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Since its inception, antisense oligonucleotides (ASOs) have been extensively used in functional genomics. Recent advancements in chemical modification and delivery technology extend its applications to the targeted management of crop pests. ASO was conceptualized in the early 1970s and first applied to inhibit retrovirus replication in chickens. The principles of ASOs rely on target complementarity by hybridizing with the mRNA and inhibiting the function. However, the application of ASO received little attention because of its high instability in the biological environment. Initial ASOs, featuring phosphorothioate linkages, laid the groundwork for subsequent advancements, by addressing challenges such as nuclease degradation and cellular uptake limitations. The second generation introduced 2'-modifications to enhance stability and specificity while allowing reversible gene regulation. Third-generation ASOs incorporated innovative chemical modifications like locked nucleic acids (LNAs), peptide nucleic acids (PNAs), phosphoramidate morpholino oligomers (PMOs), 2′-deoxy-2′-fluoro-d-arabinonucleic acid (FANA), cyclohexene nucleic acids (CeNAs), and tricyclo-DNA (tcDNA), offering enhanced efficacy and stability. ASO technology continues to evolve, promising precision medicine and customized therapies for genetic diseases. In agriculture, ASOs selectively target essential genes in pests or pathogens, which would be a novel option for providing precise crop protection and reducing reliance on conventional pesticides. Recent studies demonstrated successful ASO application in thrips, psyllids, Gypsy moths, scale insects, citrus greening, and Zebra-chip diseases. Additionally, ASOs hold the potential for improving crop yield and quality by modulating gene expression related to plant growth, development, and stress responses. They can enhance abiotic stress tolerance, regulate genes associated with specific traits, and offer alternative strategies for managing diseases, such as targeting S-genes in tomatoes for resistance against bacterial spots. These applications antecede the oligo-based pesticides that will revolutionize future agriculture by reducing pesticide uses and harmful impacts on the ecosystem. Further advancements in environmental stability, field efficacy, delivery formulation, and environmental safety would mold this technology into a novel tool for sustainable crop protection. This review highlights the fundamentals of ASO, recent advancements in design chemistry, delivery systems, validation and optimization process, and its potential in customized pest management. This would serve as a ready reckoner for future ASO research and application. [ABSTRACT FROM AUTHOR]

Comprehensive Summary: Single‐nucleotide variants (SNVs) are crucial in disease development, but their accurate detection is challenging due to their low abundance and interference from wild‐type targets. Although nucleic acid analogs like peptide nucleic acids (PNAs) have been used for SNV detection, they often lack programmable sensitivity and specificity due to poorly calculated thermodynamics and kinetics. Here, we present a computational method for calculating the stacking energy of PNA and DNA hybrids, leveraging nearest neighbor parameters. Validation against experimental data from 16 sequences under varied hybridization conditions yielded good agreement using Bland‐Altman analysis, with all data points falling within the confidence interval. Our findings indicate that PNA‐DNA hybridization is thermodynamically more stable and exhibits kinetics 140‐fold faster than DNA‐DNA hybridization for identical sequences. Utilizing this computational framework, we designed PNA toehold probes, which were screened via simulations and experiments. This combined approach facilitated the identification of highly sensitive and specific PNA toehold probes for single point mutation detection via strand displacement reaction. Our results demonstrate the successful application of PNA toehold probes for detecting point mutations with high sensitivity and specificity, achieving a selective amplification of approximately 200‐fold for variants with a variant allele frequency (VAF) of 0.5% using quantitative polymerase chain reaction. [ABSTRACT FROM AUTHOR]

The main goal of this study is to test the utility of calyculin A induced G2-PCC assay as a biodosimetry triage tool for assessing a wide range of low and acute high radiation dose exposures of photons. Towards this initiative, chromosome aberrations induced by low and high doses of x-rays were evaluated and characterized in G2-prematurely condensed chromosomes (G2-PCCs) by fluorescence in situ hybridization (FISH) using human centromere and telomere specific PNA (peptide nucleic acid) probes. A dose dependent increase in the frequency of dicentric chromosomes was observed in the G2-PCCs up to 20 Gy of x-rays. The combined yields of dicentrics and rings in the G2-PCCs showed a clear dose dependency up to 20 Gy from 0.02/cell for 0.1 Gy to 14.98/cell for 20 Gy. Centric rings were observed more frequently than acentric ring chromosomes in the G2-PCCs at all the radiation doses from 1 Gy to 20 Gy. A head-to-head comparison was also performed by FISH on the yields of chromosome aberrations induced by different doses of x-rays (0 Gy -7.5 Gy) in colcemid arrested metaphase chromosomes and calyculin A induced G2-PCCs. In general, the frequencies of dicentrics, rings and acentric fragments were slightly higher in G2-PCCs than in colcemid arrested metaphase chromosomes at all the radiation doses, but the differences were not statistically significant. To reduce the turnaround time for absorbed radiation dose estimation, attempt was made to obtain G2-PCCs by reducing the culture time to 36 hrs. The absorbed doses estimated in x-rays irradiated (0,1,2 and 4 Gy) G2-PCCs after 36 hrs of culture were grossly like that of G2-PCCs and colcemid arrested metaphase chromosomes prepared after 48 hrs of culture. Our study indicates that the shortened version of calyculin A induced G2-PCC assay coupled with the FISH staining technique can serve as an effective triage biodosimetry tool for large-scale radiological/nuclear incidents. [ABSTRACT FROM AUTHOR]

