Cortex. This technique may also contribute to quality control and standardization of herbal medicines by providing a reliable authentication tool and preventing the distribution of inauthentic adulterants. [ABSTRACT FROM AUTHOR]

DNA carries genetic information in its sequence of bases. Synthetic oligonucleotides that can sequence-specifically recognize a target gene sequence are a useful tool for regulating gene expression or detecting target genes. Among the many synthetic oligonucleotides, tail-clamp peptide nucleic acid (TC-PNA) offers advantages since it has two homopyrimidine PNA strands connected via a flexible ethylene glycol-type linker that can recognize complementary homopurine sequences via Watson-Crick and Hoogsteen base pairings and form thermally-stable PNA/PNA/DNA triplex structures. Here, we synthesized a series of TC-PNAs that can possess different lengths of azobenzenecontaining linkers and studied their binding behaviours to homopurine single-stranded DNA. Introduction of azobenzene at the N-terminus amine of PNA increased the thermal stability of PNA-DNA duplexes. Further extension of the homopyrimidine PNA strand at the N-terminus of PNA-AZO further increased the binding stability of the PNA/DNA/PNA triplex to the target homopurine sequence; however, it induced TC-PNA/DNA/TC-PNA complex formation. Among these TC-PNAs, 9W5H-C4-AZO consisting of nine Watson-Crick bases and five Hoogsteen bases tethered with a beta-alanine conjugated azobenzene linker gave a stable 1:1 TC-PNA/ssDNA complex and exhibited good mismatch recognition. Our design for TC-PNA-AZO can be utilized for detecting homopurine sequences in various genes. [ABSTRACT FROM AUTHOR]

Rapid and easy detection of a single nucleotide point mutation of bacterial genes, which is directly linked to drug susceptibility, is essential for the proper use of antimicrobial agents. Here, we established a detection method using a peptide nucleic acid mediated loop-mediated amplification (LAMP) assay for macrolide (ML)-susceptible Mycoplasma pneumoniae . This assay specifically detected the absence of missense mutations encoding the central loop of domain V in the gene encoding 23S rRNA, which can reduce the affinity for MLs and subsequently generate ML-resistant strains of M. pneumoniae . Reactions were performed at 62 °C for 60 min and targeted gene amplifications were detected by real-time turbidity with a turbidimeter and naked-eye inspection of a color change. The assay had an equivalent detection limit of 100.0 fg of DNA with the turbidimeter and showed specificity against 54 types of pathogens, whereas amplification was completely blocked, even at 100.0 pg of DNA per reaction, in the presence of point mutations at 2063A and 2064A. The expected LAMP products were confirmed through identical melting curves in real-time LAMP procedures. This method would be a simple and rapid protocol for single nucleotide polymorphism genotyping as point-of-care testing technology without amplification of the sequences carrying the point mutations 2063A and 2064A in ML-resistant M. pneumoniae strains. [ABSTRACT FROM AUTHOR]

The HERV-W family of human endogenous retroviruses represents a group of numerous sequences that show close similarity in genetic composition. It has been documented that some members of HERV-W-derived expression products are supposed to play significant role in humans' pathology, such as multiple sclerosis or schizophrenia. Other members of the family are necessary to orchestrate physiological processes (eg, ERVWE1 coding syncytin-1 that is engaged in syncytiotrophoblast formation). Therefore, an assay that would allow the recognition of particular form of HERV-W members is highly desirable. A peptide nucleic acid (PNA)-mediated technique for the discrimination between multiple sclerosis-associated retrovirus and ERVWE1 sequence has been developed. The assay uses a PNA probe that, being fully complementary to the ERVWE1 but not to multiple sclerosis-associated retrovirus (MSRV) template, shows high selective potential. Single-stranded DNA binding protein facilitates the PNA-mediated, sequence-specific formation of strand invasion complex and, consequently, local DNA unwinding. The target DNA may be then excluded from further analysis in any downstream process such as single-stranded DNA-specific exonuclease action. Finally, the reaction conditions have been optimized, and several PNA probes that are targeted toward distinct loci along whole HERV-W env sequences have been evaluated. We believe that PNA/single-stranded DNA binding protein-based application has the potential to selectively discriminate particular HERV-W molecules as they are at least suspected to play pathogenic role in a broad range of medical conditions, from psycho-neurologic disorders (multiple sclerosis and schizophrenia) and cancers (breast cancer) to that of an auto-immunologic background (psoriasis and lupus erythematosus). [ABSTRACT FROM AUTHOR]

Telomere length is an important indicator of proliferative cell history and potential. Decreasing telomere length in the cells of an immune system can indicate immune aging in immune-mediated and chronic inflammatory diseases. Quantitative fluorescent in situ hybridization (Q-FISH) of a labeled (C3TA peptide nucleic acid probe onto fixed metaphase cells followed by digital image microscopy allows the evaluation of telomere length in the arms of individual chromosomes. Computer-assisted analysis of microscopic images can provide quantitative information on the number of telomeric repeats in individual telomeres. We developed new software to estimate telomere length. The MeTeLen software contains new options that can be used to solve some Q-FISH and microscopy problems, including correction of irregular light effects and elimination of background fluorescence. The identification and description of chromosomes and chromosome regions are essential to the Q-FISH technique. To improve the quality of cytogenetic analysis after Q-FISH, we optimized the temperature and time of DNA-denaturation to get better DAPI-banding of metaphase chromosomes. MeTeLen was tested by comparing telomere length estimations for sister chromatids, background fluorescence estimations, and correction of nonuniform light effects. The application of the developed software for analysis of telomere length in patients with rheumatoid arthritis was demonstrated. [ABSTRACT FROM AUTHOR]