Detecting the FLJ22447 lncRNA in Ovarian Cancer with Cyclopentane-Modified FIT-PNAs (cpFIT-PNAs).

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

Hybrid PNA-peptide hydrogels as injectable CEST-MRI agents.

The self-assembly of peptides and peptide analogues may be exploited to develop platforms for different biomedical applications, among which CEST-MRI (chemical exchange saturation transfer magnetic resonance imaging) represents one of the most attractive techniques to be explored as a novel metal-free contrast approach in imaging acquisitions. A lysine-containing peptide sequence (LIVAGK-NH2, named K2) was thus modified by insertion, at the N-terminus, of a peptide nucleic acid (PNA) base, leading to a primary amine suitable for the signal generation. a-K2, c-K2, g-K2 and t-K2 peptides were synthesized and characterized. The c-K2 sequence displayed gelling properties and the Watson and Crick pairing, arising from its combination with g-K2, allowed a significant increase in the mechanical responsivity of the hydrogel. These matrices were able to generate a CEST signal around 2.5 ppm from water and, after assessing their cytocompatibility on GL261 (murine glioma), TS/a (murine breast carcinoma), and 3T3-NIH (murine fibroblasts) cell lines, their capability to work as implants for in vivo detection, was proved by intratumor injection in Balb/c mice inoculated with TS/a murine breast cancer cells.

Dynamics of terminal fraying-peeling and hydrogen bonds dictates the sequential vs. cooperative melting pathways of nanoscale DNA and PNA triplexes.

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

K-ras mutation detected by peptide nucleic acid-clamping polymerase chain reaction, Ki-67, S100P, and SMAD4 expression can improve the diagnostic accuracy of inconclusive pancreatic EUS-FNB specimens.

OBJECTIVES: We aimed to assess the diagnostic utility of an immunohistochemical panel including calcium-binding protein P, p53, Ki-67, and SMAD family member 4 and K-ras mutation for diagnosing pancreatic solid lesion specimens obtained by endoscopic ultrasound-guided fine-needle biopsy and to confirm their usefulness in histologically inconclusive cases. METHODS: Immunohistochemistry and peptide nucleic acid-clamping polymerase chain reaction for K-ras mutation were performed on 96 endoscopic ultrasound-guided fine-needle biopsy specimens. The diagnostic efficacy of each marker and the combination of markers was calculated. The diagnostic performances of these markers were evaluated in 27 endoscopic ultrasound-guided fine-needle biopsy specimens with histologically inconclusive diagnoses. A classification tree was constructed. RESULTS: K-ras mutation showed the highest accuracy and consistency. Positivity in more than two or three of the five markers showed high diagnostic accuracy (94.6 % and 93.6 %, respectively), and positivity for more than three markers showed the highest accuracy for inconclusive cases (92.0 %). A classification tree using K-ras mutation, Ki-67, S100P, and SMAD4 showed high diagnostic performance, with only two misclassifications in inconclusive cases. CONCLUSIONS: K-ras mutation detection via peptide nucleic acid-clamping polymerase chain reaction is a stable and accurate method for distinguishing between pancreatic ductal adenocarcinoma and non-pancreatic ductal adenocarcinoma lesions. A classification tree using K-ras mutation, Ki-67, S100P, and SMAD4 helps increase the diagnostic accuracy of cases that are histologically difficult to diagnose.

Innovative PNA-LB mediated allele-specific LAMP for KRAS mutation profiling on a compact lab-on-a-disc device.

Tailored healthcare, an approach focused on individual patients, requires integrating emerging interdisciplinary technologies to develop accurate and user-friendly diagnostic tools. KRAS mutations, prevalent in various common cancers, are crucial determinants in selecting patients for novel KRAS inhibitor therapies. This study presents a novel state-of-the-art Lab-on-a-Disc system utilizing peptide nucleic acids-loop backward (PNA-LB) mediated allele-specific loop-mediated isothermal amplification (LAMP) for detecting the frequent G12D KRAS mutation, signifying its superiority over alternative mutation detection approaches. The designed Lab-on-a-Disc system demonstrated exceptional preclinical and technical precision, accuracy, and versatility. By applying varying cutoff values to PNA- LB LAMP reactions, the assay's sensitivity and specificity were increased by 80 % and 90 %, respectively. The device's key advantages include a robust microfluidic Lab-on-a-Disc design, precise rotary control, and a cutting-edge induction heating module. These features enable multiplexing of LAMP reactions with high reproducibility and repeatability, with CV% values less than 3.5 % and 5.5 %, respectively. The device offers several methods for accurate endpoint result detection, including naked-eye observation, RGB image analysis using Python code, and time of fluorescence (Tf) values. Preclinical specificity and sensitivity, assessed using different cutoffs for Eva-Green fluorescence Tf values and pH-sensitive dyes, demonstrated comparable performance to the best standard methods. Overall, this study represents a significant step towards tailoring treatment strategies for cancer patients through precise and efficient mutation detection technologies.