Calcium phosphates (CaPs) and their substituted derivatives encompass a large number of compounds with a vast presence in nature that have aroused a great interest for decades. In particular, hydroxyapatite (HAp, Ca10(OH)2(PO4)6) is the most abundant CaP mineral and is significant in the biological world, at least in part due to being a major compound in bones and teeth. HAp exhibits excellent properties, such as safety, stability, hardness, biocompatibility, and osteoconductivity, among others. Even some of its drawbacks, such as its fragility, can be redirected thanks to another essential feature: its great versatility. This is based on the compound's tendency to undergo substitutions of its constituent ions and to incorporate or anchor new molecules on its surface and pores. Thus, its affinity for biomolecules makes it an optimal compound for multiple applications, mainly, but not only, in biological and biomedical fields. The present review provides a chemical and structural context to explain the affinity of HAp for biomolecules such as proteins and nucleic acids to generate hybrid materials. A size-dependent criterium of increasing complexity is applied, ranging from amino acids/nucleobases to the corresponding macromolecules. The incorporation of metal ions or metal complexes into these functionalized compounds is also discussed. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Detection of circulating tumor DNA (ctDNA) mutations, which are molecular biomarkers present in bodily fluids of cancer patients, can be applied for tumor diagnosis and prognosis monitoring. However, current profiling of ctDNA mutations relies primarily on polymerase chain reaction (PCR) and DNA sequencing and these techniques require preanalytical processing of blood samples, which are time‐consuming, expensive, and tedious procedures that increase the risk of sample contamination. To overcome these limitations, here the engineering of a DNA/γPNA (gamma peptide nucleic acid) hybrid nanoreporter is disclosed for ctDNA biosensing via in situ profiling and recording of tumor‐specific DNA mutations. The low tolerance of γPNA to single mismatch in base pairing with DNA allows highly selective recognition and recording of ctDNA mutations in peripheral blood. Owing to their remarkable biostability, the detached γPNA strands triggered by mutant ctDNA will be enriched in kidneys and cleared into urine for urinalysis. It is demonstrated that the nanoreporter has high specificity for ctDNA mutation in peripheral blood, and urinalysis of cleared γPNA can provide valuable information for tumor progression and prognosis evaluation. This work demonstrates the potential of the nanoreporter for urinary monitoring of tumor and patient prognosis through in situ biosensing of ctDNA mutations. [ABSTRACT FROM AUTHOR]

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 (λem, 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-CH2COOH 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. [ABSTRACT FROM AUTHOR]

Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection. Time to receive effective therapy is a primary determinant of mortality in patients with sepsis. Blood culture is the reference standard for the microbiological diagnosis of bloodstream infections, despite its low sensitivity and prolonged time to receive a pathogen detection. In recent years, rapid tests for pathogen identification, antimicrobial susceptibility, and sepsis identification have emerged, both culture-based and culture-independent methods. This rapid narrative review presents currently commercially available approved diagnostic molecular technologies in bloodstream infections, including their clinical performance and impact on patient outcome, when available. Peer-reviewed publications relevant to the topic were searched through PubMed, and manufacturer websites of commercially available assays identified were also consulted as further sources of information. We have reviewed data about the following technologies for pathogen identification: fluorescence in situ hybridization with peptide nucleic acid probes (Accelerate PhenoTM), microarray-based assay (Verigene®), multiplex polymerase chain reaction (cobas® eplex, BioFire® FilmArray®, Molecular Mouse, Unyvero BCU SystemTM), matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (Rapid MBT Sepsityper®), T2 magnetic resonance (T2Bacteria Panel), and metagenomics-based assays (Karius©, DISQVER®, Day Zero Diagnostics). Technologies for antimicrobial susceptibility testing included the following: Alfed 60 ASTTM, VITEK® REVEALTM, dRASTTM, ASTar®, Fastinov®, QuickMIC®, ResistellTM, and LifeScale. Characteristics, microbiological performance, and issues of each method are described, as well as their clinical performance, when available. [ABSTRACT FROM AUTHOR]

G-quadruplexes (G4s) are non-canonical nucleic acid secondary structures that have gathered significant interest in medicinal chemistry over the past two decades due to their unique structural features and potential roles in a variety of biological processes and disorders. Traditionally, research efforts have focused on stabilizing G4s, while in recent years, the attention has progressively shifted to G4 destabilization, unveiling new therapeutic perspectives. This review provides an in-depth overview of recent advances in the development of small molecules, starting with the controversial role of TMPyP4. Moreover, we described effective metal complexes in addition to G4-disrupting small molecules as well as good G4 stabilizing ligands that can destabilize G4s in response to external stimuli. Finally, we presented antisense strategies as a promising approach for destabilizing G4s, with a particular focus on 2′-OMe antisense oligonucleotide, peptide nucleic acid, and locked nucleic acid. Overall, this review emphasizes the importance of understanding G4 dynamics as well as ongoing efforts to develop selective G4-unfolding strategies that can modulate their biological function and therapeutic potential. [ABSTRACT FROM AUTHOR]

Cell-penetrating peptides (CPPs) are promising carriers to effectively transport antisense oligonucleotides (ASOs), including peptide nucleic acids (PNAs), into bacterial cells to combat multidrug-resistant bacterial infections, demonstrating significant therapeutic potential. Streptococcus suis, a Gram-positive bacterium, is a major bacterial pathogen in pigs and an emerging zoonotic pathogen. In this study, through the combination of super-resolution structured illumination microscopy (SR-SIM), flow cytometry analysis, and toxicity analysis assays, we investigated the suitability of four CPPs for delivering PNAs into S. suis cells: HIV-1 TAT efficiently penetrated S. suis cells with low toxicity against S. suis; (RXR)4XB had high penetration efficiency with inherent toxicity against S. suis; (KFF)3K showed lower penetration efficiency than HIV-1 TAT and (RXR)4XB; K8 failed to penetrate S. suis cells. HIV-1 TAT-conjugated PNA specific for the essential gyrase A subunit gene (TAT-anti-gyrA PNA) effectively inhibited the growth of S. suis. TAT-anti-gyrA PNA exhibited a significant bactericidal effect on serotypes 2, 4, 5, 7, and 9 strains of S. suis, which are known to cause human infections. Our study demonstrates the potential of CPP-ASO conjugates as new antimicrobial compounds for combating S. suis infections. Furthermore, our findings demonstrate that applying SR-SIM and flow cytometry analysis provides a convenient, intuitive, and cost-effective approach to identifying suitable CPPs for delivering cargo molecules into bacterial cells. [ABSTRACT FROM AUTHOR]

