CRISPR-Cas assisted diagnostics of plant viruses and challenges.

Plant viruses threaten global food security by infecting commercial crops, highlighting the critical need for efficient virus detection to enable timely preventive measures. Current techniques rely on polymerase chain reaction (PCR) for viral genome amplification and require laboratory conditions. This review explores the applications of CRISPR-Cas assisted diagnostic tools, specifically CRISPR-Cas12a and CRISPR-Cas13a/d systems for plant virus detection and analysis. The CRISPR-Cas12a system can detect viral DNA/RNA amplicons and can be coupled with PCR or isothermal amplification, allowing multiplexed detection in plants with mixed infections. Recent studies have eliminated the need for expensive RNA purification, streamlining the process by providing a visible readout through lateral flow strips. The CRISPR-Cas13a/d system can directly detect viral RNA with minimal preamplification, offering a proportional readout to the viral load. These approaches enable rapid viral diagnostics within 30 min of leaf harvest, making them valuable for onsite field applications. Timely identification of diseases associated with pathogens is crucial for effective treatment; yet developing rapid, specific, sensitive, and cost-effective diagnostic technologies remains challenging. The current gold standard, PCR technology, has drawbacks such as lengthy operational cycles, high costs, and demanding requirements. Here we update the technical advancements of CRISPR-Cas in viral detection, providing insights into future developments, versatile applications, and potential clinical translation. There is a need for approaches enabling field plant viral nucleic acid detection with high sensitivity, specificity, affordability, and portability. Despite challenges, CRISPR-Cas-mediated pathogen diagnostic solutions hold robust capabilities, paving the way for ideal diagnostic tools. Alternative applications in virus research are also explored, acknowledging the technology's limitations and challenges.

Freezing Functional Nucleic Acids: From Molecular Reactions to Surface Immobilization.

Biochemical reactions are typically slowed down by decreasing temperature. However, accelerated reaction kinetics have been observed for a long time. More recent examples have highlighted the unique role of freezing in fabricating supermaterials, degrading environmental contaminants, and accelerating bioreactions. Functional nucleic acids are DNA or RNA oligonucleotides with versatile properties, including target recognition, catalysis, and molecular co4mputing. In this review, we discuss the current observations and understanding of freezing-facilitated reactions involving functional nucleic acids. Molecular reactions such as ligation/conjugation, cleavage, and hybridization are discussed. Moreover, freezing-induced DNA-nanoparticle conjugations are introduced. Then, we describe our effect in immobilizing DNA on bulk surfaces. Finally, we address some critical questions and research opportunities in the field.

Peptide Nucleic Acids: From Origami to Editing.

Peptide nucleic acids (PNAs) combine the programmability of native nucleic acids with the robustness and ease of synthesis of a peptide backbone. These designer biomolecules have demonstrated tremendous utility across a broad range of applications, from the formation of bespoke biosupramolecular architectures to biosensing and gene regulation. Herein, we explore some of the key developments in the application of PNA in chemical biology and biotechnology in the last 5 years and present anticipated key areas of future development.

Therapeutic Use of G4-Ligands in Cancer: State-of-the-Art and Future Perspectives.

G-quadruplexes (G4s) are guanine-rich non-canonical secondary structures of nucleic acids that were identified in vitro almost half a century ago. Starting from the early 1980s, these structures were also observed in eukaryotic cells, first at the telomeric level and later in regulatory regions of cancer-related genes, in regulatory RNAs and within specific cell compartments such as lysosomes, mitochondria, and ribosomes. Because of the involvement of these structures in a large number of biological processes and in the pathogenesis of several diseases, including cancer, the interest in G4 targeting has exponentially increased in the last few years, and a great number of novel G4 ligands have been developed. Notably, G4 ligands represent a large family of heterogeneous molecules that can exert their functions by recognizing, binding, and stabilizing G4 structures in multiple ways. Regarding anti-cancer activity, the efficacy of G4 ligands was originally attributed to the capability of these molecules to inhibit the activity of telomerase, an enzyme that elongates telomeres and promotes endless replication in cancer cells. Thereafter, novel mechanisms through which G4 ligands exert their antitumoral activities have been defined, including the induction of DNA damage, control of gene expression, and regulation of metabolic pathways, among others. Here, we provided a perspective on the structure and function of G4 ligands with particular emphasis on their potential role as antitumoral agents. In particular, we critically examined the problems associated with the clinical translation of these molecules, trying to highlight the main aspects that should be taken into account during the phases of drug design and development. Indeed, taking advantage of the successes and failures, and the more recent technological progresses in the field, it would be possible to hypothesize the development of these molecules in the future that would represent a valid option for those cancers still missing effective therapies.

