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Single nucleotide polymorphism (SNP), as one of the key components of the genetic factors, is important for disease detection and early screening of hereditary diseases. Current SNP genotyping methods require laboratory instruments or long operating times. To facilitate the diagnosis of hereditary diseases, we developed a new method referred to as the LwaCas13a-based SNP genotyping platform (Cas13a platform), which is useful for detecting disease-related SNPs. We report a CRISPR/Cas13a-based SNP genotyping platform that couples recombinase-aided amplification (RAA), T7 transcription, and Leptotrichia wadei Cas13a (LwaCas13a) detection for simple and fast genotyping of human disease-related SNPs. We used this Cas13a platform to identify 17 disease-related SNPs, demonstrating that position 2 in gRNA is suitable for the introduction of additional mismatches to achieve high discrimination in genotyping across a wide range of SNP targets. The discrimination specificity of 17 SNPs was improved 3.0–35.1-fold after introducing additional mismatches at position 2 from the 5′-end. We developed a method, which has a lower risk of cross-contamination and operational complexity, for genotyping SNPs using human saliva samples in an one-pot testing that delivers results within 60 min. Compared to TaqMan probe qPCR, RFLP, AS-PCR and other SNP genotyping methods, the Cas13a platform is simple, rapid and reliable, expanding the applications of the CRISPR/Cas system in nucleic acid detection and SNP genotyping. [ABSTRACT FROM AUTHOR]

In this study, a novel electrochemical biosensor was developed for the sensitive and selective detection of the Acinetobacter baumannii gene sequence. The biosensor was created by immobilizing a capture probe specific to the A. baumannii gene on the surface of chitosan-gold modified pencil graphite electrodes. Following solid-state hybridization on the Chit-Au/PGE surface, the target DNA sequence of the A. baumannii was detected by measuring the guanine signal using square wave voltammetry (SWV). All experimental parameters impacting sensor response are examined in order to improve hybridization efficacy, and the electrochemical biosensor's performance. The limit of detection (LOD) for the A. baumannii gene sequence was calculated and found to be 1.93 nM. Three different non-complementary DNA sequences were used to evaluate the assay selectivity, but no interference effect was obtained. Additionally, the potential applicability of the biosensor to real samples was tested in artificial serum media. The suggested electrochemical test procedure is simple, approachable, and quick, making it a convenient approach for the screening of DNA sequence. [Display omitted] • Pencil graphite electrodes were modified with chitosan-Au composite. • CHIT-Au/PGEs were used for electrochemical biosensing of A. baumannii gene • The [Fe(CN) 6 ]3-/4- oxidation signal was measured by SWV technique. • The proposed electrochemical assay is simple, rapid, and accessible. [ABSTRACT FROM AUTHOR]

Programmable nuclease-based detection technology has superior sensitivity, specificity, portability, and timeliness. Interference between amplification and programmable nucleases presents a major barrier to the clinical application of programmable nuclease-based diagnosis. A one-pot assay combining amplification and nuclease cleavage could overcome issues of contamination and operation complexity. Strategies encompassing physical isolation, microfluidic chips, thermal activation, component optimization, nuclease characterization and engineering, and guide RNA modification for the development of one-pot detection are reviewed. Existing one-pot detection methods still have room for improvement, which will be necessary to promote the popularization and application of programmable nuclease-based detection technology. CRISPR/Cas and Argonaute (Ago) proteins, which target specific nucleic acid sequences, can be applied as diagnostic tools. Despite high specificity and efficiency, achieving sensitive detection often necessitates a preamplification step that involves opening the lid and multistep operation, which may elevate the risk of contamination and prove inadequate for point-of-care testing. Hence, various one-pot detection strategies have been developed that enable preamplification and sensing in a single operation. We outline the challenges of one-pot detection with Cas and Ago proteins, present several main implementation strategies, and discuss future prospects. This review offers comprehensive insights into this vital field and explores potential improvements to detection methods that will be beneficial for human health. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus generates extracellular membrane vesicles (MVs) during both in vitro culture and in vivo infection. S. aureus MVs are generated by either blebbing of the cytoplasmic membrane or explosive cell lysis. Proteins and nucleic acids encapsulated within S. aureus MVs are protected from degradation or neutralization by host factors. S. aureus MVs exhibit cytotoxicity to a variety of host cell types and induce both host innate and adaptive immune responses in animal infection models. MVs exploit different endocytic pathways for the delivery of their cargo into host cells. Deciphering the intracellular fate of S. aureus MVs after cellular entry may deepen our limited understanding of how MVs impact the host during staphylococcal infections. Staphylococcus aureus is an important bacterial pathogen that causes a wide variety of human diseases in community and hospital settings. S. aureus employs a diverse array of virulence factors, both surface-associated and secreted, to promote colonization, infection, and immune evasion. Over the past decade, a growing body of research has shown that S. aureus generates extracellular membrane vesicles (MVs) that package a variety of bacterial components, many of which are virulence factors. In this review, we summarize recent advances in our understanding of S. aureus MVs and highlight their biogenesis, cargo, and potential role in the pathogenesis of staphylococcal infections. Lastly, we present some emerging questions in the field. [ABSTRACT FROM AUTHOR]

The eukaryotic genome exhibits a predominant canonical B-DNA structure. DNA can also adopt noncanonical forms which are collectively referred to as non-B DNA. These alternative DNA structures have the potential to disturb the double helix and thereby affect gene expression. The implementation of novel cutting-edge methodologies to investigate non-B DNA motifs for global identification and profiling in plant genomes offers insights into their complex interactions with the epigenome, thereby enriching our understanding of their roles in regulating gene expression. Plants adapt their development in response to environmental changes, and the secondary and tertiary structures of nucleic acids are modulated during daily temperature oscillations and heatwaves, highlighting the importance of non-B DNA structures in gene expression regulation and plant adaptability. Regulating gene expression in plant development and environmental responses is vital for mitigating the effects of climate change on crop growth and productivity. The eukaryotic genome largely shows the canonical B-DNA structure that is organized into nucleosomes with histone modifications shaping the epigenome. Nuclear proteins and RNA interactions influence chromatin conformations and dynamically modulate gene activity. Non-B DNA conformations and their transitions introduce novel aspects to gene expression modulation, particularly in response to environmental shifts. We explore the current understanding of non-B DNA structures in plant genomes, their interplay with epigenomics and gene expression, and advances in methods for their mapping and characterization. The exploration of so far uncharacterized non-B DNA structures remains an intriguing area in plant chromatin research and offers insights into their potential role in gene regulation. [ABSTRACT FROM AUTHOR]

In vitro and ex-vivo target identification strategies often fail to predict in vivo efficacy, particularly for glioblastoma (GBM), a highly heterogenous tumor rich in resistant cancer stem cells (GSCs). An in vivo screening tool can improve prediction of therapeutic efficacy by considering the complex tumor microenvironment and the dynamic plasticity of GSCs driving therapy resistance and recurrence. This study proposes lipid nanoparticles (LNPs) as an efficient in vivo CRISPR-Cas9 gene editing tool for target validation in mesenchymal GSCs. LNPs co-delivering mRNA (mCas9) and single-guide RNA (sgRNA) were successfully formulated and optimized facilitating both in vitro and in vivo gene editing. In vitro, LNPs achieved up to 67 % reduction in green fluorescent protein (GFP) expression, used as a model target, outperforming a commercial transfection reagent. Intratumoral administration of LNPs in GSCs resulted in ∼80 % GFP gene knock-out and a 2-fold reduction in GFP signal by day 14. This study showcases the applicability of CRISPR-Cas9 LNPs as a potential in vivo screening tool in GSCs, currently lacking effective treatment. By replacing GFP with a pool of potential targets, the proposed platform presents an exciting prospect for therapeutic target validation in orthotopic GSCs, bridging the gap between preclinical and clinical research. [Display omitted] • LNPs an efficient in vitro transfection tool of CRISPR-Cas9 RNAs in GSCs, an alterantive to commercial transfecion reagents. • The CRISPR-Cas9 LNP platform enables efficient in vivo gene editing of a difficult to treat model of mesenchymal GSCs. • Proposing CRISPR-Cas9 LNPs as an in vivo screening tool for therapeutic target identification in GSCs. [ABSTRACT FROM AUTHOR]

The ability to mount an interferon-mediated innate immune response is essential in protection against neurotropic viruses, but antiviral type I interferons also have neurotoxic potential. The production of type I interferons can be triggered by self-derived nucleic acids, and the brain can be susceptible to inappropriate upregulation of type I interferon signalling. Homoeostatic dysregulation of type I interferons has been implicated in rare inborn errors of immunity (referred to as type I interferonopathies) and more common neurodegenerative disorders (eg, Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis). Recent developments include new insights into the pathogenesis of these disorders that involve dysregulated type I interferon signalling, as well as advances in their diagnosis and management. The role of type I interferons in brain cellular health suggests the future therapeutic potential of approaches that target these interferons and their signalling. [ABSTRACT FROM AUTHOR]

