Search Results (172)

RNA interference (RNAi) holds promise as a gene-silencing therapy for liver cancer but faces challenges related to siRNA instability, short half-life, and inefficient cellular uptake. In this study, we designed a self-assembling RNA nanoparticle targeting three oncogenes—hTERT, BIRC5, and FGFR1—key drivers of cancer progression. These RNA nanoparticles demonstrated enhanced stability and specificity, eliminating the need for conventional toxic delivery carriers. Functional assays revealed that the nanoparticles effectively suppressed the proliferation, migration, tumor growth and apoptosis of a Hepatocellular carcinoma cell line, Hep3B. The nanoparticles exhibited excellent safety and efficacy in xenograft model mice, without off-target toxicity. This work introduces a scalable, biocompatible RNA nanoparticle platform with multi-targeting capability, paving the way for improved RNAi-based therapeutics. Our findings offer a promising strategy for advancing personalized cancer therapies and underscore the broader potential of RNA nanotechnology in addressing complex malignancies. Full article

Background: Kenaf (Hibiscus cannabinus L.) is an important fiber crop belonging to the genus Hibiscus in the Malvaceae family. Research on its chloroplast genome holds significant importance for deciphering the evolutionary relationships of the Hibiscus species, developing genetic markers, and promoting kenaf (H. cannabinus) genetic breeding. Methods: Based on high-throughput sequencing technology, this study completed the sequencing and assembly of the kenaf (H. cannabinus) chloroplast genome. Results: (1) The kenaf (H. cannabinus) chloroplast genome exhibits a typical circular quadripartite structure with a total length of 163,019 bp, including a large single-copy region (LSC) of 90,467 bp, a small single-copy region (SSC) of 19,486 bp, and a pair of inverted repeat regions (IRa/IRb) of 26,533 bp each. The total GC content is 36.62%, among which, the IR region has the highest GC content (42.61%) and the SSC region the lowest (30.87%). (2) A total of 131 genes were annotated, including 85 mRNAs, 37 tRNAs, 8 rRNAs, and 1 pseudogene. Their functions cover photosynthesis (e.g., pet and atp family genes), self-replication (e.g., rpl, rps, and rpo family genes), and genes with unknown functions (e.g., ycf1 and ycf2). A codon usage bias analysis revealed that the relative synonymous codon usage (RSCU) value of the stop codon UAA is the highest (1.6329), and codons ending with A/U are preferentially used (e.g., GCU for alanine with RSCU = 1.778). (3) A repeat sequence analysis identified various interspersed repeat sequences (predominantly 30~31 bp in length, with a relatively high proportion in the 30~40 bp range, including forward and palindromic types) and simple sequence repeats (cpSSRs). Among them, single-base repeat SSRs account for the highest proportion (e.g., (A)8 and (T)9), and specific SSR primers were designed. (4) A comparative evolutionary analysis indicated that the Ka/Ks ratios (nonsynonymous substitution rate/synonymous substitution rate) of core chloroplast genes (e.g., rps2 and rpoC2) in kenaf (H. cannabinus) are all less than 1 (0.145~0.415), suggesting that they are under purifying selection. The collinearity similarity of chloroplast genomes between kenaf (H. cannabinus) and its closely related species reaches over 99.97%, and the IR region boundaries are relatively conserved. The phylogenetic tree shows that kenaf (H. cannabinus) clusters with closely related Hibiscus species with a 100% bootstrap value, indicating a close genetic relationship. Conclusions: This study provides basic data for the functional analysis of the kenaf (H. cannabinus) chloroplast genome, the phylogeny of Hibiscus, and the utilization of genetic resources. Full article