Quantum mechanical calculations are used to explore the thermodynamics of possible prebiotic synthesis of the building blocks of nucleic acids. Different combinations of D-ribofuranose (Ribf) and N-(2-aminoethyl)-glycine (AEG) (trifunctional connectors (TCs)); the nature of the Ribf, its anomeric form, and its ring puckering (conformation); and the nature of the nucleobases (recognition units (RUs)) are considered. The combinatorial explosion of possible nucleosides has been drastically reduced on physicochemical grounds followed by a detailed thermodynamic evaluation of alternative synthetic pathways. The synthesis of nucleosides containing N-(2-aminoethyl)-glycine (AEG) is predicted to be thermodynamically favored suggesting a possible role of AEG as a component of an ancestral proto-RNA that may have preceded today's nucleic acids. A new pathway for the building of free nucleotides (exemplified by 5'-uridine monophosphate (UMP)) and of AEG dipeptides is proposed. This new pathway leads to a spontaneous formation of free UMP assisted by an AEG nucleoside in an aqueous environment. This appears to be a workaround to the "water problem" that prohibits the synthesis of nucleotides in water. [ABSTRACT FROM AUTHOR]

This article explores the use of metagenomics in studying plant diseases and understanding microbial communities. It discusses the challenges of traditional methods and the benefits of metagenomics in uncovering the diversity and composition of plant-associated microbiomes. The article provides best practices for generating metagenomic workflows and emphasizes the importance of experimental design and sequencing rationale. It also discusses the use of different sequencing technologies and the need for proper databases in classifying metagenomic data. The document includes a list of references and a glossary of terms related to plant science and microbiology, which can be helpful for researchers studying plant diseases and genetic diversity in plants. [Extracted from the article]

Purpose: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive tumor with a poor prognosis that poses challenges for diagnosis using traditional tissuebased techniques. DNA methylation alterations have emerged as potential and promising biomarkers for PDAC. In this study, we aimed to assess the diagnostic potential of a novel DNA methylation assay based on epigenetic-specific peptide nucleic acid (Epi-sPNA) in both tissue and plasma samples for detecting PDAC. Materials and methods: The study involved 46 patients with PDAC who underwent surgical resection. Epi-TOP pancreatic assay was used to detect PDAC-specific epigenetic biomarkers. The Epi-sPNA allowed accurate and rapid methylation analysis without bisulfite sample processing. Genomic DNA extracted from paired normal pancreatic and PDAC tissues was used to assess the diagnostic efficacy of epigenetic biomarkers for PDAC. Subsequent validation was conducted on cell-free DNA (cfDNA) extracted from plasma samples, with 10 individuals represented in each group: PDAC, benign pancreatic cystic neoplasm, and healthy control. Results: The combination of seven epigenetic biomarkers (HOXA9, TWIST, WT1, RPRM, BMP3, NPTX2, and BNC1) achieved 93.5% sensitivity and 96.7% specificity in discerning normal pancreatic from PDAC tissues. Plasma cfDNA, analyzed using these markers and KRAS mutations, exhibited a substantial 90.0% sensitivity, 95.0% specificity, and an overall 93.3% accuracy for discriminating PDAC. Notably, cancer antigen 19-9 and carcinoembryonic antigen both had an accuracy of 90.0%. Conclusion: Our study suggests that analyzing seven differentially methylated genes with KRAS mutations in cfDNA using the novel Epi-TOP pancreatic assay is a potential blood-based biomarker for the diagnosis of PDAC. [ABSTRACT FROM AUTHOR]

High-throughput sequencing has become a prominent tool to assess plant-associated microbial diversity. Still, some technical challenges remain in characterising these communities, notably due to plant and fungal DNA co-amplification. Fungal-specific primers, Peptide Nucleic Acid (PNA) clamps, or adjusting PCR conditions are approaches to limit plant DNA contamination. However, a systematic comparison of these factors and their interactions, which could limit plant DNA contamination in the study of plant mycobiota, is still lacking. Here, three primers targeting the ITS2 region were evaluated alone or in combination with PNA clamps both on nettle (Urtica dioica) root DNA and a mock community. PNA clamps did not improve the richness or diversity of the fungal communities but increased the number of fungal reads. Among the tested factors, the most significant was the primer pair. Specifically, the 5.8S-Fun/ITS4-Fun pair exhibited a higher OTU richness but fewer fungal reads. Our study demonstrates that the choice of primers is critical for limiting plant and fungal DNA co-amplification. PNA clamps increase the number of fungal reads when ITS2 is targeted but do not result in higher fungal diversity recovery at high sequencing depth. At lower read depths, PNA clamps might enhance microbial diversity quantification for primer pairs lacking fungal specificity. [ABSTRACT FROM AUTHOR]

Prey metabarcoding has become a popular tool in molecular ecology for resolving trophic interactions at high resolution, from various sample types and animals. To date, most predator–prey studies of small‐sized animals (<1 mm) have met the problem of overabundant predator DNA in dietary samples by adding blocking primers/peptide nucleic acids. These primers aim to limit the PCR amplification and detection of the predator DNA but may introduce bias to the prey composition identified by interacting with sequences that are similar to those of the predator. Here we demonstrate the use of an alternative method to explore the prey of small marine copepods using whole‐body DNA extracts and deep, brute force metabarcoding of an 18S rDNA fragment. After processing and curating raw data from two sequencing runs of varying depths (0.4 and 5.4 billion raw reads), we isolated 1.3 and 52.2 million prey reads, with average depths of ~15,900 and ~120,000 prey reads per copepod individual, respectively. While data from both sequencing runs were sufficient to distinguish dietary compositions from disparate seasons, locations, and copepod species, greater sequencing depth led to better separation of clusters. As computation and sequencing are becoming ever more powerful and affordable, we expect the brute force approach to become a general standard for prey metabarcoding, as it offers a simple and affordable solution to consumers that is impractical to dissect or unknown to science. [ABSTRACT FROM AUTHOR]