PNA-Functionalized, Silica Nanowires-Filled Glass Microtube for Ultrasensitive and Label-Free Detection of miRNA-21.

MicroRNAs (miRNAs) are endogenous and noncoding single-stranded RNA molecules with a length of approximately 18-25 nucleotides, which play an undeniable role in early cancer screening. Therefore, it is very important to develop an ultrasensitive and highly specific method for detecting miRNAs. Here, we present a bottom-up assembly approach for modifying glass microtubes with silica nanowires (SiNWs) and develop a label-free sensing platform for miRNA-21 detection. The three-dimensional (3D) networks formed by SiNWs make them abundant and highly accessible sites for binding with peptide nucleic acid (PNA). As a receptor, PNA has no phosphate groups and exhibits an overall electrically neutral state, resulting in a relatively small repulsion between PNA and RNA, which can improve the hybridization efficiency. The SiNWs-filled glass microtube (SiNWs@GMT) sensor enables ultrasensitive, label-free detection of miRNA-21 with a detection limit as low as 1 aM at a detection range of 1 aM-100 nM. Noteworthy, the sensor can still detect miRNA-21 in the range of 102-108 fM in complex solutions containing 1000-fold homologous interference of miRNAs. The high anti-interference performance of the sensor enables it to specifically recognize target miRNA-21 in the presence of other miRNAs and distinguish 1-, 3-mismatch nucleotide sequences. Significantly, the sensor platform is able to detect miRNA-21 in the lysate of breast cancer cell lines (e.g., MCF-7 cells and MDA-MB-231 cells), indicating that it has good potential in the screening of early breast cancers.

Zinc N,N-bis(2-picolyl)amine Chelates Show Substitution-Dependent Cleavage of Phosphodiesters in Models as Well as of PNAzyme-RNA Bulges.

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

Effects of PNA Sequence and Target Site Selection on Function of a 4.5S Non-Coding RNA.

Peptide nucleic acid (PNA) based antisense strategy is a promising therapeutic approach to specifically inhibit target gene expression. However, unlike protein coding genes, identification of an ideal PNA binding site for non-coding RNA is not straightforward. Here, we compare the inhibitory activities of PNA molecules that bind a non-coding 4.5S RNA called SRP RNA, a key component of the bacterial signal recognition particle (SRP). A 9-mer PNA (PNA9) complementary to the tetraloop region of the RNA was more potent in inhibiting its interaction with the SRP protein, compared to an 8-mer PNA (PNA8) targeting a stem-loop. PNA9, which contained a homo-pyrimidine sequence could form a triplex with the complementary stretch of RNA in vitro as confirmed using a fluorescent derivative of PNA9 (F-PNA13). The RNA-PNA complex formation resulted in inhibition of SRP function with PNA9 and F-PNA13, but not PNA8 highlighting the importance of target site selection. Surprisingly, F-PNA13 which was more potent in inhibiting SRP function in vitro, showed weaker antibacterial activity compared to PNA9 likely due to poor cell penetration of the longer PNA. Our results underscore the importance of suitable target site selection and optimum PNA length to develop better antisense molecules against non-coding RNA.

Hybrid peptide-PNA monomers as building blocks for the fabrication of supramolecular aggregates.

Advantages like biocompatibility, biodegradability and tunability allowed the exploitation of peptides and peptidomimetics as versatile therapeutic or diagnostic agents. Because of their selectivity towards transmembrane receptors or cell membranes, peptides have also been identified as suitable molecules able to deliver in vivo macromolecules, proteins or nucleic acids. However, after the identification of the homodimer diphenylalanine (FF) as an aggregative motif inside the Aß1-42 polypeptide, short and ultrashort peptides have been studied as building blocks for the fabrication of supramolecular, ordered nanostructures for applications in biotechnological, biomedical and industrial fields. In this perspective, many hybrid molecules that combine FF with other chemical entities have been synthesized and characterized. Two novel hybrid derivatives (tFaF and cFgF), in which the FF homodimer is alternated with the peptide-nucleic acid (PNA) heterodimer "g-c" (guanine-cytosine) or "a-t" (adenine-thymine) and their dimeric forms (tFaF)2 and (cFgF)2 were synthesized. The structural characterization performed by circular dichroism (CD), Fourier transform infrared (FTIR) and fluorescence spectroscopies highlighted the capability of all the FF-PNA derivatives to self-assemble into ß-sheet structures. As a consequence of this supramolecular organization, the resulting aggregates also exhibit optoelectronic properties already reported for other similar nanostructures. This photoemissive behavior is promising for their potential applications in bioimaging.