Solid Lipid Nanoparticles for Pulmonary Delivery of Biopharmaceuticals: A Review of Opportunities, Challenges, and Delivery Applications.

Biopharmaceuticals such as nucleic acids, proteins, and peptides constitute a new array of treatment modalities for chronic ailments. Invasive routes remain the mainstay of administering biopharmaceuticals due to their labile nature in the biological environment. However, it is not preferred for long-term therapy due to the lack of patient adherence and clinical suitability. Therefore, alternative routes of administration are sought to utilize novel biopharmaceutical therapies to their utmost potential. Nanoparticle-mediated pulmonary delivery of biologics can facilitate both local and systemic disorders. Solid lipid nanoparticles (SLNs) afford many opportunities as pulmonary carriers due to their physicochemical stability and ability to incorporate both hydrophilic and hydrophobic moieties, thus allowing novel combinatorial drug/gene therapies. These applications include pulmonary infections, lung cancer, and cystic fibrosis, while systemic delivery of biomolecules, like insulin, is also attractive for the treatment of chronic ailments. This Review explores physiological and particle-associated factors affecting pulmonary delivery of biopharmaceuticals. It compares the advantages and limitations of SLNs as pulmonary nanocarriers along with design improvements underway to overcome these limitations. Current research illustrating various SLN designs to deliver proteins, peptides, plasmids, oligonucleotides, siRNA, and mRNA is also summarized.

High-content tailoring strategy to improve the multifunctionality of functional nucleic acids.

Functional nucleic acids (FNAs) have attracted increasing attention in recent years due to their diverse physiological functions. The understanding of their conformational recognition mechanisms has advanced through nucleic acid tailoring strategies and sequence optimization. With the development of the FNA tailoring techniques, they have become a methodological guide for nucleic acid repurposing. Therefore, it is necessary to systematize the relationship between FNA tailoring strategies and the development of nucleic acid multifunctionality. This review systematically categorizes eight types of FNA multifunctionality, and introduces the traditional FNA tailoring strategy from five aspects, including deletion, substitution, splitting, fusion and elongation. Based on the current state of FNA modification, a new generation of FNA tailoring strategy, called the high-content tailoring strategy, was unprecedentedly proposed to improve FNA multifunctionality. In addition, the multiple applications of rational tailoring-driven FNA performance enhancement in various fields were comprehensively summarized. The limitations and potential of FNA tailoring and repurposing in the future are also explored in this review. In summary, this review introduces a novel tailoring theory, systematically summarizes eight FNA performance enhancements, and provides a systematic overview of tailoring applications across all categories of FNAs. The high-content tailoring strategy is expected to expand the application scenarios of FNAs in biosensing, biomedicine and materials science, thus promoting the synergistic development of various fields.

DNA Anchoring Strength Directly Correlates with Spherical Nucleic Acid-Based HPV E7 Cancer Vaccine Potency.

Vaccination for cancers arising from human papillomavirus (HPV) infection holds immense potential, yet clinical success has been elusive. Herein, we describe vaccination studies involving spherical nucleic acids (SNAs) incorporating a CpG adjuvant and a peptide antigen (E711-19) from the HPV-E7 oncoprotein. Administering the vaccine to humanized mice induced immunity-dependent on the oligonucleotide anchor chemistry (cholesterol vs (C12)9). SNAs containing a (C12)9-anchor enhanced IFN-γ production >200-fold, doubled memory CD8+ T-cell formation, and delivered more than twice the amount of oligonucleotide to lymph nodes in vivo compared to a simple admixture. Importantly, the analogous construct with a weaker cholesterol anchor performed similar to admix. Moreover, (C12)9-SNAs activated 50% more dendritic cells and generated T-cells cytotoxic toward an HPV+ cancer cell line, UM-SCC-104, with near 2-fold greater efficiency. These observations highlight the pivotal role of structural design, and specifically oligonucleotide anchoring strength (which correlates with overall construct stability), in developing efficacious therapeutic vaccines.