Ultrafine Gold nanoparticles (Au NPs) functionalized with various biomolecules constitute an alternative to antibodies as anti-amyloidogenic agents. However, generating stable ultrafine Au NPs with high surface activity is challenging. Here, the capacity of phosphate groups in biomolecules is used to stabilize Au NPs. The characteristics of Au NPs decorated with adenosine mono-, di-, and tri-phosphate are compared as well as adenosine and peptide nucleic acid-containing adenosine as controls. Among them, ATP-Au NPs are found to be superior having small size (2-4 nm) and stability (for several months) when analysed by spectroscopy and electron microscopy. Spectroscopy analysis also revealed that each ATP-stabilized Au NP is decorated with 7-8 molecules of ATP. ThT binding analysis and TEM imaging showed that the ATP-Au NPs efficiently prevented amyloid fibril formation in vitro by Aβ-42, α-Synuclein as well as by the Glucosylceramide metabolite, and disaggregated their pre-formed fibrils. NMR analysis revealed the interaction of the ATP-Au NPs with the amyloid fibrils. The ATP-Au NPs are safe toward cultured SH-SY5Y cells and when co-incubated with α-Synuclein amyloids inhibited their cytotoxicity and readily enter the cells to inhibit formation of amyloid fibrils within them. The results indicates the pharmacological potentials of ATP decorated Au NPs. [ABSTRACT FROM AUTHOR]

This article discusses the synthesis of 2-aminopyridine-modified peptide nucleic acids (PNAs) and their potential applications in research and diagnostics. PNAs are known for their high affinity and sequence specificity in binding to nucleic acids. The article describes the chemical modifications made to improve the properties of PNAs, such as replacing cytosine with pseudoisocytosine and using 2-aminopyridine as an alternative. The authors also outline the optimized synthetic route to produce the modified PNA monomers. The article provides detailed procedures and recommendations for the synthesis of PNAs using different synthesizers. Overall, the article offers valuable insights for researchers interested in the synthesis and applications of modified PNAs. [Extracted from the article]

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

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 17th 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. [ABSTRACT FROM AUTHOR]

The document is a contents list for the journal Chemical Communications, published by The Royal Society of Chemistry. It includes articles on various topics such as direct borohydride fuel cells, composite electrode performance, peptide nucleic acids, and self-assembly of peptide nanomaterials. The journal also features communications on subjects like photocatalytic hydrogen production, liquid-crystalline circularly polarised fluorescent emitters, and electrochemical detection of glutathione. The document provides a comprehensive overview of the articles included in the journal issue. [Extracted from the article]

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. [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 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]

Proprotein convertase subtilisin/kexin 9 (PCSK9) is a protein that plays a key role in the metabolism of low-density lipoprotein (LDL) cholesterol. The gain-of-function mutations of the PCSK9 gene lead to a reduced number of surface LDL receptors by binding to them, eventually leading to endosomal degradation. This, in turn, is the culprit of hypercholesterolemia, resulting in accelerated atherogenesis. The modern treatment for hypercholesterolemia encompasses the use of biological drugs against PCSK9, like monoclonal antibodies and gene expression modulators such as inclisiran—a short, interfering RNA (siRNA). Peptide nucleic acid (PNA) is a synthetic analog of nucleic acid that possesses a synthetic peptide skeleton instead of a phosphate–sugar one. This different structure determines the unique properties of PNA (e.g., neutral charge, enzymatic resistance, and an enormously high affinity with complementary DNA and RNA). Therefore, it might be possible to use PNA against PCSK9 in the treatment of hypercholesterolemia. We sought to explore the impact of three selected PNA oligomers on PCSK9 gene expression. Using a cell-free transcription/translation system, we showed that one of the tested PNA strands was able to reduce the PCSK9 gene expression down to 74%, 64%, and 68%, as measured by RT–real-time PCR, Western blot, and HPLC, respectively. This preliminary study shows the high applicability of a cell-free enzymatic environment as an efficient tool in the initial evaluation of biologically active PNA molecules in the field of hypercholesterolemia research. This cell-free approach allows for the omission of the hurdles associated with transmembrane PNA transportation at the early stage of PNA selection. [ABSTRACT FROM AUTHOR]

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

MicroRNAs are small ribonucleotides that act as key gene regulators. Their altered expression is often associated with the onset and progression of several human diseases, including cancer. Given their potential use as biomarkers, there is a need to find detection methods for microRNAs suitable for use in clinical setting. Field-effect-transistor-based biosensors (bioFETs) appear to be valid tools to detect microRNAs, since they may reliably quantitate the specific binding between the immobilized probe and free target in solution through an easily detectable electrical signal. We have investigated the detection of human microRNA 155 (miR-155) using an innovative capturing probe constituted by a synthetic peptide nucleic acid (PNA), which has the advantage to form a duplex even at ionic strengths approaching the physiological conditions. With the aim to develop an optimized BioFET setup, the interaction kinetics between miR-155 and the chosen PNA was preliminarily investigated by using surface plasmon resonance (SPR). By exploiting both these results and our custom-made bioFET system, we were able to attain a low-cost, real-time, label-free and highly specific detection of miR-155 in the nano-molar range. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Microbiome engineering is an emerging research field that aims to design an artificial microbiome and modulate its function. In particular, subtractive modification of the microbiome allows us to create an artificial microbiome without the microorganism of interest and to evaluate its functions and interactions with other constituent bacteria. However, few techniques that can specifically remove only a single species from a large number of microorganisms and can be applied universally to a variety of microorganisms have been developed. Antisense peptide nucleic acid (PNA) is a potent designable antimicrobial agent that can be delivered into microbial cells by conjugating with a cell-penetrating peptide (CPP). Here, we tested the efficacy of the conjugate of CPP and PNA (CPP-PNA) as microbiome modifiers. The addition of CPP-PNA specifically inhibited the growth of Escherichia coli and Pseudomonas putida in an artificial bacterial consortium comprising E. coli, P. putida, Pseudomonas fluorescens, and Lactiplantibacillus plantarum. Moreover, the growth inhibition of P. putida promoted the growth of P. fluorescens and inhibited the growth of L. plantarum. These results indicate that CPP-PNA can be used not only for precise microbiome engineering but also for analyzing the growth relationships among constituent microorganisms in the microbiome. [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]