Animal diseases are a major concern to animal welfare, human health and the global economy. Early detection, prevention and control of these animal diseases are crucial to ensure sustainability of livestock sector, to reduce farm losses and protecting public health. Points of care (POC) devices are small, portable instruments that provide rapid results thus reduce the risk of disease transmission and enable early intervention. CRISPR based diagnostics offer more accurate and efficient solution for monitoring animal health due to their quick response, can detect very low level of pathogenic organism or disease markers and specificity. These diagnostics are particularly useful in the in area with limited resources or access to common diagnostic methods, especially in developing countries. The ability of electrochemical sensors to detect accurately very low analyte concentration makes them suitable for POC diagnostics and field application. CRISPR base electrochemical biosensors show great potential in revolutionizing disease detection and diagnosis including animal health. However, challenges, such as achieving selectivity and sensitivity, need to be addressed to enhance the competitiveness of these biosensors. Currently, most CRISPR based bioassay research focuses on nucleic acid target detection, but researchers exploring to monitor small organic/inorganic non-nucleic acid molecules like toxins and proteins. Emerging diagnostics would be centered on CRISPR-Cas system will offer great potential as an accurate, specific and effective means to identify microorganism, virus, toxins, small molecules, peptides and nucleic acid related to various animal health disorders particularly when integrated into electrochemical biosensing platform. • Overviewed the potential of electrochemical CRISPR based diagnostics for monitoring animal health. • Application of cis-clevage activity of CRISPR- Cas system for Electrochemical bioassay has reviewed. • Emphasized the application of Electrochemical CRISPR-Cas system in clinical diagnosis and food safety. [ABSTRACT FROM AUTHOR]

Approximately 25% of newly diagnosed AML patients display an internal tandem duplication (ITD) in the fms-like tyrosine kinase 3 (FLT3) gene. Although both multi-targeted and FLT3 specific tyrosine kinase inhibitors (TKIs) are being utilized for clinical therapy, drug resistance, short remission periods, and high relapse rates are challenges that still need to be tackled. RNA interference (RNAi), mediated by short interfering RNA (siRNA), presents a mechanistically distinct therapeutic platform with the potential of personalization due to its gene sequence-driven mechanism of action. This study explored the use of a non-viral approach for delivery of FLT3 siRNA (siFLT3) in FLT3-ITD positive AML cell lines and primary cells as well as the feasibility of combining this treatment with drugs currently used in the clinic. Treatment of AML cell lines with FLT3 siRNA nanocomplexes resulted in prominent reduction in cell proliferation rates and induction of apoptosis. Quantitative analysis of relative mRNA transcript levels revealed downregulation of the FLT3 gene, which was accompanied by a similar decline in FLT3 protein levels. Moreover, an impact on leukemic stem cells was observed in a small pool of primary AML samples through significantly reduced colony numbers. An absence of a molecular response post-treatment with lipopolymer/siFLT3 complexes in peripheral blood mononuclear cells, obtained from healthy individuals, denoted a passive selectivity of the complexes towards malignant cells. The effect of combining lipopolymer/siFLT3 complexes with daunorubucin and FLT3 targeting TKI gilteritinib led to a significant augmentation of anti-leukemic activity. These findings demonstrate the promising potential of RNAi implemented with lipopolymer complexes for AML molecular therapy. The study prospectively supports the addition of RNAi therapy to current treatment modalities available to target the heterogeneity prevalent in AML. We show that a clinically validated target, the FLT3 gene, can be eradicated in leukemia cells using non-viral RNAi. We validated these lipopolymers as effective vehicles to deliver nucleic acids to leukemic cells. The potency of the lipopolymers was superior to that of the 'gold-standard' delivery agent, lipid nanoparticles (LNPs), which are not effective in leukemia cells at clinically relevant doses. Mechanistic studies were undertaken to probe structure-function relationships for effective biomaterial formulations. Cellular and molecular responses to siRNA treatment have been characterized in cell models, including leukemia patient-derived cells. The use of the siRNA therapy with clinically used chemotherapy was demonstrated. [Display omitted] [ABSTRACT FROM AUTHOR]

Formycin A (FOR) and pyrazofurin A (PYR) are nucleoside analogs with antiviral and antitumor properties. They are known to interfere with nucleic acid metabolism, but their direct effect on transcription is less understood. We explored how RNA polymerases (RNAPs) from bacteria, mitochondria, and viruses utilize FOR, PYR, and oxidized purine nucleotides. All tested polymerases incorporated FOR in place of adenine and PYR in place of uridine. FOR also exhibited surprising dual-coding behavior, functioning as a cytosine substitute, particularly for viral RNAP. In contrast, 8-oxoadenine and 8-oxoguanine were incorporated in place of uridine in addition to their canonical Watson-Crick codings. Our data suggest that the interconversion of canonical anti and alternative syn conformers underlies dual-coding abilities of FOR and oxidized purines. Structurally distinct RNAPs displayed varying abilities to utilize syn conformers during transcription. By examining base pairings that led to substrate incorporation and the entire spectrum of geometrically compatible pairings, we have gained new insights into the nucleobase selection processes employed by structurally diverse RNAPs. These insights may pave the way for advancements in antiviral therapies. [ABSTRACT FROM AUTHOR]

During transcription initiation, the holoenzyme of bacterial RNA polymerase (RNAP) specifically recognizes promoters using a dedicated σ factor. During transcription elongation, the core enzyme of RNAP interacts with nucleic acids mainly nonspecifically, by stably locking the DNA template and RNA transcript inside the main cleft. Here, we present a synthetic DNA aptamer that is specifically recognized by both core and holoenzyme RNAPs from extremophilic bacteria of the Deinococcus-Thermus phylum. The aptamer binds RNAP with subnanomolar affinities, forming extremely stable complexes even at high ionic strength conditions, blocks RNAP interactions with the DNA template and inhibits RNAP activity during transcription elongation. We propose that the aptamer binds at a conserved site within the downstream DNA-binding cleft of RNAP and traps it in an inactive conformation. The aptamer can potentially be used for structural studies to reveal RNAP conformational states, affinity binding of RNAP and associated factors, and screening of transcriptional inhibitors. • Aptamer T5 forms highly stable complexes with Thermus - Deinococcus RNA polymerases. • Aptamer T5 binds both core and holoenzyme forms of RNA polymerase. • Aptamer T5 can precipitate RNA polymerase from cell lysates. • Aptamer T5 inhibits the interactions of RNA polymerase with the DNA template. [ABSTRACT FROM AUTHOR]

Intracellular delivery of biologicals such as peptides, proteins, and nucleic acids presents a great opportunity for innovative therapeutics. However, the endosome entrapment remains a major bottleneck in the intracellular delivery of biomacromolecules, largely limiting their therapeutic potential. Here, we converted a cell-penetrating peptide (CPP), low molecular weight protamine (LMWP), to endosomal escape peptides (EEPs) by masking LMWP with a pH-responsive counter-ionic peptide. The resulting masked CPPs (mLMWP and mLMWP2) effectively promoted the escape of peptide/protein cargoes from endosomes into the cytoplasm. Consequential lysosome repair and lysophagy were initiated upon the endolysosomal leakage. Minimal reactive oxygen species (ROS) elevation or cell death was observed. Based on mLMWP2, we constructed an intracellular protein delivery system containing an antibody as a targeting module, mLMWP2 as an endosomal escape module, and the desired protein cargo. With the HER2-targeting delivery system, we efficiently translocated cyclization recombination enzyme (Cre) and BH3-interacting domain death agonist (BID) into the cytosol of HER2+ cells to exert their biological activity. Thereby, the modular delivery system shows its potential as a promising tool for scientific studies and therapeutic applications. A cell-penetrating peptide protected by a pH-dependent electrostatic mask is capable of promoting endosome-to-cytosol transport of peptide/protein cargo upon antibody-mediated endocytosis. [Display omitted] [ABSTRACT FROM AUTHOR]

Cyclic nucleic acids are biologically stable against nucleic acid exonucleases due to the absence of 5′ and 3′ termini. Studies of cyclic nucleic acids mainly focus on cyclic single-stranded nucleic acids. Cyclic single-stranded nucleic acids are further divided into circular RNA (circRNA) and circular single-stranded DNA (cssDNA). The synthesis methods of circRNA include lasso-driven cyclization, intron-paired cyclization, intron cyclization, intron complementary pairing-driven cyclization, RNA-binding protein-driven cyclization, and artificial synthesis depending on the source. Its main role is to participate in gene expression and the treatment of some diseases. Circular single-stranded DNA is mainly synthesized by chemical ligation, template-directed enzyme ligation, and new techniques for the efficient preparation of DNA single loops and topologies based on CircLigase. It is mainly used in rolling circle amplification (RCA) technology and in the bioprotection of circular aptamers and second messengers. This review focuses on the types, synthesis methods, and applications of cyclic single-stranded nucleic acids, providing a reference for further research on cyclic single-stranded nucleic acids. • Circular single-stranded nucleic acids can be divided into circular single-stranded DNA (cssDNA) and circular RNA (circRNA). • Using circRNA is a new intervention of PKR related autoimmune diseases. • Natural circRNA can act as a miRNA sponge to regulate its activity. • Golden Gate assembly can generate user-defined cssDNA. • CssDNA can be used as a biosensor to detect Marine toxicity. [ABSTRACT FROM AUTHOR]