Tetranuclear Polypyridylruthenium(II) Complexes as Selective Nucleic Acid Stains for Flow Cytometric Analysis of Monocytic and Epithelial Lung Carcinoma Large Extracellular Vesicles.

Selective staining of extracellular vesicles (EVs) is a major challenge for diagnostic and therapeutic applications. Herein, the EV labeling properties of a new class of tetranuclear polypyridylruthenium(II) complexes, Rubb7-TNL and Rubb7-TL, as phosphorescent stains are described. These new stains have many advantages over standard stains to detect and characterize EVs, including: high specificity for EV staining versus cell staining; high phosphorescence yields; photostability; and a lack of leaching from EVs until incorporation with target cells. As an example of their utility, large EVs released from control (basal) or lipopolysaccharide (LPS)-stimulated THP-1 monocytic leukemia cells were studied as a model of immune system EVs released during bacterial infection. Key findings from EV staining combined with flow cytometry were as follows: (i) LPS-stimulated THP-1 cells generated significantly larger and more numerous large EVs, as compared with those from unstimulated cells; (ii) EVs retained native EV physical properties after staining; and (iii) the new stains selectively differentiated intact large EVs from artificial liposomes, which are models of cell membrane fragments or other lipid-containing debris, as well as distinguished two distinct subpopulations of monocytic EVs within the same experiment, as a result of biochemical differences between unstimulated and LPS-stimulated monocytes. Comparatively, the staining patterns of A549 epithelial lung carcinoma-derived EVs closely resembled those of THP-1 cell line-derived EVs, which highlighted similarities in their selective staining despite their distinct cellular origins. This is consistent with the hypothesis that these new phosphorescent stains target RNA within the EVs.

A Bioswitchable MiRNA Delivery System: Tetrahedral Framework DNA-Based miRNA Delivery System for Applications in Wound Healing.

The human body's primary line of defense, the skin, is especially prone to harm. Although microRNA (miRNA)-based therapies have attracted increasing attention for skin wound healing, their applications remain limited owing to a range of issues. Tetrahedral framework DNA (tFNA), a nanomaterial possessing nucleic acid characteristics, exhibits an excellent biocompatibility, in addition to anti-inflammatory and transdermal delivery capabilities, and can accelerate skin wound healing. Due to its potential to exert synergistic action with therapeutic miRNA, tFNA has been considered an ideal vehicle for miRNA therapy. The design and synthesis of a bioswitchable miRNA delivery system (BiRDS) is reported, which contains three miRNAs as well as a nucleic acid core to maximize the loading capacity while preserving the characteristics of tFNA. A high stability, excellent permeability of cells as well as tissues and good biological compatibility are demonstrated. By selectively inhibiting heparin-binding epidermal growth factor (HB-EGF), the BiRDS can inhibit the NF-κB pathway while simultaneously controlling the PTEN/Akt pathway. As a result, the BiRDS helps wound healing go through the inflammation to the proliferative phase. This study demonstrates the advantages of the BiRDS in miRNA-based therapy and provides new research ideas for the treatment of skin-related diseases.

From Structure to Application: The Evolutionary Trajectory of Spherical Nucleic Acids.

Since the proposal of the concept of spherical nucleic acids (SNAs) in 1996, numerous studies have focused on this topic and have achieved great advances. As a new delivery system for nucleic acids, SNAs have advantages over conventional deoxyribonucleic acid (DNA) nanostructures, including independence from transfection reagents, tolerance to nucleases, and lower immune reactions. The flexible structure of SNAs proves that various inorganic or organic materials can be used as the core, and different types of nucleic acids can be conjugated to realize diverse functions and achieve surprising and exciting outcomes. The special DNA nanostructures have been employed for immunomodulation, gene regulation, drug delivery, biosensing, and bioimaging. Despite the lack of rational design strategies, potential cytotoxicity, and structural defects of this technology, various successful examples demonstrate the bright and convincing future of SNAs in fields such as new materials, clinical practice, and pharmacy.

Functionalized nanoparticles to deliver nucleic acids to the brain for the treatment of Alzheimer's disease.