The prebiological anomeric selectivity of ribonucleosides is a key phenomenon in the understanding of the RNA world hypothesis and the origin of life. While each ribonucleoside can have two anomers (α or β), ribonucleosides naturally occur in the β form, while α anomers are extremely rare. Guanosine, a canonical ribonucleoside, binds to borate and self-assembles into G4-quartets, enabling the formation of borate–guanosine hydrogels. These macrostructures, exhibiting elevated thermal robustness and self-healing properties, have been suggested as plausible frameworks for the syntheses of prebiological molecules. Moreover, their external layers could have prevented degradation of compounds by aggressive primitive radiation and reduced molecular dispersion. Herein it is proposed that anomeric selectivity may have occurred due to the different 3D organization and stereochemical environment formed by each borate–guanosine anomer and subsequent formation of borate–guanosine hydrogels. DFT was applied to the optimization of α and β anomeric structures in four steps, from borate–guanosine diesters to G4 structures. The results obtained suggest that β-syn-guanosine (the most stable structure) is the only anomer that forms a planar G4-quartet with borate, capable of self-assembling into a hydrogel. Given the properties of borate–guanosine hydrogels, this could explain why β-guanosine is currently the sole anomer present in living organisms. Full article

Alterations in the expression of the Klotho gene have been associated with chronic kidney disease (CKD), and its potential as an early diagnostic biomarker is currently under active investigation. In this work, we report the development of a highly sensitive, label-free electrochemical DNA-based biosensor for the detection of a 100 mer DNA fragment corresponding to a partial region of Klotho mRNA. The proposed bioplatform integrates mixed self-assembled monolayers (SAMs) and gold nanoparticles for efficient DNA immobilization within a sandwich-type configuration, coupled with impedimetric detection. Different SAM architectures were evaluated by cyclic voltammetry and electrochemical impedance spectroscopy, with the binary monolayer composed of 1-hexadecanethiol (HDT) and the capture probe (CP) exhibiting the best analytical performance. The use of gold nanoparticle-modified screen-printed carbon electrodes (AuNPs–SPCEs) resulted in a 1.4-fold increase in the signal-to-noise ratio compared to screen-printed gold electrodes. Additionally, the incorporation of a blocking step using bovine serum albumin (BSA–HDT–CP–AuNPs–SPCE) enhanced the sensitivity by 1.6-fold compared to the unblocked system. The genosensor displayed a linear response in the concentration range of 3 × 10⁻¹⁰ to 7.5 × 10⁻⁸ M, achieving a detection limit of 0.09 nM. Relative standard deviations below 7.5% were obtained for different Klotho concentrations, confirming high intra-assay and intermediary precision. Selectivity assays demonstrated negligible signals for non-complementary sequences, while recovery experiments in spiked human serum samples yielded satisfactory values between 96.5% and 103.4%. Full article

Surface modification of nucleic acid-based electrochemical biosensors has been at the forefront of research since their inception. Effective modification ensures the optimization of the sensitivity, specificity, and stability of modern biosensors. Recent advances in DNA nanotechnology have enabled the development of novel electrochemical biosensor interfaces with precise assembly and high biocompatibility. In this review, we explore three strategies for enhancing biosensor performance: the integration of tetrahedral DNA nanostructures (TDNs), self-assembled monolayers (SAMs), and DNA-based hydrogels. TDNs offer well-defined geometry and controlled spatial presentation of capture probes, significantly reducing background noise and improving target accessibility. SAMs provide a robust and tunable platform for anchoring these nanostructures, enabling reproducible and chemically stable interfaces. DNA hydrogels serve as a responsive and flexible scaffold capable of signal amplification and analyte retention. These surface architectures enhance sensitivity and minimize non-specific adsorption (NSA). We discuss recent applications and experimental outcomes, highlighting how each component is driving the next generation of nucleic acid-based biosensors. Full article

Botrytis cinerea, a necrotrophic fungal pathogen responsible for gray mold, poses a severe threat to over 1400 plant species, causing significant pre- and postharvest losses worldwide. RNA interference (RNAi)-based strategies, particularly spray-induced gene silencing (SIGS), have emerged as environmentally friendly alternatives to chemical fungicides. However, the application of naked double-stranded RNA (dsRNA) suffers from poor stability and low cellular uptake. In this study, we engineered a self-assembling triangular RNA nanoparticle, termed Bc-triangle, targeting four virulence genes of B. cinerea—BcDCL1, BcPPI10, BcNMT1 and BcBAC. The nanostructure was designed using RNA origami principles and produced in Escherichia coli. Functional assays demonstrated that Bc-triangle significantly inhibited conidial germination and mycelial growth in vitro, and markedly reduced disease severity in plants. Compared with linear dsRNA, Bc-triangle showed superior persistence and efficacy, with lesion area reduction sustained up to 10 days post-spraying. qRT-PCR analysis revealed substantial downregulation of the target genes, especially BcNMT1, indicating enhanced RNAi activation. These findings establish RNA nanotechnology as a powerful platform for transgene-free, programmable, and sustainable control of fungal pathogens in crop production. Full article