Lipid-based complex injectables are renowned for their effectiveness in delivering drugs, with many approved products. While significant strides have been made in formulating nanosystems for small molecular weight drugs, a pivotal breakthrough emerged with the recognition of lipid nanoparticles as a promising platform for delivering nucleic acids. This finding has paved the way for tackling long-standing challenges in molecular and delivery aspects (e.g., mRNA stability, intracellular delivery) that have impeded the clinical translation of gene therapy, especially in the realm of immunotherapy. Nonetheless, developing and implementing new lipid-based delivery systems pose significant challenges, as industrial manufacturing of these formulations often involves complex, multi-batch processes, giving rise to issues related to scalability, stability, sterility, and regulatory compliance. To overcome these obstacles, embracing the principles of quality-by-design (QbD) is imperative. Furthermore, adopting cutting-edge manufacturing and process analytical tools (PAT) that facilitate the transition from batch to continuous production is essential. Herein, the key milestones and insights derived from the development of currently approved lipid- nanosystems will be explored. Additionally, a comprehensive and critical overview of the latest technologies and regulatory guidelines that underpin the creation of more efficient, scalable, and flexible manufacturing processes for complex lipid-based nanoformulations will be provided. [Display omitted] • Lipid-based complex injectables reliably deliver drugs with many approved products. • Lipid nanoparticles significantly enhance nucleic acid delivery for gene therapy. • Multi-batch processes challenge scalability, stability, and compliance. • Quality-by-Design ensures consistent product quality in complex manufacturing. • Process Analytical Tools enhance efficiency and flexibility in manufacturing. [ABSTRACT FROM AUTHOR]

Basing on logical assumptions and necessary steps of complexification along biological evolution, we propose here an evolutionary path from molecules to cells presenting four ages and three major transitions. At the first age, the basic biomolecules were formed and become abundant. The first transition happened with the event of a chemical symbiosis between nucleic acids and peptides worlds, which marked the emergence of both life and the process of organic encoding. FUCA, the first living process, was composed of self-replicating RNAs linked to amino acids and capable to catalyze their binding. The second transition, from the age of FUCA to the age of progenotes, involved the duplication and recombination of proto-genomes, leading to specialization in protein production and the exploration of protein to metabolite interactions in the prebiotic soup. Enzymes and metabolic pathways were incorporated into biology from protobiotic reactions that occurred without chemical catalysts, step by step. Then, the fourth age brought origin of organisms and lineages, occurring when specific proteins capable to stackle together facilitated the formation of peptidic capsids. LUCA was constituted as a progenote capable to operate the basic metabolic functions of a cell, but still unable to interact with lipid molecules. We present evidence that the evolution of lipid interaction pathways occurred at least twice, with the development of bacterial-like and archaeal-like membranes. Also, data in literature suggest at least two paths for the emergence of DNA biosynthesis, allowing the stabilization of early life strategies in viruses, archaeas and bacterias. Two billion years later, the eukaryotes arouse, and after 1,5 billion years of evolution, they finally learn how to evolve multicellularity via tissue specialization. [ABSTRACT FROM AUTHOR]

Targeted novel treatment approaches for GBM using 4-amino cyanine and miRNA inhibitors. [Display omitted] • Developing novel therapeutic strategies is a promising approach for GBM. • ncRNAs play important roles in GBM pathogenesis, proliferation, and metastasis. • This review highlights the roles of miRNAs in GBM pathogenesis. • GBM treatment by combining 4-amino cyanine, and miRNAs-related Inhibitors. • Highlights the key challenges in miRNA therapy that need to be overcome. Glioblastoma (GBM) poses a formidable challenge in oncology due to its aggressive nature and dismal prognosis, with average survival rates around 15 months despite conventional treatments. This review proposes a novel therapeutic strategy for GBM by integrating microRNA (miRNA) therapy with 4-amino cyanine molecules possessing near-infrared (NIR) properties. miRNA holds promise in regulating gene expression, particularly in GBM, making it an attractive therapeutic target. 4-amino cyanine molecules, especially those with NIR properties, have shown efficacy in targeted tumor cell degradation. The combined approach addresses gene expression regulation and precise tumor cell degradation, offering a breakthrough in GBM treatment. Additionally, the review explores noncoding RNAs classification and characteristics, highlighting their role in GBM pathogenesis. Advanced technologies such as antisense oligonucleotides (ASOs), locked nucleic acids (LNAs), and peptide nucleic acids (PNAs) show potential in targeting noncoding RNAs therapeutically, paving the way for precision medicine in GBM. This synergistic combination presents an innovative approach with the potential to advance cancer therapy in the challenging landscape of GBM. [ABSTRACT FROM AUTHOR]

Small interfering RNAs (siRNAs) have the ability to induce selective gene silencing, although siRNAs are vulnerable to degradation in vivo. Various active pharmaceutical ingredients (APIs) are currently used as effective therapeutics in the treatment of cancer. However, routes of administration are limited due to their physicochemical and biopharmaceutical properties. This research aimed to develop oral pharmaceutical formulations based on self-nanoemulsifying drug delivery systems (SNEDDS) for optimal transport and co-delivery of siRNAs related to cancer and APIs. Formulations were developed using optimal mixing design (Design-Expert 11 software) for SNEDDS loading with siRNA (water/oil emulsion), API (oil/water emulsion), and siRNA-API (multiphase water/oil/water emulsion). The final formulations were characterized physicochemically and biologically. The nanosystems less than 50 nm in size had a drug loading above 48 %. The highest drug release occurred at intestinal pH, allowing drug protection in physiological fluids. SNEDDS-siRNA-APIs showed a twofold toxicity effect than APIs in solution and higher transfection and internalization of siRNA in cancer cells with respect to free siRNAs. In the duodenum, higher permeability was observed with SNEDDS-API than with the API solution, as determined by ex-vivo fluorescence microscopy. The multifunctional formulation based on SNEDDS was successfully prepared, siRNA, hydrophobic chemotherapeutics (doxorubicin, valrubicin and methotrexate) and photosensitizers (rhodamine b and protoporphyrin IX) agents were loaded, using a chitosan-RNA core, and Labrafil® M 1944 CS, Cremophor® RH40, phosphatidylcholine shell, forming stable hybrid SNEDDS as multiphasic emulsion, suitable as co-delivery system with a potent anticancer activity. [ABSTRACT FROM AUTHOR]

Understanding the diversity of DNA structure and functions in biology requires tools to study this biomolecule selectively and thoroughly. Fluorescence methods are powerful technique for non-invasive research. Due to the low quantum yield, the intrinsic fluorescence of nucleotides has not been considered for use in the detection and differentiation of nucleic acid bases. Here, we have studied the influence of protonation of nucleotides on their fluorescence properties. We show that protonation of ATP and GTP leads to enhanced intrinsic fluorescence. Fluorescence enhancement at acidic pH has been observed for double-stranded DNA and single-stranded oligonucleotides. The formation of G4 secondary structures apparently protected certain nucleotides from protonation, resulting in less pronounced fluorescence enhancement. Furthermore, acid-induced depurination under protonation was less noticeable in G4 structures than in double-stranded and single-stranded DNA. We show that changes in the intrinsic fluorescence of guanine can be used as a sensitive sensor for changes in the structure of the DNA and for the protonation of specific nucleotides. • Protonation of purine nucleoside triphosphates leads to enhanced intrinsic fluorescence. • The intrinsic fluorescence of protonated purines depends on the DNA structure. • Formation of G4 structures protects certain guanines from protonation. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are small highly conserved non-coding RNA molecules that orchestrate a wide range of biological processes through post-transcriptional regulation of gene expression. During development, miRNAs play a key role in driving embryo patterning and morphogenesis in a specific and stage-dependent manner. Here, we investigated whether sperm from bulls with different fertilizing ability in vitro influence blastocyst quality and miRNA content. Results demonstrate that blastocysts obtained using sperm from high fertility sires (H group) display significantly greater cleavage and blastocyst development as well as greater transcript abundance in blastocysts for the developmental competence markers CDX2 , KRT8 , NANOG , OCT4 , PLAC8 , PTGS2 , SOX17 , and SOX2 , compared to blastocysts generated using sperm from low fertility sires (L group). In parallel, high throughput deep sequencing and differential expression studies revealed that H blastocysts exhibit a greater miRNA content compared to L blastocysts, with hsa-miR-4755–5p and hsa-miR-548d-3p uniquely detected in the H group, and greater abundance of hsa-miR-1225–3p in the H group. Gene ontology (GO) and KEGG pathway analyses indicated that the 3 differentially expressed miRNAs identified are involved in the regulation of many biological mechanisms with a key role in aspects of early embryo development, including transcriptional regulation, cellular biosynthesis, nucleic acid metabolism, cellular differentiation, apoptosis, cytoskeleton remodeling, cell-to-cell interactions, and endocytosis. Overall, our results indicate that sperm fertilizing ability influences blastocyst developmental ability and miRNA content. In addition, we demonstrate an association between blastocyst quality and miRNA content, thus suggesting the possibility to score miRNA expression as biomarkers for improved routine embryo selection technologies to support assisted reproductive efforts. • Sperm fertilizing ability influences blastocyst developmental ability and miRNA content. • Paternal influence extends its effects through early embryogenesis and may impact on the further development. • Blastocysts obtained from H group exhibit a greater miRNA content compared to blastocysts obtained from Lgroup. • Differentially expressed miRNAs are involved in the regulation of many biological mechanisms with a key role in aspects of early embryo development. [ABSTRACT FROM AUTHOR]