Growing evidence suggests that the exposure of bisphenol A (BPA), an endocrine disruptor that commonly present in the environment, can impair reproduction. However, conflicting results have been reported, and the underlying mechanism has not been fully understood. In this study, 3-week-old male mice were oral exposed to 50 mg/kg/d BPA or equivalent corn oil for 28 days. Their testis and epididymis were then collected for morphology examination by HE stains. The number of sperm was counted, and the morphology was analyzed by PNA (peptide nucleic acid) and pap staining. Fertilization capacity and successful rate were analyzed after mating with wide-type females. Spermatid DNA damage and apoptosis were evaluated by DFI, γH2AX stain, and TUNEL assay. RNA sequencing analysis was conducted to identify differentially expressed genes in testicular tissue of mice exposed to BPA. RNA interference was used to verify the regulatory mechanism of BPA exposure on gene expression in GC-2 cells. Our data showed that the total number of sperm was decreased and the morphology was impaired in BPA-exposed mice. In addition, the serum testosterone level and fertilization efficiency were also reduced. Mechanism studies showed that BPA could suppress the expression of PCBP2, a key regulatory gene in spermatid development, by activating the EZH2/H3K27me3. In conclusion, we found that BPA exposure can impair spermatid development via affecting key gene expression that is at least partially due to epigenetic modification. [ABSTRACT FROM AUTHOR]

Global reports of novel SARS-CoV-2 variants and recurrence cases continue despite substantial vaccination campaigns, raising severe concerns about COVID-19. While repurposed drugs offer some treatment options for COVID-19, notably, nucleoside inhibitors like Remdesivir stand out as curative therapies for COVID-19 that are approved by the US Food and Drug Administration (FDA). The emergence of highly contagious SARS-CoV-2 variants underscores the imperative for antiviral drugs adaptable to evolving viral mutations. RNA-dependent RNA polymerase (RdRp) plays a key role in viral genome replication. Currently, inhibiting viral RdRp function remains a pivotal strategy to tackle the notorious virus. Peptide nucleic acid (PNA) therapy shows promise by effectively targeting specific genome regions, reducing viral replication, and inhibiting infection. In our study, we designed PNA antisense oligomers conjugated with cell-penetrating peptides (CPP) aiming to evaluate their antiviral effects against RdRp target using structure-guided drug design, which involves molecular docking simulations, drug likeliness and pharmacokinetic evaluations, molecular dynamics simulations, and computing binding free energy. The in silico analysis predicts that chemically modified PNAs might act as antisense molecules in order to disrupt ribosome assembly at RdRp's translation start site, and their chemically stable and neutral backbone might enhance sequence-specific RNA binding interaction. Notably, our findings demonstrate that PNA-peptide conjugates might be the most promising inhibitors of SARS-CoV-2 RdRp, with superior binding free energy compared to Remdesivir in the current COVID-19 medication. Specifically, PNA-CPP-1 could bind simultaneously to the active site residues of RdRp protein and sequence-specific RdRp-RNA target in order to control viral replication. [ABSTRACT FROM AUTHOR]

Natural products and analogues are a source of antibacterial drug discovery. Considering drug resistance levels emerging for antibiotics, identification of bacterial metalloenzymes and the synthesis of selective inhibitors are interesting for antibacterial agent development. Peptide nucleic acids are attractive antisense and antigene agents representing a novel strategy to target pathogens due to their unique mechanism of action. Antisense inhibition and development of antisense peptide nucleic acids is a new approach to antibacterial agents. Due to the increased resistance of biofilms to antibiotics, alternative therapeutic options are necessary. To develop antimicrobial strategies, optimised in vitro and in vivo models are needed. In vivo models to study biofilm-related respiratory infections, device-related infections: ventilator-associated pneumonia, tissue-related infections: chronic infection models based on alginate or agar beads, methods to battle biofilm-related infections are discussed. Drug delivery in case of antibacterials often is a serious issue therefore this review includes overview of drug delivery nanosystems. [ABSTRACT FROM AUTHOR]

Ideal drug carriers feature a high loading capacity to minimize the exposure of patients with excessive, inactive carrier materials. The highest imaginable loading capacity could be achieved by nanocarriers, which are assembled from the therapeutic cargo molecules themselves. Here, we describe peptide nucleic acid (PNA)-based zirconium (Zr) coordination nanoparticles which exhibit very high PNA loading of > 94 % w/w. This metal-organic hybrid nanomaterial class extends the enormous compound space of coordination polymers towards bioactive oligonucleotide linkers. The architecture of single- or double-stranded PNAs was systematically varied to identify design criteria for the coordination driven self-assembly with Zr(IV) nodes at room temperature. Aromatic carboxylic acid functions, serving as Lewis bases, and a two-step synthesis process with preformation of Z r 6 O 4 (O H) 4 turned out to be decisive for successful nanoparticle assembly. Confocal laser scanning microscopy confirmed that the PNA-Zr nanoparticles are readily internalized by cells. PNA-Zr nanoparticles, coated with a cationic lipopeptide, successfully delivered an antisense PNA sequence for splicing correction of the β -globin intron mutation IVS2-705 into a functional reporter cell line and mediated splice-switching via interaction with the endogenous mRNA splicing machinery. The presented PNA-Zr nanoparticles represent a bioactive platform with high design flexibility and extraordinary PNA loading capacity, where the nucleic acid constitutes an integral part of the material, instead of being loaded into passive delivery systems. [ABSTRACT FROM AUTHOR]

Four new isoorotamide (Io)‐containing PNA nucleobases have been designed for A−U recognition of double helical RNA. New PNA monomers were prepared efficiently and incorporated into PNA nonamers for binding A−U in a PNA:RNA2 triplex. Isothermal titration calorimetry and UV thermal melting experiments revealed slightly improved binding affinity for singly modified PNA compared to known A‐binding nucleobases. Molecular dynamics simulations provided further insights into binding of Io bases in the triple helix. Together, the data revealed interesting insights into binding modes including the notion that three Hoogsteen hydrogen bonds are unnecessary for strong selective binding of an extended nucleobase. Cationic monomer Io8 additionally gave the highest affinity observed for an A‐binding nucleobase to date. These results will help inform future nucleobase design toward the goal of recognizing any sequence of double helical RNA. [ABSTRACT FROM AUTHOR]