Brain-targeted gene delivery across the blood-brain barrier (BBB) is a significant challenge in the 21st century for the healthcare sector, particularly in developing an effective treatment strategy against Alzheimer's disease (AD). The Internal architecture of the brain capillary endothelium restricts bio-actives entry into the brain. Additionally, therapy with nucleic acids faces challenges like vulnerability to degradation by nucleases and potential immune responses. Functionalized nanocarrier-based gene delivery approaches have resulted in safe and effective platforms. These nanoparticles (NPs) have demonstrated efficacy in protecting nucleic acids from degradation, enhancing transport across the BBB, increasing bioavailability, prolonging circulation time, and regulating gene expression of key proteins involved in AD pathology. We provided a detailed review of several nanocarriers and targeting ligands such as cell-penetrating peptides (CPPs), endogenous proteins, and antibodies. The utilization of functionalized NPs extends beyond a singular system, serving as a versatile platform for customization in related neurodegenerative diseases. Only a few numbers of bioactive regimens can go through the BBB. Thus, exploring functionalized NPs for brain-targeted gene delivery is of utmost necessity. Currently, genes are considered high therapeutic potential molecules for altering any disease-causing gene. Through surface modification, nanoparticulate systems can be tailored to address various diseases by replacing the target-specific molecule on their surface. This review article presents several nanoparticulate delivery systems, such as lipid NPs, polymeric micelles, exosomes, and polymeric NPs, for nucleic acids delivery to the brain and the functionalization strategies explored in AD research.

Distinct features of ribonucleotides within genomic DNA in Aicardi-Goutières syndrome ortholog mutants of Saccharomyces cerevisiae.

Ribonucleoside monophosphates (rNMPs) are abundantly found within genomic DNA of cells. The embedded rNMPs alter DNA properties and impact genome stability. Mutations in ribonuclease (RNase) H2, a key enzyme for rNMP removal, are associated with the Aicardi-Goutières syndrome (AGS), a severe neurological disorder. Here, we engineered orthologs of the human RNASEH2A-G37S and RNASEH2C-R69W AGS mutations in yeast Saccharomyces cerevisiae: rnh201-G42S and rnh203-K46W. Using the ribose-seq technique and the Ribose-Map bioinformatics toolkit, we unveiled rNMP abundance, composition, hotspots, and sequence context in these AGS-ortholog mutants. We found a high rNMP presence in the nuclear genome of rnh201-G42S-mutant cells, and an elevated rCMP content in both mutants, reflecting preferential cleavage of RNase H2 at rGMP. We discovered unique rNMP patterns in each mutant, showing differential activity of the AGS mutants on the leading or lagging replication strands. This study guides future research on rNMP characteristics in human genomes with AGS mutations.

DNA nanoassembly based turn-on amplification probe for sensitive colorimetric CRISPR/Cas12a-mediated detection of pathogen DNA.

Clustered regularly interspaced short palindromic repeat (CRISPR) system has been explored as an efficient tool for nucleic acid diagnostics. However, it normally needs instrumentation or produces turn-off signals. Herein, a bulged Y-shape DNA (Y-DNA) nanoassembly was designed and synthesized as a novel turn-on probe. A CRISPR/Cas12a and Y-DNA probe mediated colorimetric assay (named as CYMCOA) strategy was developed for visual detection of pathogen DNA. Upon activating Cas12a with pathogen DNA, the Y-DNA bulge is catalytically trans-cleaved, releasing the G-quadruplex sequence embedded in the Y-DNA nanoassembly as a peroxidase-like DNAzyme. Visible signals with chromogen substrates are thus produced. The CYMCOA strategy was combined with recombinase polymerase amplification (RPA), an isothermal amplification technique, in detecting Helicobacter pylori (Hp) bacteria and SARS-CoV-2 N plasmids as two model pathogens. The bioassay has very excellent detection sensitivity and specificity, owing to the triple cascade amplification reactions and the very low mismatch tolerance. The lower limit of detection values were 0.16 cfu⋅mL-1, 1.5 copies⋅µL-1, and 0.17 copies⋅µL-1 for Hp bacteria, Hp plasmids, and SARS-CoV-2 N plasmids respectively. The detection is fast and accurate. The colorimetric bioassay strategy provides to be a simple, accurate, fast and instrumentation-free platform for nucleic acids detections in various settings, including crude and emergent situations.