Assays that utilize nucleic acid amplification are prone to contamination from a variety of sources, including both target organisms and amplicon. Implementing effective systems for contamination monitoring as well as laboratory best practices are essential to any quality management system for molecular microbiology testing. A comprehensive contamination monitoring toolbox will be introduced here that includes guidance for environment swabbing, positivity rate monitoring, use of process controls, and monitoring complaints. As prevention of contamination is preferable, best practices for laboratory design, workflow, and personnel training will also be discussed. Lastly, a suggested plan for managing a contamination event will be proposed. The laboratory director should carefully assess the specific risks associated with testing performed in the laboratory and create a written plan for contamination monitoring, prevention, and management of contamination events. [ABSTRACT FROM AUTHOR]

The full potential of ionizable lipid nanoparticles (LNPs) as an in vivo nucleic acid delivery platform has not yet been realized given that LNPs primarily accumulate in the liver following systemic administration, limiting their success to liver-centric conditions. The engineering of LNPs with antibody targeting moieties can enable extrahepatic tropism by facilitating site-specific LNP tethering and driving preferential LNP uptake into receptor-expressing cell types via receptor-mediated endocytosis. Obstetric conditions stemming from placental dysfunction, such as preeclampsia, are characterized by overexpression of cellular receptors, including the epidermal growth factor receptor (EGFR), making targeted LNP platforms an exciting potential treatment strategy for placental dysfunction during pregnancy. Herein, an EGFR antibody-conjugated LNP (aEGFR-LNP) platform was developed by engineering LNPs with increasing densities of antibody functionalization. aEGFR-LNPs were screened in vitro in immortalized placental trophoblasts and in vivo in non-pregnant and pregnant mice and compared to non-targeted formulations for extrahepatic, antibody-targeted mRNA LNP delivery to the placenta. Our top performing LNP with an intermediate density of antibody functionalization (1:5 aEGFR-LNP) mediated a ∼twofold increase in mRNA delivery in murine placentas and a ∼twofold increase in LNP uptake in EGFR-expressing trophoblasts compared to non-targeted counterparts. These results demonstrate the potential of antibody-conjugated LNPs for achieving extrahepatic tropism, and the ability of aEGFR-LNPs in promoting mRNA delivery to EGFR-expressing cell types in the placenta. [Display omitted] • Strain-promoted azide-DBCO cycloaddition produces stable antibody-conjugated LNPs • Pregnancy alters EGFR antibody-conjugated LNP biodistribution in vivo • Intermediate EGFR antibody density enhances mRNA LNP delivery to the placenta • EGFR antibody-conjugated LNPs enhance uptake in EGFR-expressing trophoblasts [ABSTRACT FROM AUTHOR]

Inherited genetic disorders of the liver pose a significant public health burden. Liver transplantation is often limited by the availability of donor livers and the exorbitant costs of immunosuppressive therapy. To overcome these limitations, nucleic acid therapy provides a hopeful alternative that enables gene repair, gene supplementation, and gene silencing with suitable vectors. Though viral vectors are the most efficient and preferred for gene therapy, pre-existing immunity debilitating immune responses limit their use. As a potential alternative, lipid nanoparticle-mediated vectors are being explored to deliver multiple nucleic acid forms, including pDNA, mRNA, siRNA, and proteins. Herein, we discuss the broader applications of lipid nanoparticles, from protein replacement therapy to restoring the disease mechanism through nucleic acid delivery and gene editing, as well as multiple preclinical and clinical studies as a potential alternative to liver transplantation. Lipid nanoparticles serve as delivery vehicles to transport therapeutic genes or gene-editing tools into target cells, which allows for correction of genetic mutations, introduction of therapeutic genes, or modulation of gene expression. [Display omitted] [ABSTRACT FROM AUTHOR]

Mitochondrial double-stranded RNA (dsRNA) can form spontaneously in mitochondria, blocking mitochondrial gene expression and triggering an immune response. A recent study by Kim, Tan, et al. identified a safeguard mechanism in which NOP2/Sun RNA methyltransferase 4 (NSUN4)-mediated RNA methylation (m5C) recruits the RNA degradation machinery to prevent dsRNA formation. [ABSTRACT FROM AUTHOR]

Due to the rapid increase in the world's population, many developing countries are facing malnutrition problems, including famine and food insecurity. Particularly, the deficiency of protein sources becomes a serious problem for human and animal nutrition. In this context, Single Cell Proteins, could be exploited as an alternative source of unconventional proteins. The aim of the study was to investigate SCP production and composition by Cupriavidus necator under various environmental conditions, temperature and pH values. A mono-factorial approach was implemented using batch bioreactor cultures under well-controlled conditions. Results were compared in terms of bacterial growth and SCP composition (proteins, nucleic acids, amino acids and elemental formula). Complementary analyses were performed by flow cytometry to study cell morphology, membrane permeability and the presence of Poly(3-hydroxybutyrate) (PHB) production. Our data confirmed the ability of C. necator to produce high amount of proteins (69 % DW at 30 °C and pH7). The results showed that temperature and pH independently impact SCP production and composition. This impact was particularly observed at the highest temperature (40 °C) and also the lowest pH value (pH5) providing lower growth rates, cell elongation, changes in granularity and lower amounts of proteins (down to 44 % DW at pH5) and nucleic acids. These low percentages were related to the production of PHB production (up to 44 % DW at 40 °C) which is the first report of a PHB accumulation in C. necator under nutrient unlimited conditions. • Higher amount of Proteins (69 % DW) obtained at 30°C and pH7 • PHB accumulation under nutrient unlimited conditions at 40°C or pH5 • Cells morphology change at 40°C or pH5 [ABSTRACT FROM AUTHOR]

Successful development of nucleic acid therapeutics requires effective manufacturing strategies tailored to the production and purification of RNA and DNA. Membrane processes can be used for initial clarification, removal of smaller host cell proteins and transcription factors, concentration and buffer exchange, and sterile filtration. Transmission of DNA and RNA in ultrafiltration is governed by both the effective size and elongational flexibility of the nucleic acids. Sterile filtration of DNA/RNA therapeutics can present unique challenges due to their large size and shear sensitivity. Nucleic acid therapeutics have the potential to revolutionize the biopharmaceutical industry, providing highly effective vaccines and novel treatments for cancers and genetic disorders. The successful commercialization of these therapeutics will require development of manufacturing strategies specifically tailored to the purification of nucleic acids. Membrane technologies already play a critical role in the downstream processing of nucleic acid therapeutics, ranging from clarification to concentration to selective purification. This review provides an overview of how membrane systems are currently used for nucleic acid purification, while highlighting areas of future need and opportunity, including adoption of membranes in continuous bioprocessing. [ABSTRACT FROM AUTHOR]

Electrotransfer is an effective non-viral strategy to deliver exogenous cargo such as nucleic acids and proteins into living cells in ex vivo and in vivo scenarios. Next-generation electrotransfer strategies aim at enhancing efficiency through localized electroporation and hybrid methods involving microfluidics, mechanoporation, and sonoporation. In addition, there is a focus on creating affordable, single-use electroporation devices that will potentially expand the global reach of DNA vaccination. Continuous advances in electrotransfer, and the integration of these next-generation electroporation techniques into in utero and in vivo applications, hold the promise of significantly improving gene-editing efficiencies in these scenarios. Promising results from clinical trials utilizing DNA electrotransfer highlight its favorable safety profile and indicate encouraging prospects for its broader application. Electrotransfer of nucleic acids and proteins has become crucial in biotechnology for gene augmentation and genome editing. This review explores the applications of electrotransfer in both ex vivo and in vivo scenarios, emphasizing biomedical uses. We provide insights into completed clinical trials and successful instances of nucleic acid and protein electrotransfer into therapeutically relevant cells such as immune cells and stem and progenitor cells. In addition, we delve into emerging areas of electrotransfer where nanotechnology and deep learning techniques overcome the limitations of traditional electroporation. [ABSTRACT FROM AUTHOR]