The detection of trace microRNAs (miRNA) is crucial for early cancer diagnosis. Here, a sensitive biosensor for miRNA directive detection was put forward, which was based on RNA‐templated palladium nanoparticles (PdNPs) signal amplification strategy. Uncharged peptide nucleic acids (PNA) probe was used to capture the target miRNA(T‐RNA) specifically. Subsequently, a large amount of palladium ions absorbed on T‐RNA strands via electrostatic forces, and the palladium nanocluster is rapidly generated based on chemical reduction. Obviously, RNA metallization method enables biosensor to be carried out efficiently at room temperature also simplifies the preparation and detection of the sensor. Under optimal conditions, this method can analyze the concentration of miRNA in the range of 10 fM to 1 nM, and the detection limit is 3.33 fM. At the same time, this method has strong anti‐interference ability in serum samples, which provides a broad prospect for the early screening and diagnosis of cancer. [ABSTRACT FROM AUTHOR]

Chemical Communications is a journal published by The Royal Society of Chemistry that focuses on the chemical sciences. The current issue covers topics such as astrochemistry and advances in circularly polarized luminescence materials. The journal is published 100 times a year and is available in both print and electronic formats. The document also provides information for authors who want to submit their work for publication, including submission instructions and guidelines for reproducing published contributions. Additionally, it includes copyright information and permissions required for reproducing the publication. A list of previously published feature articles and communications is also provided. [Extracted from the article]

A sophisticated high-order framework nucleic acid (FNA) was engineered for the targeted delivery and responsive release of environment tolerant antisense peptide nucleic acids (asPNAs). The dendritic FNA-asPNAs system was constructed via simple one-pot modular assembly and demonstrated a good synergistic effect with chemotherapy on drug resistant cancer cells. [ABSTRACT FROM AUTHOR]

The use of template molecules as chemical scaffolds that significantly influence the course of the reaction has recently been intensively studied. Peptide nucleic acids (PNA) are molecules that mimic natural nucleic acids. They are a promising matrix in such reactions because they possess high affinity and specificity in their interactions. The manner of PNA interaction is predictable based on sequence complementarity. Recently, we report the visible light-induced metathesis reaction in peptides containing a diselenide bond. Herein, we present an efficient and straightforward method of the visible light-driven diselenide-based metathesis of peptide–nucleic acid conjugates. Compared to a similar photochemical transformation in peptides, a significant increase in the metathesis efficiency was obtained due to the template effect. [ABSTRACT FROM AUTHOR]

The exceptional hybridization properties of peptide nucleic acids (PNAs) coupled with the ease of their synthesis has made this artificial nucleic acid mimetic a desirable platform for diagnostics, therapeutics and supramolecular engineering. PNA backbone modifications have been extensively explored to finetune physicochemical properties and for conjugation of functional molecules. Here, we detail the synthesis of a universal γ‐propargyl‐PNA backbone from serine, and its acylation with the four DNA canonical nucleobases. The availability of serine as d or l enantiomer provide simple accesses to PNA oligomers for hybridization with natural oligonucleotides or for orthogonal hybridization circuitry. We show that late‐stage conjugation enables optimization of the physicochemical properties. This approach is appealing due to its orthogonality to Fmoc‐SPPS, its flexibility and ease for introducing diversity by on‐resin copper(I)‐catalyzed azide‐alkyne cycloaddition (CuAAC). We exemplified the utility of these novel monomers with PNA based hybridization chain reactions (HCRs). [ABSTRACT FROM AUTHOR]

Introduction: Antisense oligonucleotides (ASOs) with therapeutic potential have recently been reported to target the SARS-CoV-2 genome. Peptide nucleic acids (PNAs)-based ASOs have been regarded as promising drug candidates, but intracellular delivery has been a significant obstacle. Here, we present novel modified PNAs, termed OPNAs, with excellent cell permeability that disrupt the RNA genome of SARS-CoV-2 and HCoV-OC43 by introducing cationic lipid moiety onto the nucleobase of PNA oligomer backbone. Methods: HCT-8 cells and Caco-2 cells were treated with 1 μM antisense OPNAs at the time of viral challenge and the Viral RNA levels were measured by RT-qPCR three days post infection. Results: NSP 14 targeting OPNA 5 and 11, reduced the viral titer to a half and OPNA 530, 531 and 533 lowered viral gene expression levels to less than 50% of control by targeting the 5' UTR region. Several modifications (oligo size and position, etc.) were introduced to enhance the efficacy of selected OPNAs. Improved OPNAs exhibited a dose-dependent reduction in viral replication and nucleoprotein (NP) protein. When a mixture of oligomers was applied to infected cells, viral titer and NP levels decreased by more than eightfold. Discussion: In this study, we have developed a modified PNA ASO platform with exceptional chemical stability, high binding affinity, and cellular permeability. These findings indicate that OPNAs are a promising platform for the development of antivirals to combat future pandemic viral infections that do not require a carrier. [ABSTRACT FROM AUTHOR]

Photonic crystals (PCs) exhibit promising structural coloration properties and possess extensive application prospects in diverse optical fields. However, state-of-the-art inorganic or polymeric PCs show limited adaptivity as their configurations are fixed once formed. Herein, bio-organic adaptive PCs are fabricated via drop-casting of amphiphilic guanine-based peptide nucleic acid self-assembled microspheres. The high formation activation energy of up to 81.8 kJ·mol−1 suggests that the self-assembly step dominates the entire process. Therefore, the configurations along with the structural coloration of the supramolecular PCs are sensitive to self-assembly influencing parameters, showing temperature-encoded structural color evolution and solvent polarity-dependent solvatochromism. Our findings demonstrate that the supramolecular PCs are adaptive, thus showing promising potential for detection of organic solvents of different polarities in a visual and real-time manner for environmental protection or optical applications. [ABSTRACT FROM AUTHOR]