An integrated LC-MS/MS platform for noninvasive urinary nucleus acid adductomics: A pilot study for tobacco exposure.

Tobacco smoke exposure significantly increases the level of global nucleoside damage. To evaluate all aspects of nucleic acid (NA) modifications, NA adductomics analyzes DNA, RNA and nucleobase adducts and provides comprehensive data. Liquid chromatography-tandem triple quadrupole mass spectrometry (LC-QQQ-MS/MS) and LC-Zeno-TOF-MS/MS were employed to screen for DNA, RNA and nucleobase adducts, as part of the analytical platform that was designed to combine high sensitivity and high resolution detection. We identified and distinguished urine nucleoside adducts via precursor ion and neutral loss scanning. A total of 245 potential adducts were detected, of which 28 were known adducts. The smoking group had significantly higher concentrations of nucleoside adducts in rat urine than the control group, based on MRM scanning, which was then used to perform relative quantitative analysis of these adducts. Urine nucleoside adducts were further confirmed using LC-Zeno-TOF-MS/MS. This highlights the potential of untargeted detection methods to provide comprehensive data on both known and unknown adducts. These approaches can be used to investigate the interactions among oxidative and alkylation stresses, and epigenetic modifications caused by exposure to tobacco smoke.

Unlocking the therapeutic potential of locked nucleic acids through lipid nanoparticle delivery.

Locked nucleic acids (LNAs) are a subtype of antisense oligonucleotides (ASOs) that are characterized by a bridge within the sugar moiety. LNAs owe their robustness to this chemical modification, which as the name suggests, locks it in one conformation. This perspective includes two components: a general overview on ASOs from one side and on delivery issues focusing on lipid nanoparticles (LNPs) on the other side. Throughout, a screening of the ongoing clinical trials involving ASOs is given, as well as a take on the versatility and challenges of using LNAs. Finally, we highlight the potential of LNPs as carriers for the successful delivery of LNAs.

Tetrahedral Framework Nucleic Acid-Based Delivery of Astaxanthin Suppresses Chondrocyte Pyroptosis and Modulates Oxidative Stress for the Treatment of Osteoarthritis.

Worldwide, osteoarthritis (OA) is regarded as the most widespread, distressing, and limiting chronic disease that affects degenerative joints. Currently, there is no treatment available to modify the progression of OA. The pathogenesis of OA is significantly linked with oxidative stress and pyroptosis. Astaxanthin (Ast) is a natural ketocarotenoid pigment with potent antioxidant activity and is shown to effectively alleviate cartilage damage in OA. However, its bioavailability is greatly limited due to poor water solubility, high sensitivity to light, temperature, and pH. In this study, Ast-loaded tetrahedral framework nucleic acids (tFNAs) or tFNA/Ast complexes (TAC) for Ast delivery are developed. Compared with free Ast and tFNA alone, TAC exhibits improved drug stability and cellular uptake. Most importantly, TAC effectively protects chondrocytes against oxidative stress-induced pyroptosis while promoting extracellular matrix anabolism by chondrocytes, and ultimately alleviates cartilage damage in a mouse destabilization of the medial meniscus (DMM) model. Thus, TAC holds great promise for the treatment of OA patients.

Optimized lipid nanoparticles (LNPs) for organ-selective nucleic acids delivery in vivo.

Nucleic acid therapeutics offer tremendous promise for addressing a wide range of common public health conditions. However, the in vivo nucleic acids delivery faces significant biological challenges. Lipid nanoparticles (LNPs) possess several advantages, such as simple preparation, high stability, efficient cellular uptake, endosome escape capabilities, etc., making them suitable for delivery vectors. However, the extensive hepatic accumulation of LNPs poses a challenge for successful development of LNPs-based nucleic acid therapeutics for extrahepatic diseases. To overcome this hurdle, researchers have been focusing on modifying the surface properties of LNPs to achieve precise delivery. The review aims to provide current insights into strategies for LNPs-based organ-selective nucleic acid delivery. In addition, it delves into the general design principles, targeting mechanisms, and clinical development of organ-selective LNPs. In conclusion, this review provides a comprehensive overview to provide guidance and valuable insights for further research and development of organ-selective nucleic acid delivery systems.

siRNA-mediated reduction of a circulating protein in swine using lipid nanoparticles.