[Display omitted] Chemo-immunotherapy, in which chemotherapeutic drugs activate immune system to suppress tumor growth and metastases, has great potential for clinical application. However, insufficient immunogenic cell death and serious side effects caused by tumor multidrug resistance and non-specific drug distribution, as well as inadequate and dysfunctional immune cells, greatly impair the effectiveness of chemo-immunotherapy. Herein, taking advantage of the functional diversity and structural programmability of nucleic acids, a DNA-based bioorthogonal nanoagonist is constructed to initiate and augment immune responses for robust chemo-immunotherapy. Benefiting from polyvalent targeting and template effects of DNA, the tailor-made nanoagonist shows prior bioorthogonal catalytic performance. Chemotherapeutic drug is bioorthogonally synthesized in situ under the catalysis of the nanoagonist, maximizing immunogenic cell death and minimizing systemic toxicity. The large amount of antigen and damage-associated molecular patterns released from dying tumor cells effectively initiates antitumor immunity. Meanwhile, the integration of high density of immunologic adjuvant can more effectively stimulate immune cells. The combination of bioorthogonal catalytic drug synthesis and immunostimulatory effect of DNA adjuvant not only destroys local primary tumors, but also eliminates distal metastasis. Moreover, the nanoagonist triggered the immune memory effect. The work extends the application of bioorthogonal chemistry to immunotherapy and provides a safe and powerful strategy for cancer chemo-immunotherapy. [ABSTRACT FROM AUTHOR]

Metabolism is a key regulator of immune cell function. Tricarboxylic acid cycle rewiring and metabolites have immunoregulatory roles. Fumarate hydratase (FH) and fumarate are now known to regulate cytokine production. Recent findings suggest FH loss leads to mitochondrial nucleic acid release, leading to type I interferon production. Tricarboxylic acid (TCA) cycle metabolites have been implicated in modulating signalling pathways in immune cells. Notable examples include succinate and itaconate, which have pro- and anti-inflammatory roles, respectively. Recently, fumarate has emerged as having specific roles in macrophage activation, regulating the production of such cytokines as interleukin (IL)-10 and type I interferons (IFNs). Fumarate hydratase (FH) has been identified as a control point. Notably, FH loss in different models and cell types has been found to lead to DNA and RNA release from mitochondria which are sensed by cytosolic nucleic acid sensors including retinoic acid-inducible gene (RIG)-I, melanoma differentiation-associated protein (MDA)5, and cyclic GMP-AMP synthase (cGAS) to upregulate IFN-β production. These findings may have relevance in the pathogenesis and treatment of diseases associated with decreased FH levels such as systemic lupus erythematosus (SLE) or FH-deficient kidney cancer. Tricarboxylic acid (TCA) cycle metabolites have been implicated in modulating signalling pathways in immune cells. Notable examples include succinate and itaconate, which have pro- and anti-inflammatory roles respectively. Recently, fumarate has emerged as having specific roles in macrophage activation, regulating the production of such cytokines as interleukin (IL)-10 and type I interferons (IFNs). Fumarate hydratase (FH) has been identified as a control point. Notably, FH loss in different models and cell types has been found to lead to DNA and RNA release from mitochondria which are sensed by cytosolic nucleic acid sensors including retinoic acid-inducible gene (RIG)-I, melanoma differentiation-associated protein (MDA)5, and cyclic GMP-AMP synthase (cGAS) to upregulate IFN-β production. These findings may have relevance in the pathogenesis and treatment of diseases associated with decreased FH levels such as systemic lupus erythematosus or FH-deficient kidney cancer. [ABSTRACT FROM AUTHOR]

The advent of lipid nanoparticles (LNPs) containing ionizable cationic lipids has enabled the encapsulation, stabilization, and intracellular delivery of nucleic acid payloads, leading to FDA-approved siRNA-based therapy and mRNA-based vaccines. Other nucleic acid-based therapeutic modalities, including protein replacement and CRISPR-mediated gene knockout and editing, are being tested in clinical trials, in many cases, for the treatment of liver-related diseases. However, to fully exploit these therapies beyond the liver, improvements in their delivery to extrahepatic targets are needed. Towards this end, both active targeting strategies based on targeting ligands grafted onto LNPs and passive targeting relying on physicochemical LNP parameters such as surface composition, charge, and size are being evaluated. Often, the latter strategy depends on the interaction of LNPs with blood components, forming what is known as the biomolecular corona. Here, I discuss potential challenges related to current LNP-based targeting strategies and the studies of the biomolecular corona on LNPs. I propose potential solutions to overcome some of these obstacles and present approaches currently being tested in preclinical and clinical studies, which face fewer biological barriers than traditional organ-targeting approaches. [Display omitted] • Presents an overview of LNP-based strategies for extrahepatic RNA delivery. • Highlights potential challenges with the LNP strategies and approaches to overcome some of them. • Stresses inherent challenges associated with biomolecular corona studies of LNPs. • Highlights that LNPs are a minor subpopulation of nanoparticles in human plasma. • Advocates innovative approaches to drive progress in the LNP field. [ABSTRACT FROM AUTHOR]

The combination of chemotherapy and gene therapy holds great promise for the treatment and eradication of tumors. However, due to significant differences in physicochemical properties between chemotherapeutic agents and functional nucleic acid drugs, direct integration into a single nano-agent is hindered, impeding the design and construction of an effective co-delivery nano-platform for synergistic anti-tumor treatments. In this study, we have developed an mRNA-responsive two-in-one nano-drug for effective anti-tumor therapy by the direct self-assembly of 2′-fluoro-substituted antisense DNA against P-glycoprotein (2′F-DNA) and chemo drug paclitaxel (PTX). The 2′-fluoro modification of DNA could significantly increase the interaction between the therapeutic nucleic acid and the chemotherapeutic drug, promoting the successful formation of 2′F-DNA/PTX nanospheres (2′F-DNA/PTX NSs). Due to the one-step self-assembly process without additional carrier materials, the prepared 2′F-DNA/PTX NSs exhibited considerable loading efficiency and bioavailability of PTX. In the presence of endogenous P-glycoprotein mRNA, the 2′F-DNA/PTX NSs were disassembled. The released 2′F-DNA could down-regulate the expression of P-glycoprotein, which decreased the multidrug resistance of tumor cells and enhanced the chemotherapy effect caused by PTX. In this way, the 2′F-DNA/PTX NSs could synergistically induce the apoptosis of tumor cells and realize the combined anti-tumor therapy. This strategy might provide a new tool to explore functional intracellular co-delivery nano-systems with high bioavailability and exhibit potential promising in the applications of accurate diagnosis and treatment of tumors. [Display omitted] [ABSTRACT FROM AUTHOR]

Immunotherapy is currently a standard of care in the treatment of many malignancies. However, predictable side effects caused by systemic administration of highly immunostimulatory molecules have been a serious concern within this field. Intratumoural expression or silencing of immunogenic and immunoinhibitory molecules using nucleic acid-based approaches such as plasmid DNA (pDNA) and small interfering RNA (siRNA), respectively, could represent a next generation of cancer immunotherapy. Here, we employed lipid nanoparticles (LNPs) to deliver either non-specific pDNA and siRNA, or constructs targeting two prominent immunotherapeutic targets OX40L and indoleamine 2,3-dioxygenase-1 (IDO), to tumours in vivo. In the B16F10 mouse model, intratumoural delivery of LNP-formulated non-specific pDNA and siRNA led to strong local immune activation and tumour growth inhibition even at low doses due to the pDNA immunogenic nature. Replacement of these non-specific constructs by pOX40L and siIDO resulted in more prominent immune activation as evidenced by increased immune cell infiltration in tumours and tumour-draining lymph nodes. Consistently, pOX40L alone or in combination with siIDO could prolong overall survival, resulting in complete tumour regression and the formation of immunological memory in tumour rechallenge models. Our results suggest that intratumoural administration of LNP-formulated pDNA and siRNA offers a promising approach for cancer immunotherapy. [Display omitted] • I.t injection of non-specific LNPs led to strong local immune activation and tumour growth inhibition even at low doses. • pOX40L + siIDO LNPs induced more prominent immune activation than non-specific pDNA+siRNA LNPs. • pOX40L or pOX40L + siIDO LNPs prolonged survival, led to complete tumour regression and immunological memory generation. • I.t injection of LNP-formulated pDNA and siRNA offers a promising approach for cancer immunotherapy. [ABSTRACT FROM AUTHOR]