A Biotinylated cpFIT-PNA Platform for the Facile Detection of Drug Resistance to Artemisinin in Plasmodium falciparum.

The evolution of drug resistance to many antimalarial drugs in the lethal strain of malaria (Plasmodium falciparum) has been a great concern over the past 50 years. Among these drugs, artemisinin has become less effective for treating malaria. Indeed, several P. falciparum variants have become resistant to this drug, as elucidated by specific mutations in the pfK13 gene. This study presents the development of a diagnostic kit for the detection of a common point mutation in the pfK13 gene of P. falciparum, namely, the C580Y point mutation. FIT-PNAs (forced-intercalation peptide nucleic acid) are DNA mimics that serve as RNA sensors that fluoresce upon hybridization to their complementary RNA. Herein, FIT-PNAs were designed to sense the C580Y single nucleotide polymorphism (SNP) and were conjugated to biotin in order to bind these molecules to streptavidin-coated plates. Initial studies with synthetic RNA were conducted to optimize the sensing system. In addition, cyclopentane-modified PNA monomers (cpPNAs) were introduced to improve FIT-PNA sensing. Lastly, total RNA was isolated from red blood cells infected with P. falciparum (WT strain - NF54-WT or mutant strain - NF54-C580Y). Streptavidin plates loaded with either FIT-PNA or cpFIT-PNA were incubated with the total RNA. A significant difference in fluorescence for mutant vs WT total RNA was found only for the cpFIT-PNA probe. In summary, this study paves the way for a simple diagnostic kit for monitoring artemisinin drug resistance that may be easily adapted to malaria endemic regions.

Peptide-Based Flavivirus Biosensors: From Cell Structure to Virological and Serological Detection Methods.

In tropical and developing countries, mosquito-borne diseases by flaviviruses pose a serious threat to public health. Early detection is critical for preventing their spread, but conventional methods are time-consuming and require skilled technicians. Biosensors have been developed to address this issue, but cross-reactivity with other flaviviruses remains a challenge. Peptides are essentially biomaterials used in diagnostics that allow virological and serological techniques to identify flavivirus selectively. This biomaterial originated as a small protein consisting of two to 50 amino acid chains. They offer flexibility in chemical modification and can be easily synthesized and applied to living cells in the engineering process. Peptides could potentially be developed as robust, low-cost, sensitive, and selective receptors for detecting flaviviruses. However, modification and selection of the receptor agents are crucial to determine the effectiveness of binding between the targets and the receptors. This paper addresses two potential peptide nucleic acids (PNAs) and affinity peptides that can detect flavivirus from another target-based biosensor as well as the potential peptide behaviors of flaviviruses. The PNAs detect flaviviruses based on the nucleotide base sequence of the target's virological profile via Watson-Crick base pairing, while the affinity peptides sense the epitope or immunological profile of the targets. Recent developments in the functionalization of peptides for flavivirus biosensors are explored in this Review by division into electrochemical, optical, and other detection methods.

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

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

Reflections on a Copenhagen-Minneapolis Axis in Bioorganic Chemistry.

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.

A New Peptide Nucleic Acid Fluorescence In Situ Hybridization Probe for the Specific Detection of Salmonella Species in Food Matrices.

Salmonella spp. is among the most central etiological agents in foodborne bacterial disorders. To identify Salmonella spp., numerous new molecular techniques have been developed conversely to the traditional culture-based methods. In this work, a new peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) method was developed for the specific detection of Salmonella species, allowing a faster analysis compared with the traditional methods (ISO 6579-1: 2017). The method was optimized based on a novel PNA probe (SalPNA1692) combined with a blocker probe to detect Salmonella in food samples through an assessment of diverse-rich and selective enrichment broths. Our findings indicated that the best outcome was obtained using a 24-h pre-enrichment step in buffered peptone water, followed by RambaQuick broth selective enrichment for 16 h. For the enrichment step performance validation, fresh ground beef was artificially contaminated with two ranges of concentration of inoculum: a low level (0.2-2 colony-forming units [CFUs]/25 g) and a high level (2-10 CFUs/25 g). The new PNA-FISH method presented a specificity of 100% and a detection limit of 0.5 CFU/25 g of food sample, which confirms the great potential of applying PNA probes in food analysis.