Virus-like particles (VLPs) formed as a result of self-assembly of viral capsid proteins are widely used as a platform for antigen presentation in vaccine development. However, since the inclusion of a foreign peptide into the capsid protein can alter its spatial structure and interfere with VLP assembly, such insertions are usually limited to short peptides. In this study, we have demonstrated the potential of capsid protein (CP) of single-stranded RNA phage PQ465 to present long peptides using green fluorescent protein (GFP) as a model. GFP was genetically linked to either the N- or C-terminus of PQ465 CP. Hybrid proteins were expressed in Escherichia coli and Nicotiana benthamiana plants. Spherical virus-like particles (~35 nm according to transmission electron microscopy) were successfully formed by both N- and C-terminal fusions expressed in E. coli, and by plant-produced CP with GFP fused to the C-terminus. ELISA revealed that GFP in VLPs was accessible for specific antibodies suggesting that it is exposed on the surface of PQ465-GFP particles. VLPs carrying GFP were recognized by anti-CP antibodies with less efficiency than VLPs formed by empty CP, which indicates shielding of the CP core in PQ465-GFP particles. Therefore, PQ465 CP can be used as a chimeric VLP platform for the display of relatively large protein antigens, which can operate in bacterial and plant expression systems. Full article

The success of mRNA-based SARS-CoV-2 vaccines has been confirmed in both preclinical and clinical settings. However, the development of safe and efficient mRNA vaccine delivery platforms remains challenging. In this report, PBAE-G-B-SS-modified CaO₂ nanofibers and Au@OVA@Cu@Dendrobium huoshanense polysaccharides were employed to establish novel self-assembling polymeric micelles (CaO₂@Au@OVA@Cu@DHPs) capable of serving as both an adjuvant and a delivery system for mRNA vaccines. In vitro, CaO₂@Au@OVA@Cu@DHPs nanoparticles (NPs) were conducive to effective macrophage antigen uptake and efficient antigen processing. In vivo, P-CaO₂@Au@OVA@Cu@DHPs NP administration was associated with a reduction in the ovalbumin (OVA) release rate that was conducive to the sustained induction of long-term immunity and to the production of higher levels of different IgG subtypes, suggesting that these effects were attributable to enhanced antigen uptake by antigen-presenting cells. Overall, these present data highlight the promise of these P-CaO₂@Au@OVA@Cu@DHPs NPs as an effective and safe platform amenable to vaccine delivery through their ability to provide robust adjuvant activity and sustained antigen release capable of eliciting long-term immunological memory while potentiating humoral and cellular immune responses. Full article

Podocytopathy, characterized by proteinuria, contributes significantly to kidney diseases, with hypertension playing a key role in damaging podocytes and the glomerular filtration barrier (GFB). The lack of functional in vitro models, however, impedes research and treatment development for hypertensive podocytopathy. We established a novel constant pressure-driven podocyte-on-chip model, utilizing our previously developed dynamic staining self-assembly cell array chip (SACA chip) and 3D printing. This platform features a differentiated podocyte monolayer under controlled hydrostatic pressures, mimicking the epithelial side of the GFB. Using this platform, we investigated mechanical force-dependent permeability to three sizes of fluorescent dextran under varying hydrostatic pressures, comparing the results with a puromycin aminonucleoside (PAN)-induced injury model. We observed that external pressures induced size-dependent permeability changes and altered cell morphology. Higher pressures led to greater macromolecule infiltration, especially for larger dextran (70 kDa, 500 kDa). Mature podocytes exhibited immediate, pressure-dependent cytoskeleton rearrangements, with better recovery at lower pressures (20 mmHg) but irreversible injury at higher pressures (40, 60 mmHg). These morphological changes were also corroborated by dynamic mRNA expression of cytoskeleton-associated proteins, Synaptopodin and ACTN4. This platform offers a promising in vitro tool for investigating the pathomechanisms of hypertension-induced podocytopathy, performing on-chip studies of the GFB, and conducting potential drug screening. Full article