Recent developments in artificial nucleic acid and drug delivery systems present possibilities for the symbiotic engineering of therapeutic oligonucleotides, such as antisense oligonucleotides (ASOs) and small interfering ribonucleic acids (siRNAs). Employing these technologies, triplex-forming oligonucleotides (TFOs) or peptide nucleic acids (PNAs) can be applied to the development of symbiotic genome-targeting tools as well as a new class of oligonucleotide drugs, which offer conceptual advantages over antisense as the antigene target generally comprises two gene copies per cell rather than multiple copies of mRNA that are being continually transcribed. Further, genome editing by TFOs or PNAs induces permanent changes in the pathological genes, thus facilitating the complete cure of diseases. Nuclease-based gene-editing tools, such as zinc fingers, CRISPR-Cas9, and TALENs, are being explored for therapeutic applications, although their potential off-target, cytotoxic, and/or immunogenic effects may hinder their in vivo applications. Therefore, this review is aimed at describing the ongoing progress in TFO and PNA technologies, which can be symbiotic genome-targeting tools that will cause a near-future paradigm shift in drug development. [ABSTRACT FROM AUTHOR]

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]

The R132H isocitrate dehydrogenase one (IDH1) mutation is a prognostic biomarker present in a subset of gliomas and is associated with heightened survival when paired with aggressive surgical resection. In this study, we establish proof-of-principle for rapid colorimetric detection of the IDH1-R132H mutation in tumor samples in under 1 hour without the need for a nucleic acid extraction. Colorimetric peptide nucleic acid loop-mediated isothermal amplification (CPNA-LAMP) utilizes 4 conventional LAMP primers, a blocking PNA probe complementary to the wild-type sequence, and a self-annealing loop primer complementary to the single nucleotide variant to only amplify the DNA sequence containing the mutation. This assay was evaluated using IDH1-WT or IDH1-R132H mutant synthetic DNA, wild-type or IDH1-R132H mutant U87MG cell lysates, and tumor lysates from archived patient samples in which the IDH1 status was previously determined using immunohistochemistry (IHC). Reactions were performed using a hot water bath and visually interpreted as positive by a pink-to-yellow color change. Results were subsequently verified using agarose gel electrophoresis. CPNA-LAMP successfully detected the R132H single nucleotide variant, and results from tumor lysates yielded 100% concordance with IHC results, including instances when the single nucleotide variant was limited to a portion of the tumor. Importantly, when testing the tumor lysates, there were no false positive or false negative results. [ABSTRACT FROM AUTHOR]

Background: Non-coding microRNAs (miRNAs) play critical roles in tumor progression and hold great promise as therapeutic agents for multiple cancers. MicroRNA 29a (miR-29a) is a tumor suppressor miRNA that inhibits cancer cell growth and tumor progression in non-small cell lung cancer. Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6), which plays an important role in lung cancer progression, has been identified as a target of miR-29a. Here, we evaluated the therapeutic efficacy of a peptide vector capable of delivering miR-29a intracellularly using the acidic tumor microenvironment in a lung adenocarcinoma xenograft mouse model. Methods: A miRNA delivery vector was constructed by tethering the peptide nucleic acid form of miR-29a to a peptide with a low pH-induced transmembrane structure (pHLIP) to enable transport of the miRNAs across the plasma membrane. Tumor suppressive effects of pHLIP-miR29a on lung adenocarcinoma development in vivo were assessed using a BALB/c xenograft model injected with A549 cells. Results: Incubation of A549 cells with pHLIP-miR-29a at an acidic pH downregulated endogenous CEACAM6 expression and reduced cell viability. Intravenous injection of the mice with pHLIP-miR-29a inhibited tumor growth by up to 18.1%. Intraperitoneal injection of cisplatin reduced tumor volume by 29.9%. Combined pHLIP-miR-29a + cisplatin treatment had an additive effect, reducing tumor volume up to 39.7%. Conclusions: Delivery of miR-29a to lung adenocarcinoma cells using a pHLIP-mediated method has therapeutic potential as a unique cancer treatment approach. [ABSTRACT FROM AUTHOR]

Antisense peptide nucleic acids (PNAs) inhibit bacterial growth in several infection models. Since PNAs are not spontaneously taken up by bacteria, they are often conjugated to carriers such as cell-penetrating peptides (CPPs) in order to improve translocation. Hydrophobic counterions such as pyrenebutyrate (PyB) have been shown to facilitate translocation of peptides over natural and artificial membranes. In this study, the capability of PyB to support translocation of CPP-coupled antisense PNAs into bacteria was investigated in Streptococcus pyogenes and Streptococcus pneumoniae. PyB enhanced the antimicrobial activity of CPP-conjugated antisense PNAs in S. pyogenes. The most significant effect of PyB was observed in combination with K8-conjugated anti-gyrA PNAs. In contrast, no significant effect of PyB on the antimicrobial activity of CPP-conjugated PNAs in S. pneumoniae was detected. Uptake of K8-FITC into S. pyogenes, Escherichia coli, and Klebsiella pneumoniae could be improved by pre-incubation with PyB, indicating that PyB supports the antimicrobial effect of CPP-antisense PNAs in S. pyogenes by facilitating the translocation of peptides across the bacterial membrane. [ABSTRACT FROM AUTHOR]

Ideal drug carriers feature a high loading capacity to minimize the exposure of patients with excessive, inactive carrier materials. The highest imaginable loading capacity could be achieved by nanocarriers, which are assembled from the therapeutic cargo molecules themselves. Here, we describe peptide nucleic acid (PNA)-based zirconium (Zr) coordination nanoparticles which exhibit very high PNA loading of > 94 % w/w. This metal-organic hybrid nanomaterial class extends the enormous compound space of coordination polymers towards bioactive oligonucleotide linkers. The architecture of single- or double-stranded PNAs was systematically varied to identify design criteria for the coordination driven self-assembly with Zr(IV) nodes at room temperature. Aromatic carboxylic acid functions, serving as Lewis bases, and a two-step synthesis process with preformation of Z r 6 O 4 (O H) 4 turned out to be decisive for successful nanoparticle assembly. Confocal laser scanning microscopy confirmed that the PNA-Zr nanoparticles are readily internalized by cells. PNA-Zr nanoparticles, coated with a cationic lipopeptide, successfully delivered an antisense PNA sequence for splicing correction of the β -globin intron mutation IVS2-705 into a functional reporter cell line and mediated splice-switching via interaction with the endogenous mRNA splicing machinery. The presented PNA-Zr nanoparticles represent a bioactive platform with high design flexibility and extraordinary PNA loading capacity, where the nucleic acid constitutes an integral part of the material, instead of being loaded into passive delivery systems. [ABSTRACT FROM AUTHOR]