Genetic manipulation of animal models is a fundamental research tool in biology and medicine but is challenging in large animals. In rodents, models can be readily developed by knocking out genes in embryonic stem cells or by knocking down genes through in vivo delivery of nucleic acids. Swine are a preferred animal model for studying the cardiovascular and immune systems, but there are limited strategies for genetic manipulation. Lipid nanoparticles (LNPs) efficiently deliver small interfering RNA (siRNA) to knock down circulating proteins, but swine are sensitive to LNP-induced complement activation-related pseudoallergy (CARPA). We hypothesized that appropriately administering optimized siRNA-LNPs could knock down circulating levels of plasminogen, a blood protein synthesized in the liver. siRNA-LNPs against plasminogen (siPLG) reduced plasma plasminogen protein and hepatic plasminogen mRNA levels to below 5% of baseline values. Functional assays showed that reducing plasminogen levels modulated systemic blood coagulation. Clinical signs of CARPA were not observed, and occasional mild and transient hepatotoxicity was present in siPLG-treated animals at 5 h post-infusion, which returned to baseline by 7 days. These findings advance siRNA-LNPs in swine models, enabling genetic engineering of blood and hepatic proteins, which can likely expand to proteins in other tissues in the future.

Application of CytoPath®Easy Vials in Cervical Cancer Screening: Self-Sampling Approach.

Context: CytoPath®Easy kit (DiaPath S.p.A.) offers a major advantage compared to other commercially available kits available for the screening of cervical cancer, as it does not require additional equipment for sample processing. Using this methodology, collected epithelial cells are immersed in a preservative liquid before setting as a thin layer on a slide via gravity sedimentation. Aims: To evaluate the suitability of the CytoPath®Easy kit for the processing of cervical samples, detection of pre-neoplastic lesions, and nucleic preservation and extraction for HR-HPV diagnosis. Materials and Methods: A total of 242 self-sampled cervical specimens were utilized, with 192 collected in CytoPath®Easy vials and 50 collected and processed using the ThinPrepTM for comparative analysis. The samples underwent processing, Papanicolaou staining, and microscopic evaluation for morphological parameters. The extracted nucleic acids were assessed for purity and integrity, and the detection of high-risk human papillomavirus (HR-HPV) was carried out using the Alinitym HR HPV system kit (Abbott Laboratórios Lda). Results: Both methods demonstrated effective performance, enabling the morphological assessment of the cervical epithelium. Statistical analysis indicated that ThinPrepTM yielded significantly better results in terms of cellularity. Conversely, CytoPath®Easy exhibited superior performance in terms of the quantity of extracted DNA and its degree of purification. Concerning the time consumed during processing, both methods were comparable, with the CytoPath®Easy methodology standing out for its cost-effectiveness, as it does not necessitate additional instruments and consumables. Conclusions: The novel CytoPath®Easy methodology proves effective in preserving both nucleic acids and cell morphology characteristics, two crucial features for cervical cancer screening.

A novel synthesis method of magnetic Janus particles for wastewater applications.

HYPOTHESIS: Magnetic particles are widely used in many adsorption and removal processes. Among the many types of magnetic colloids, magnetic Janus particles offer significant possibilities for the effective removal of several components from aqueous solutions. Nevertheless, the synthesis of structures integrating different types of materials requires scalable fabrication processes to overcome the limitations of the available methodologies. Herein, we hypothesized a fabrication process for dual-surface functionalized magnetic Janus particles. EXPERIMENTS: The primary silica particles with surface-attached amine groups are further asymmetrically modified by iron oxide nanoparticles, exploiting Pickering emulsion and electroless deposition techniques. The dual surface functionality of the particles is designed for its versatility and demonstrated in two wastewater-related applications. FINDINGS: We show that our design can simultaneously remove chromium (VI) and phenol from aqueous solution. The fabricated magnetic-responsive Janus particles are also an effective adsorbent for genomic Deoxyribonucleic acid (DNA) and show superior performance to commercial magnetic beads. Thus, this study provides a novel platform for designing magnetic Janus particles with multifunctional surfaces for wastewater treatment applications.