Evaluation and cultivation method of high-tech value patents for mechanical products.

Evaluation of high value patents is essential for the enterprise's technical layout and innovative product design. The existing research on the patent value needs the support of a large number of professional statistical information and is difficult to directly reflect the technical value. Since technological innovation is the fundamental means to enhance the sustainable competitiveness of enterprises. Therefore, a high-tech value patent evaluation and cultivation method for engineering designers need to be proposed. Firstly, the patent samples based on design methodology are retrieved and the indicators for evaluating technical value are summarized and the rationality of the evaluation indicators is verifier through empirical study based on improved evidence theory. Secondly, based on principal component analysis and factor analysis, a high-tech value patent evaluation and cultivation method is proposed. Finally, the proposed method is applied to identify the high-tech value patents in the cutting machine industry, and structural improvement is made based on this patent to demonstrate the cultivation process of high-tech value patents. The proposed method provides a clear guiding direction for the cultivation of high novelty patents and enterprise innovative product design. The method can effectively assist the product R&D activities of engineering designers and enhance the sustainable competitiveness of enterprises from a technological perspective.

Peptide nucleic acid-assisted generation of targeted double-stranded DNA breaks with T7 endonuclease I.

Gene-editing technologies have revolutionized biotechnology, but current gene editors suffer from several limitations. Here, we harnessed the power of gamma-modified peptide nucleic acids (γPNAs) to facilitate targeted, specific DNA invasion and used T7 endonuclease I (T7EI) to recognize and cleave the γPNA-invaded DNA. Our data show that T7EI can specifically target PNA-invaded linear and circular DNA to introduce double-strand breaks (DSBs). Our PNA-Guided T7EI (PG-T7EI) technology demonstrates that T7EI can be used as a programmable nuclease capable of generating single or multiple specific DSBs in vitro under a broad range of conditions and could be potentially applied for large-scale genomic manipulation. With no protospacer adjacent motif (PAM) constraints and featuring a compact protein size, our PG-T7EI system will facilitate and expand DNA manipulations both in vitro and in vivo, including cloning, large-fragment DNA assembly, and gene editing, with exciting applications in biotechnology, medicine, agriculture, and synthetic biology.

Detection of miR-155 Using Peptide Nucleic Acid at Physiological-like Conditions by Surface Plasmon Resonance and Bio-Field Effect Transistor.

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.

The Application of Peptide Nucleic Acids (PNA) in the Inhibition of Proprotein Convertase Subtilisin/Kexin 9 (PCSK9) Gene Expression in a Cell-Free Transcription/Translation System.

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.

Combined antimicrobial effect of two peptide nucleic acids against Staphylococcus aureus and S. pseudintermedius veterinary isolates.

BACKGROUND: Staphylococcus aureus and S. pseudintermedius are the major etiological agents of staphylococcal infections in humans, livestock, and companion animals. The misuse of antimicrobial drugs has led to the emergence of antimicrobial-resistant Staphylococcus spp., including methicillin-resistant S. aureus (MRSA) and methicillin-resistant S. pseudintermedius (MRSP). One novel therapeutic approach against MRSA and MRSP is a peptide nucleic acid (PNA) that can bind to the target nucleotide strands and block expression. Previously, two PNAs conjugated with cell-penetrating peptides (P-PNAs), antisense PNA (ASP)-cmk and ASP-deoD, targeting two essential genes in S. aureus, were constructed, and their antibacterial activities were analyzed. OBJECTIVES: This study analyzed the combined antibacterial effects of P-PNAs on S. aureus and S. pseudintermedius clinical isolates. METHODS: S. aureus ATCC 29740 cells were treated simultaneously with serially diluted ASP-cmk and ASP-deoD, and the minimal inhibitory concentrations (MICs) were measured. The combined P-PNA mixture was then treated with S. aureus and S. pseudintermedius veterinary isolates at the determined MIC, and the antibacterial effect was examined. RESULTS: The combined treatment of two P-PNAs showed higher antibacterial activity than the individual treatments. The MICs of two individual P-PNAs were 20 and 25 µM, whereas that of the combined treatment was 10 µM. The application of a combined treatment to clinical Staphylococcus spp. revealed S. aureus isolates to be resistant to P-PNAs and S. pseudintermedius isolates to be susceptible. CONCLUSIONS: These observations highlight the complexity of designing ASPs with high efficacy for potential applications in treating staphylococcal infections in humans and animals.

A comparative analysis of peptide-delivered antisense antibiotics using diverse nucleotide mimics.

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