Using lipid nanocarriers to deliver the mRNA of a specific antigen to immune cells is a powerful innovative approach to rapidly develop new safe and effective vaccines. Understanding and optimizing the mixing process necessary for mRNA lipid nanoparticles (LNPs) is the focus of this review. The first objective is to review the fundamentals of microfluidic and turbulent fluid-mixing basics needed to understand the mixing process. The mRNA LNP self-assembly flash nanoprecipitation/self-assembly process will be discussed. Then, some important experimental nanoparticle studies which are the basis for the current understanding of microfluidic and turbulent mRNA LNP mixing process will be reviewed. Finally, the current commercially available LNP mixing technology will be summarized. There appears to be no universally “best” mixing process for formulating nanoparticles or mRNA LNPs. Both chaotic advection and turbulent flow microfluidic mixing devices, using the proper parameters for each device, will formulate similar mRNA LNP vaccines during development research. However, the low fluid output of microfluidic devices may not be practicable at higher fluid flow rates. Larger-scale turbulent mixing devices are more suitable for clinical-scale mRNA LNP production. Full article

Background/Objectives: SARS-CoV-2 vaccine candidates comprising the receptor binding domain (RBD) of the spike protein have been shown to confer protection against infection. Previous research evaluating vaccine candidates with SARS-CoV-2 RBD fused to ferritin (RBD-ferritin) and other scaffolds suggested that multimeric assemblies of RBD can enhance antigen presentation to improve the potency and breadth of immune responses. Though RBDs directly fused to a self-assembling scaffold can be delivered as messenger RNA (mRNA) formulated with lipid nanoparticles (LNPs), reports of SARS-CoV-2 vaccine candidates that combine these approaches remain scarce. Methods: Here, we designed RBD fused to AP205 or TIP60 self-assembling nanoparticles following a search of available structures focused on several scaffold properties. RBD-AP205 and RBD-TIP60 were tested for antigenicity following transfection and for immunogenicity and neutralization potency when delivered as mRNA in mice, with RBD-ferritin as a direct comparator. Results: All scaffolded RBD constructs were readily secreted to transfection supernatant and showed antigenicity in ELISA, though clear heterogeneity in assembly was observed. RBD-AP205 and RBD-TIP60 also exhibited robust antibody binding and neutralization titers in mice that were comparable to those elicited by RBD-ferritin or a full-length membrane-bound spike. Conclusions: These data suggest that AP205 and TIP60 can present RBD as effectively as ferritin and induce similar immune responses. By describing additional scaffolds for multimeric display that accommodate mRNA delivery platforms, this work can provide new tools for future vaccine design efforts. Full article

Fibrotic disorders pose a significant global health burden due to limited treatment options, creating an urgent need for novel therapeutic strategies. Amphiregulin (AREG), a low-affinity ligand for the epidermal growth factor receptor (EGFR), has emerged as a key mediator of fibrogenesis through dual signaling pathways. Unlike high-affinity EGFR ligands, AREG induces sustained signaling that activates downstream effectors and promotes the integrin-mediated activation of transforming growth factor (TGF)-β. This enables both canonical and non-canonical EGFR signaling pathways that contribute to fibrosis. Elevated AREG expression correlates with disease severity across multiple organs, including the lungs, kidneys, liver, and heart. The therapeutic targeting of AREG has shown promising antifibrotic and anticancer effects, suggesting a dual-benefit strategy. The increasing recognition of the shared mechanisms between fibrosis and cancer further supports the development of unified treatment approaches. The inhibition of AREG has been shown to sensitize fibrotic tumor microenvironments to chemotherapy, enhancing combination therapy efficacy. Targeted therapies, such as Self-Assembled-Micelle inhibitory RNA (SAMiRNA)-AREG, have demonstrated enhanced specificity and favorable safety profiles in preclinical studies and early clinical trials. Personalized treatment based on AREG expression may improve clinical outcomes, establishing AREG as a promising precision medicine target for both fibrotic and malignant diseases. This review aims to provide a comprehensive understanding of AREG biology and evaluate its therapeutic potential in fibrosis and cancer. Full article