Mitochondria are membrane-bound organelles of endosymbiotic origin with limited protein-coding capacity. The import of nuclear-encoded proteins and nucleic acids is required and essential for maintaining organelle mass, number, and activity. As plant mitochondria do not encode all the necessary tRNA types required, the import of cytosolic tRNA is vital for organelle maintenance. Recently, two mitochondrial outer membrane proteins, named Tric1 and Tric2, for tRNA import component, were shown to be involved in the import of cytosolic tRNA. Tric1/2 binds tRNAala via conserved residues in the C-terminal Sterile Alpha Motif (SAM) domain. Here we report the X-ray crystal structure of the Tric1 SAM domain. We identified the ability of the SAM domain to form a helical superstructure with six monomers per helical turn and key amino acid residues responsible for its formation. We determined that the oligomerization of the Tric1 SAM domain may play a role in protein function whereby mutation of Gly241 introducing a larger side chain at this position disrupted the oligomer and resulted in the loss of RNA binding capability. Furthermore, complementation of Arabidopsis thaliana Tric1/ 2 knockout lines with a mutated Tric1 failed to restore the defective plant phenotype. AlphaFold2 structure prediction of both the SAM domain and Tric1 support a cyclic pentameric or hexameric structure. In the case of a hexameric structure, a pore of sufficient dimensions to transfer tRNA across the mitochondrial membrane is observed. Our results highlight the importance of oligomerization of Tric1 for protein function. [ABSTRACT FROM AUTHOR]

Adoptive cell therapy using chimeric antigen receptor (CAR) T cells is effective against B cell malignancies; however, the complex manufacturing process and financial realities constrain the scalability of the approach. The in vivo generation of CAR T cells, and possibly other immune cells, using off-the-shelf products therefore has numerous logistical and functional advantages. In preclinical models, in vivo gene delivery using nanoparticles or viral vectors has yielded CAR T cells with therapeutic equivalency to ex vivo generated CAR T cells. Ongoing research efforts are attempting to determine how to best target and leverage effector cells of interest (T cells, macrophages etc.), understand how direct in vivo CAR generation interfaces with other immune cells, and optimize design elements of the viral vectors or nanoparticle and nucleic acid formulations. T cells modified to express intelligently designed chimeric antigen receptors (CARs) are exceptionally powerful therapeutic agents for relapsed and refractory blood cancers and have the potential to revolutionize therapy for many other diseases. To circumvent the complexity and cost associated with broad-scale implementation of ex vivo manufactured adoptive cell therapy products, alternative strategies to generate CAR T cells in vivo by direct infusion of nanoparticle-formulated nucleic acids or engineered viral vectors under development have received a great deal of attention in the past few years. Here, we outline the ex vivo manufacturing process as a motivating framework for direct in vivo strategies and discuss emerging data from preclinical models to highlight the potency of the in vivo approach, the applicability for new disease indications, and the remaining challenges associated with clinical readiness, including delivery specificity, long term efficacy, and safety. [ABSTRACT FROM AUTHOR]

Facile construction of a fully biodegradable spherical nucleic acid (SNA) nanoplatform is highly desirable for clinical translations but remains rarely explored. We developed herein the first polycarbonate-based biodegradable SNA nanoplatform for self-codelivery of a chemotherapeutic drug, doxorubicin (DOX), and a human liver-specific miR122 for synergistic chemo-gene therapy of hepatocellular carcinoma (HCC). Ring-opening polymerization (ROP) of a carbonate monomer leads to a well-defined polycarbonate backbone for subsequent DOX conjugation to the pendant side chains via acidic pH-cleavage Schiff base links and miR122 incorporation to the chain termini via click coupling, affording an amphiphilic polycarbonate-DOX-miR122 conjugate, PBis-Mpa 30 -DOX-miR122 that can self-assemble into stabilized SNA. Besides the desired biodegradability, another notable merit of this nanoplatform is the use of miR122 not only for gene therapy but also for enhanced innate immune response. Together with the ICD-triggering effect of DOX, PBis-Mpa 30 -DOX-miR122 SNA-mediated DOX and miR122 codelivery leads to synergistic immunogenicity enhancement, resulting in tumor growth inhibition value (TGI) of 98.1 % significantly higher than those of the groups treated with only drug or gene in a Hepa1-6-tumor-bearing mice model. Overall, this study develops a useful strategy toward biodegradable SNA construction, and presents a drug and gene-based self-codelivery SNA with synergistic immunogenicity enhancement for efficient HCC therapy. Facile construction of a fully biodegradable SNA nanoplatform is useful for in vivo applications but remains relatively unexplored likely due to the synthetic challenge. We report herein construction of a polycarbonate-based SNA nanoplatform for co-delivering a chemotherapeutic drug, DOX, and a human liver-specific miR-122 for synergistic HCC treatment. In addition to the desired biodegradability properties, this SNA nanoplatform integrates DOX-triggered ICD and miR-122-enhanced innate immunity for simultaneously activating adaptive and innate immunities, which leads to potent antitumor efficiency with a TGI value of 98.1 % in a Hepa1-6-tumor-bearing mice model. [Display omitted] [ABSTRACT FROM AUTHOR]

• AS LNA qPCR can accurately identify VP1-E 295 K containing samples in sPV2 batches. • The developed AS LNA qPCR for E 295 K mutation is highly sensitive. • LNA probes used are highly specific in targeting known mutations. • AS LNA qPCR is likely applicable to other vaccine strains, serotypes, and mutations. Sabin Inactivated Poliovirus Vaccine (sIPV) has become one of the preferred vaccination options for the last step in the Poliovirus eradication program. Sequencing of poliovirus samples is needed during the manufacturing of poliovirus vaccines to assure the safety and immunogenicity of these vaccines. Next-generation sequencing analysis is the current costly and time-consuming gold standard for monitoring the manufacturing processes. We developed a low-cost and quick, highly sensitive, and allele-specific locked nucleic acid-probe-based reverse transcription quantitative PCR alternative that can accurately detect mutations in poliovirus vaccine samples during process development, scaling up, and release. Using the frequently in vitro occurring and viral replication-impacting VP1-E 295 K mutation as a showcase, we show that this technology can accurately detect E 295 K mutations in poliovirus 2 samples to similar levels as NGS. The qPCR technology was developed employing a synthetic dsDNA fragment-based standard curve containing mixes of E 295 K-WT (wildtype) and Mut (mutant) synthetic dsDNA fragments ranging from 1 × 107 copies/µL to 1 × 102 copies/µL to achieve a linear correlation with R2 > 0.999, and PCR efficiencies of 95–105 %. Individual standard concentration levels achieved accuracies of ≥92 % (average 96 %) and precisions of ≤17 % (average 3.3 %) RSD. Specificity of locked nucleic acid (LNA)-probes was confirmed in the presence and absence of co-mutations in the probe-binding region. Application of the developed assay to Sabin Poliovirus type 2 production run samples, illustrated a linear relationship with an R2 of 0.994, and an average accuracy of 97.2 % of the variant (allele)-specific AS LNA qPCR result, compared to NGS. The assay showed good sensitivity for poliovirus samples, containing E 295 K mutation levels between 0 % and 95 % (quantification range). In conclusion, the developed AS LNA qPCR presents a valuable low-cost, and fast tool, suitable for the process development and quality control of polio vaccines. [ABSTRACT FROM AUTHOR]

Nanocarriers protect RNAs from enzymatic degradation after application onto plants for sustained protection against viruses and insects. Nanocarriers enabled controlled delivery of nucleic acids to plant cells, including slow and sustained release, subcellular organ targeting delivery, stimulated release, and endosome escape. The nanoparticle-mediated delivery system has high versatility, in terms of applicability to all plant species and the capacity to deliver alternative forms of nucleic acids and proteins. Plant biotechnology plays a crucial role in developing modern agriculture and plant science research. However, the delivery of exogenous genetic material into plants has been a long-standing obstacle. Nanoparticle-based delivery systems are being established to address this limitation and are proving to be a feasible, versatile, and efficient approach to facilitate the internalization of functional RNA and DNA by plants. The nanoparticle-based delivery systems can also be designed for subcellular delivery and controlled release of the biomolecular cargo. In this review, we provide a concise overview of the recent advances in nanocarriers for the delivery of biomolecules into plants, with a specific focus on applications to enhance RNA interference, foreign gene transfer, and genome editing in plants. [ABSTRACT FROM AUTHOR]

In this study, a homogeneous polysaccharide, named, DRMP-1 was isolated from the mycelium of Dictyophora rubrovolvata by water extraction and alcohol precipitation, and its structure and activity were studied. The molecular weight of DRMP-1 is 2.33 × 106 Da, and it does not contain protein and nucleic acid. DRMP-1 is composed of mannose, glucose and galactose with a molar ratio of 0.04: 1: 0.04. Through methylation and NMR analysis, DRMP-1 is mainly composed of 1 → 6)-α-Gal p -(1 →, T-α-D-Glc p , →1)-α-D-Man p , → 6)-β-D-Glc p -(1 →, → 4, 6)- α-D-Man p -(1 →, → 4)- α-D-Glc p -(1 →, → 3)-β-D-Glc p -(1 → and → 4, 6)- α-D-Glc p -(1 →. In addition, the immunomodulative activity of DRMP-1 was evaluated by establishing the RAW264.7 cell model. The results showed that DRMP-1 could significantly increase phagocytosis activity and NO production more than twice as much as that of the blank group, and could increase intracellular ACP, LSZ and SOD activities. Therefore, it is concluded that the polysaccharide of Dictyophora rubrovolvata mycelium may be a potential immunomodulator. [Display omitted] • A new polysaccharide DRMP-1 was extracted from Dictyophora rubrodictya mycelia. • The chemical structure of DRMP-1 was characterized. • DRMP-1 has immune activity and can be used as a natural immunomodulator. [ABSTRACT FROM AUTHOR]