Nucleic acid probes are valuable tools in biology and chemistry and are indispensable for PCR amplification of DNA, RNA quantification and visualization, and downregulation of gene expression. Recently, triplex-forming oligonucleotides (TFO) have received increased attention due to their improved selectivity and sensitivity in recognizing purine-rich double-stranded RNA regions at physiological pH by incorporating backbone and base modifications. For example, triplex-forming peptide nucleic acid (PNA) oligomers have been used for imaging a structured RNA in cells and inhibiting influenza A replication. Although a handful of programs are available to identify triplex target sites (TTS) in DNA, none are available that find such regions in structured RNAs. Here, we describe TFOFinder, a Python program that facilitates the identification of intramolecular purine-only RNA duplexes that are amenable to forming parallel triple helices (pyrimidine/purine/pyrimidine) and the design of the corresponding TFO(s). We performed genome- and transcriptome-wide analyses of TTS in Drosophila melanogaster and found that only 0.3% (123) of total unique transcripts (35,642) show the potential of forming 12-purine long triplex forming sites that contain at least one guanine. Using minimization algorithms, we predicted the secondary structure(s) of these transcripts, and using TFOFinder, we found that 97 (79%) of the identified 123 transcripts are predicted to fold to form at least one TTS for parallel triple helix formation. The number of transcripts with potential purine TTS increases when the strict search conditions are relaxed by decreasing the length of the probe or by allowing up to two pyrimidine inversions or 1-nucleotide bulge in the target site. These results are encouraging for the use of modified triplex forming probes for live imaging of endogenous structured RNA targets, such as pre-miRNAs, and inhibition of target-specific translation and viral replication. Author summary: Nucleic acid molecules are most often encountered in living organisms as double-stranded (DNA) or single-stranded (RNA). However, when meeting certain sequence requirements, they can also form complex structures in which three (triplex) of four (quadruplex) strands will interact. Important biological roles were reported for short intramolecular RNA triplexes and more recently it was shown that noncoding RNAs can control gene expression via intermolecular triplex formation with double-stranded DNA. Current algorithms identify double-stranded DNA regions, as well as single-stranded RNA regions that can form a triplex, but no programs are available to identify such regions in a structured RNA. We wrote TFOFinder, a Python program to design probes that are predicted to form intermolecular triplexes with structured regions of a given RNA target. These probes can be used for imaging structured RNAs in physiological conditions or for target-specific translation inhibition. We first analyze the fruit fly transcriptome for RNAs that show the potential to form triplexes and predict the secondary structure of all hits. Using our program, we take into consideration the structure of each target and find that most of these hits are predicted to contain regions amenable to forming triplexes. [ABSTRACT FROM AUTHOR]

Numerous novel methods to detect foodborne pathogens have been extensively developed to ensure food safety. Among the important foodborne bacteria, Bacillus cereus was identified as a pathogen of concern that causes various food illnesses, leading to interest in developing effective detection methods for this pathogen. Although a standard method based on culturing and biochemical confirmative test is available, it is time- and labor-intensive. Alternative PCR-based methods have been developed but lack high-throughput capacity and ease of use. This study, therefore, attempts to develop a robust method for B. cereus detection by leveraging the highly specific pyrrolidinyl peptide nucleic acids (PNAs) as probes for a bead array method with multiplex and high-throughput capacity. In this study, PNAs bearing prolyl-2-aminocyclopentanecarboxylic acid (ACPC) backbone with groEL, motB, and 16S rRNA sequences were covalently coupled with three sets of fluorescently barcoded beads to detect the three B. cereus genes. The developed acpcPNA-based bead array exhibited good selectivity where only signals were detectable in the presence of B. cereus, but not for other species. The sensitivity of this acpcPNA-based bead assay in detecting genomic DNA was found to be 0.038, 0.183 and 0.179 ng for groEL, motB and 16S rRNA, respectively. This performance was clearly superior to its DNA counterpart, hence confirming much stronger binding strength of acpcPNA over DNA. The robustness of the developed method was further demonstrated by testing artificially spiked milk and pickled mustard greens with minimal interference from food metrices. Hence, this proof-of-concept acpcPNA-based bead array method has been proven to serve as an effective alternative nucleic acid-based method for foodborne pathogens. [ABSTRACT FROM AUTHOR]

One of the most appealing approaches for regulating gene expression, named the "microRNA therapeutic" method, is based on the regulation of the activity of microRNAs (miRNAs), the intracellular levels of which are dysregulated in many diseases, including cancer. This can be achieved by miRNA inhibition with antimiRNA molecules in the case of overexpressed microRNAs, or by using miRNA-mimics to restore downregulated microRNAs that are associated with the target disease. The development of new efficient, low-toxic, and targeted vectors of such molecules represents a key topic in the field of the pharmacological modulation of microRNAs. We compared the delivery efficiency of a small library of cationic calix[4]arene vectors complexed with fluorescent antimiRNA molecules (Peptide Nucleic Acids, PNAs), pre-miRNA (microRNA precursors), and mature microRNAs, in glioma- and colon-cancer cellular models. The transfection was assayed by cytofluorimetry, cell imaging assays, and RT-qPCR. The calix[4]arene-based vectors were shown to be powerful tools to facilitate the uptake of both neutral (PNAs) and negatively charged (pre-miRNAs and mature microRNAs) molecules showing low toxicity in transfected cells and ability to compete with commercially available vectors in terms of delivery efficiency. These results could be of great interest to validate microRNA therapeutics approaches for future application in personalized treatment and precision medicine. [ABSTRACT FROM AUTHOR]