The virus-first theory presents a model in which viral lineages emerged before cells. This proposal aims to give the theory greater relevance by offering a plausible evolutionary framework that explains both (i) the origin of viruses from prebiotic chemistry and (ii) how viruses contributed to the emergence of cells. Here, we propose that viruses should be understood as a distinct class of ribonucleoprotein (RNP) systems, some of which evolved directly from the RNP-world. In our model, simple progenotes produced capsid-like particles through the evolution of a single gene encoding a self-assembling peptide. This allowed the formation of icosahedral shells around RNA genomes, as observed today in certain viral families whose capsids consist of ~60 identical subunits derived from a single gene product. These early capsids enabled mobility and protection, representing key intermediates toward biological complexity. Over time, some of those populations acquired additional peptides and evolved more elaborate architectures. Finally, the incorporation of lipid-binding domains in those capsid-like peptides allowed the formation of proteolipidic membranes akin to those found in modern cells. This model provides a gradualistic and logically coherent evolutionary path from the RNP-world to the emergence of cellular life, emphasizing the foundational role of viruses in early evolution. Full article

The synthesis of novel biodegradable polymers as non-viral vectors remains one of the challenging tasks in the field of gene delivery. In this study, the synthesis of the polysaccharide-g-polypeptide copolymers, namely, hyaluronic acid-g-polylysine (HA-g-PLys), using a copper-free strain-promoted azide-alkyne cycloaddition reaction was proposed. For this purpose, hyaluronic acid was modified with dibenzocyclooctyne moieties, and poly-L-lysine with a terminal azido group was obtained using ring-opening polymerization of N-carboxyanhydride of the corresponding protected amino acid, initiated with the amino group azido-PEG₃-amine. Two HA-g-PLys samples with different degrees of grafting were synthesized, and the structures of all modified and synthesized polymers were confirmed using ¹H NMR and FTIR spectroscopy. The HA-g-PLys samples obtained were able to form nanoparticles in aqueous media due to self-assembly driven by electrostatic interactions. The binding of DNA and model siRNA by copolymers to form polyplexes was analyzed using ethidium bromide, agarose gel electrophoresis, and SybrGreen I assays. The hydrodynamic diameter of polyplexes was ˂300 nm (polydispersity index, PDI ˂ 0.3). The release of a model fluorescently-labeled oligonucleotide in the complex biological medium was significantly higher in the case of HA-g-PLys as compared to that in the case of PLys-based polyplexes. In addition, the cytotoxicity in normal and cancer cells, as well as the ability of HA-g-PLys to facilitate intracellular delivery of anti-GFP siRNA to NIH-3T3/GFP+ cells, were evaluated. Full article

Magnesium ions (Mg²⁺) play an important role in animal health, with their concentration in the bloodstream serving as a key indicator for hypomagnesemia diagnosis. In this study, a flexible hydrophobic paper-based microfluidic field-effect biosensor was developed for point-of-care Mg²⁺ detection, which integrated flexible hydrophobic paper, semiconducting single-walled carbon nanotubes (SWNTs) and a Mg²⁺-specific RNA-cleaving DNAzyme(RCD)-based DNA nanostructure. Flexible hydrophobic paper was synthesized by using cellulose paper and octadecyltrichlorosilane, improving mechanical strength and decreasing biological interference. To achieve high sensitivity, the Mg²⁺-specific RCD was functionalized with SWNTs, and then repeatedly self-assembled two different Y-shaped DNAs to construct a DNA nanostructure based on a similar DNA origami technique. This proposed biosensor exhibited a linear detection range from 1 μM to 1000 μM, with a detection limit of 0.57 μM, demonstrating its great stability, selectivity, and anti-interference performance. This innovative design offers promising potential for Mg²⁺ monitoring in real applications. Full article