Short tandem repeats are inherently unstable during DNA replication depending on repeat length, and the expansion of the repeat length in the human genome is responsible for repeat expansion disorders. Pentanucleotide AAGGG and ACAGG repeat expansions in intron 2 of the gene encoding replication factor C subunit 1 (RFC1) cause cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS) and other phenotypes of late-onset cerebellar ataxia. Herein, we reveal the structural polymorphism of the RFC1 repeats associated with CANVAS in vitro. Single-stranded AAGGG repeat DNA formed a hybrid-type G-quadruplex, whereas its RNA formed a parallel-type G-quadruplex with three layers. The RNA of the ACAGG repeat formed hairpin structure comprising C-G and G-C base pairs with A:A and GA:AG mismatched repeats. Furthermore, both pathogenic repeat RNAs formed more rigid structures than those of the nonpathogenic repeat RNAs. These findings provide novel insights into the structural polymorphism of the RFC1 repeats, which may be closely related to the disease mechanism of CANVAS. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) have unique features for drug delivery and offer a potential solution to the stability issues and on-target off-tissue adverse effects that are associated with biologic drugs. Cutting-edge research findings have demonstrated the effectiveness of the endogenous method for loading EVs with large biological drugs. Advances in understanding EV biogenesis have enabled researchers to exploit molecules involved in cargo selection and loading pathways to enhance the efficiency of biologic drug loading inside EVs. Numerous research articles employing the endogenous loading method for EVs have obtained promising results in preclinical in vivo experiments, which demonstrates the applicability of EVs across a spectrum of therapeutic contexts. The use of extracellular vesicles (EVs) for drug delivery is being widely explored by scientists from several research fields. To fully exploit their therapeutic potential, multiple methods for loading EVs have been developed. Although exogenous methods have been extensively utilized, in recent years the endogenous method has gained significant attention. This approach, based on parental cell genetic engineering, is suitable for loading large therapeutic biomolecules such as proteins and nucleic acids. We review the most commonly used EV loading methods and emphasize the inherent advantages of the endogenous method over the others. We also examine the most recent advances and applications of this innovative approach to inform on the diverse therapeutic opportunities that lie ahead in the field of EV-based therapies. [ABSTRACT FROM AUTHOR]

Nucleosides and nucleic acids are abundant in our diet, but their nutritional value has been poorly investigated. Nucleosides maintain the viability of cells and organs when glucose is limited. Specific nucleoside phosphorylases, including uridine phosphorylase 1/2 (UPP1/2), thymidine phosphorylase (TYMP), and purine nucleoside phosphorylase (PNP), catalyze nucleoside cleavage and release of the ribose moiety, which can enter the glycolytic pathway, thus fueling central carbon metabolism. UPP1/2 integrate genetic and environmental signals to regulate uridine catabolism. Energy production from nucleosides has major implications for metabolism, immunity, and cancer. From our daily nutrition and synthesis within cells, nucleosides enter the bloodstream and circulate throughout the body and tissues. Nucleosides and nucleotides are classically viewed as precursors of nucleic acids, but recently they have emerged as a novel energy source for central carbon metabolism. Through catabolism by nucleoside phosphorylases, the ribose sugar group is released and can provide substrates for lower steps in glycolysis. In environments with limited glucose, such as at sites of infection or in the tumor microenvironment (TME), cells can use, and may even require, this alternative energy source. Here, we discuss the implications of these new findings in health and disease and speculate on the potential new roles of nucleosides and nucleic acids in energy metabolism. [ABSTRACT FROM AUTHOR]

Significant advances have been made in our understanding of osteoarthritis (OA) pathogenesis; however, no disease-modifying therapies have been identified. This review will summarize the gene therapy landscape, its initial successes for OA, and possible challenges using recent studies and examples of gene therapies in clinical trials. This narrative review has three major sections: 1) vector systems for OA gene therapy, 2) current and emerging targets for OA gene therapy, and 3) considerations and future directions. Gene therapy is the strategy by which nucleic acids are delivered to treat and reverse disease progression. Specificity and prolonged expression of these nucleic acids are achieved by manipulating promoters, genes, and vector systems. Certain vector systems also allow for the development of combinatorial nucleic acid strategies that can be delivered in a single intraarticular injection – an approach likely required to treat the complexity of OA pathogenesis. Several viral and non-viral vector-based gene therapies are in clinical trials for OA, and many more are being evaluated in the preclinical arena. In a post-coronavirus disease 2019 (COVID-19) era, the future of gene therapy for OA is certainly promising; however, the majority of preclinical validation continues to focus heavily on post-traumatic models and changes in only cartilage and subchondral bone. To ensure successful translation, new candidates in the preclinical arena should be examined against all joint tissues as well as pain using diverse models of injury-, obesity-, and age-induced disease. Lastly, consideration must be given to strategies for repeat administration and the cost of treatment owing to the chronic nature of OA. [ABSTRACT FROM AUTHOR]

Microbial pathogens cause persistent infections by forming biofilms and producing numerous virulence factors. Bacterial extracellular vesicles (BEVs) are nanostructures produced by various bacterial species vital for molecular transport. BEVs include various components, including lipids (glycolipids, LPS, and phospholipids), nucleic acids (genomic DNA, plasmids, and short RNA), proteins (membrane proteins, enzymes, and toxins), and quorum-sensing signaling molecules. BEVs play a major role in forming extracellular polymeric substances (EPS) in biofilms by transporting EPS components such as extracellular polysaccharides, proteins, and extracellular DNA. BEVs have been observed to carry various secretory virulence factors. Thus, BEVs play critical roles in cell-to-cell communication, biofilm formation, virulence, disease progression, and resistance to antimicrobial treatment. In contrast, BEVs have been shown to impede early-stage biofilm formation, disseminate mature biofilms, and reduce virulence. This review summarizes the current status in the literature regarding the composition and role of BEVs in microbial infections. Furthermore, the dual functions of BEVs in eliciting and suppressing biofilm formation and virulence in various microbial pathogens are thoroughly discussed. This review is expected to improve our understanding of the use of BEVs in determining the mechanism of biofilm development in pathogenic bacteria and in developing drugs to inhibit biofilm formation by microbial pathogens. Bacterial extracellular vesicles (BEVs) are nanostructures formed by membrane blebbing and explosive cell lysis. It is essential for transporting lipids, nucleic acids, proteins, and quorum-sensing signaling molecules. BEVs play an important role in the formation of the biofilm's extracellular polymeric substances (EPS) by transporting its components, such as extracellular polysaccharides, proteins, and extracellular DNA. Furthermore, BEVs shield genetic material from nucleases and thermodegradation by packaging it during horizontal gene transfer, contributing to the transmission of bacterial adaptation determinants like antibiotic resistance. Thus, BEVs play a critical role in cell-to-cell communication, biofilm formation, virulence enhancement, disease progression, and drug resistance. In contrast, BEVs have been shown to prevent early-stage biofilm, disperse mature biofilm, and reduce virulence characteristics. [Display omitted] [ABSTRACT FROM AUTHOR]

[Display omitted] A water-soluble cationic porphyrin containing N -methyl- pyridinium fragments and an indole residue, namely, 5-[4-(1-methylindol-2-yl)phenyl]-10,15,20-tris(1-methyl- pyridinium-3-yl)porphyrin triiodide, has been synthesized. The porphyrin forms sedimentation unstable complexes with low and high molecular weight nucleic acids. This ability to precipitate nucleic acids can be used in laboratory, clinical and research practice for isolating nucleic acids. [ABSTRACT FROM AUTHOR]

