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.

Nucleic acid sensors based on a peptide nucleic acid (PNA) probe have seen a surge in interest since their discovery in the 1990s, and after the patent protecting them expired in 2013. The appeal of PNA as capture and/or sensing probes as an alternative to standard DNA or RNA oligonucleotides originates from their superior chemical stability and affinity for complementary oligonucleotides, as well as their increased responsiveness to single base mismatches. The implementation of PNA probes onto optical and electrochemical sensors has showed great promise although progress has been hampered by issues mostly associated with surface chemistry, probe accessibility and non-specific binding. Herein, we report on a systematic comparison between various PNA immobilisation strategies on carbon substrates based on both covalent and non-covalent chemistries. Besides the use of standard electrochemical techniques to characterise the extent of surface modification, the ability of immobilised PNAs to engage in chemical interactions with freely diffusing molecules was also investigated. Using original chemical tags, this study provides a unique insight into the impact of immobilisation chemistries on PNA\'s (bio)availability. Rapid immobilisation of biotinylated PNA oligomers on screen-printed carbon electrode (SPCE) coated with adsorbed polystreptavidin (pSA) demonstrated highest efficiency and ease in the preparation process. An original nucleic acid sensor using this immobilisation chemistry is reported that is based on a sandwich assay between a surface bound PNA capture probe and a freely diffusing electrochemically active PNA sensing probe.

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

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.

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.

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.

Two reports by Dhuri et al. and Oyaghire et al., respectively, show that, through installing chiral centers at the backbone of the artificial nucleic acid, peptide nucleic acid (PNA), enhanced miRNA targeting and genome modification can be achieved, with important implications in fighting cancers and β-thalassemia.

Targeting oncogenes at the genomic DNA level can open new avenues for precision medicine. Significant efforts are ongoing to target oncogenes using RNA-targeted and protein-targeted platforms, but no progress has been made to target genomic DNA for cancer therapy. Here, we introduce a gamma peptide nucleic acid (γPNA)-based genomic DNA-targeted platform to silence oncogenes in vivo. γPNAs efficiently invade the mixed sequences of genomic DNA with high affinity and specificity. As a proof of concept, we establish that γPNA can inhibit c-Myc transcription in multiple cell lines. We evaluate the in vivo efficacy and safety of genomic DNA targeting in three pre-clinical models. We also establish that anti-transcription γPNA in combination with histone deacetylase inhibitors and chemotherapeutic drugs results in robust antitumor activity in cell-line- and patient-derived xenografts. Overall, this strategy offers a unique therapeutic platform to target genomic DNA to inhibit oncogenes for cancer therapy.

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.

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.