After the decline of the COVID-19 pandemic, health systems were challenged by the simultaneous prevalence of different respiratory viruses causing a wide overlap in symptoms. This increased the demand for multi-virus diagnostic tests which require suitable pre-analytical workflow solutions in order to receive valid diagnostic results. In this context, the effects of specimen storage duration and temperature on the RNA/DNA copy number stability of influenza A/B, RSV A/B, SARS-CoV-2 and adenovirus were examined for four commercially available transport swab systems and saliva collection devices. The respiratory viruses were more stable in the saliva collection devices than in the transport swab systems when stored at RT or 37 °C for up to 96 h. Moreover, no differences between viral nucleic acid stability of enveloped and non-enveloped viruses were observed. The infectivity of all enveloped viruses could be inactivated by the saliva collection device from PreAnalytiX. The Norgen saliva device completely inactivated influenza A/B, while RSV A/B were partially inactivated. The non-enveloped adenovirus was inactivated by a reduction factor of 10E+ 4 in both saliva collection devices. All respiratory viruses remained infectious in the transport swab systems. Two possible transport medium additives were tested which inactivated or strongly reduced viral replication of tested enveloped viruses but had no effect on the non-enveloped adenovirus. Finally the implementation of multi-target detection procedures involving a direct amplification approach was successfully tested by spike-in of all enveloped viruses simultaneously into transport swab systems. This fast and reproducible setup presents a valuable solution for future implementations in multi-virus testing strategies. • Specimen collection, storage, transport and handling influence diagnostic results. • Multi-target detection assays could aid rapid identification of causative pathogens. • Tested saliva collection devices inactivated respiratory viruses used here. • Tested swab systems do not inactivate respiratory viruses used here. • Inactivating additives can reduce the risk of infection for healthcare personnel. [ABSTRACT FROM AUTHOR]

The human genome's nucleotide sequence variation, such as single nucleotide mutations, can cause numerous genetic diseases. However, detecting nucleic acids accurately and rapidly in complex biological samples remains a major challenge. While natural deoxyribonucleic acid (DNA) has been used as biorecognition probes, it has limitations like poor specificity, reproducibility, nuclease-induced enzymatic degradation, and reduced bioactivity on solid surfaces. To address these issues, we introduce a stable and reliable biosensor called graphene oxide (GO)- threose nucleic acid (TNA). It comprises chemically modified TNA capture probes on GO for detecting and imaging target nucleic acids in vitro and in vivo , distinguishing single nucleobase mismatches, and monitoring dynamic changes in target microRNA (miRNA). By loading TNA capture probes onto the GO substrate, the GO-TNA sensing platform for nucleic acid detection demonstrates a significant 88-fold improvement in the detection limit compared to TNA probes alone. This platform offers a straightforward preparation method without the need for costly and labor-intensive isolation procedures or complex chemical reactions, enabling real-time analysis. The stable TNA-based GO sensing nanoplatform holds promise for disease diagnosis, enabling rapid and accurate detection and imaging of various disease-related nucleic acid molecules at the in vivo level. The study's significance lies in the development of the GO-TNA biosensor, which addresses limitations in nucleic acid detection. By utilizing chemically modified nucleic acid analogues, the biosensor offers improved reliability and specificity, distinguishing single nucleobase mismatches and avoiding false signals. Additionally, its ability to detect and image target nucleic acids in vivo facilitates studying disease mechanisms. The simplified preparation process enhances practicality and accessibility, enabling real-time analysis. The biosensor's potential applications extend beyond healthcare, contributing to environmental analysis and food safety. Overall, this study's findings have substantial implications for disease diagnosis, biomedical research, and diverse applications, advancing nucleic acid detection and its impact on various fields. [Display omitted] [ABSTRACT FROM AUTHOR]

Physical-based gene delivery via biolistic methods (such as the Helios gene gun) involve precipitation of nucleic acids onto microparticles and direct transfection through cell membranes of exposed tissue (e.g. skin) by high velocity acceleration. The glycosaminoglycan (GAG)-binding enhanced transduction (GET) system exploits novel fusion peptides consisting of cell-binding, nucleic acid condensing, and cell-penetrating domains, which enable enhanced transfection across multiple cell types. In this study, we combined chemical (GET) and physical (gene gun) DNA delivery systems, and hypothesized the combination would generate enhanced distribution and effective uptake in cells not initially transfected by biolistic penetration. Physicochemical characterization, optimization of bullet contents and transfection experiments in vitro in cell monolayers and engineered tissue demonstrated these formulations transfected efficiently, including DC2.4 dendritic cells. We incorporated these formulations into a biolistic format for gene gun by forming fireable dry bullets obtained via lyophilization (freeze drying). This system is simple and with enhanced scalability compared to conventional methods to generate bullets. Flushed GET bullet contents retained their ability to mediate transfection (17-fold greater and 13-fold greater reporter gene expression than standard spermidine bullets in the absence and presence of serum, respectively). Fired GET bullets in vitro (in cells and collagen gels) and in vivo (mice) showed increased reporter gene transfection compared to untreated controls, whilst maintaining cell viability in vitro and having no obvious toxicity in vivo. Lastly, a SARS-CoV-2 plasmid DNA vaccine with spike (S) protein-receptor binding domain (S-RBD) was delivered by gene gun using GET bullets. Specific T cell and antibody responses comparable to the conventional system were generated. The non-physical and physical combination of GET‑gold-DNA carriers using gene gun shows potential as an alternative DNA delivery method that is scalable for mass deployable vaccination and intradermal gene delivery. [Display omitted] [ABSTRACT FROM AUTHOR]

Infectious diseases are one of the world's leading causes of morbidity. Their rapid spread emphasizes the need for accurate and fast diagnostic methods for large-scale screening. Here, we describe a robust method for the detection of pathogens based on microscale thermophoresis (MST). The method involves the hybridization of a fluorescently labeled DNA probe to a target RNA and the assessment of thermophoretic migration of the resulting complex in solution within a 2 to 30-time window. We found that the thermophoretic migration of the nucleic acid-based probes is primarily determined by the fluorescent molecule used, rather than the nucleic acid sequence of the probe. Furthermore, a panel of uniformly labeled probes that bind to the same target RNA yields a more responsive detection pattern than a single probe, and moreover, can be used for the detection of specific pathogen variants. In addition, intercalating agents (ICA) can be used to alter migration directionality to improve detection sensitivity and resolving power by several orders of magnitude. We show that this approach can rapidly diagnose viral SARS-CoV2, influenza H1N1, artificial pathogen targets, and bacterial infections. Furthermore, it can be used for anti-microbial resistance testing within 2 h, demonstrating its diagnostic potential for early pathogen detection. [ABSTRACT FROM AUTHOR]

Cytosolic long dsRNA, among the most potent proinflammatory signals, is recognized by melanoma differentiationassociated protein 5 (MDA5). MDA5 binds dsRNA cooperatively forming helical filaments. ATP hydrolysis byMDA5 fulfills a proofreading function by promoting dissociation of shorter endogenous dsRNs from MDA5 while allowing longer viral dsRNAs to remain bound leading to activation of interferon-ß responses. Here, we show that adjacent MDA5 subunits in MDA5-dsRNA filaments hydrolyze ATP cooperatively, inducing cooperative filament disassembly. Consecutive rounds of ATP hydrolysis amplify the filament footprint, displacing tightly bound proteins from dsRNA. Our electron microscopy and biochemical assays show that LGP2 binds to dsRNA at internal binding sites through noncooperative ATP hydrolysis. Unlike MDA5, LGP2 has low nucleic acid selectivity and can hydrolyze GTP and CTP as well as ATP. Binding of LGP2 to dsRNA promotes nucleation of MDA5 filament assembly resulting in shorter filaments. Molecular modeling identifies an internally bound MDA5-LGP2-RNA complex, with the LGP2 C-terminal tail forming the key contacts with MDA5. These contacts are specifically required for NTP-dependent internal RNA binding. We conclude that NTPase-dependent binding of LGP2 to internal dsRNA sites complements NTPase-independent binding to dsRNA ends, via distinct binding modes, to increase the number and signaling output of MDA5-dsRNA complexes. [ABSTRACT FROM AUTHOR]

Colorectal cancer is one of the most common cancers, and its incidence rate keeps increasing worldwide. Epidermal growth factor receptor (EGFR) is highly expressed in colorectal cancer cells, and its activation leads to the activation of downstream kinase pathways to promote cell growth, resulting in tumour growth and progression. EGFR is expressed in only small amounts in normal cells, however, it is overexpressed in tumour cells and is a potential tumour marker, thus the detection of which is of great importance. This work proposes a novel and highly sensitive electrochemical immunosensor based on nucleic acid aptamer recognition and silver deposition for EGFR detection. The key novelty of this work is the use of SiO2 nanospheres to enhance the sensitivity. First, SiO 2 nanospheres were synthesized and modified on the surface of a gold electrode using a self-assembly process. This provided a large surface area to allow high loading of capture antibodies. The antibody was then immobilized on the SiO 2 nanosphere modified electrode surface to form an immunosandwich complex with the antigen and biotin-labelled nucleic acid aptamer. Afterwards, the affinity-labelled alkaline phosphatase was immobilized onto the electrode with the specific reaction of biotin and affinity. The obtained product was subjected to silver deposition in the substrate solution. The large surface area provided by the SiO 2 nanospheres allowed significantly improved sensitivity. Experiments were performed to optimize the concentration of immobilized antibodies and biotin-labelled nucleic acid aptamer. The experimental results show a good linearity of EGFR concentration in the range of 1 to 1000 ng/mL, with the lowest detection limit up to 0.06 ng/mL. This method has high sensitivity and stability, which allows it to have a broad application prospects in the clinical field. [ABSTRACT FROM AUTHOR]