Search Results (103)

Circular RNAs (circRNAs) have emerged as a transformative class of RNA therapeutics, distinguished by their closed-loop structure conferring nuclease resistance, reduced immunogenicity, and sustained translational activity. While challenges in pharmacokinetic control and manufacturing standardization require resolution, emerging synergies between computational design tools and modular delivery platforms are accelerating clinical translation. In this review, we synthesize recent advances in circRNA therapeutics, with a focused analysis of their stability and immunogenic properties in vaccine and drug development. Notably, key synthesis strategies, delivery platforms, and AI-driven optimization methods enabling scalable production are discussed. Moreover, we summarize preclinical and emerging clinical studies that underscore the potential of circRNA in vaccine development and protein replacement therapies. As both a promising expression vehicle and programmable regulatory molecule, circRNA represents a versatile platform poised to advance next-generation biologics and precision medicine. Full article

This review summarizes the research progress in the co-delivery of natural products (NPs) and small RNAs in cancer therapy. NPs such as paclitaxel, camptothecin, and curcumin possess multi-target antitumor effects, but their applications are limited by drug resistance and non-specific distribution. Small RNAs can achieve precise antitumor effects through gene regulation, yet their delivery efficiency is low, and they are prone to degradation by nucleases. Nanomaterial-based drug delivery systems (nano-DDSs) provide an efficient platform for the co-delivery of both, which can enhance the targeting of their delivery and improve the synergistic antitumor effects simultaneously. The mechanisms of the antitumor action of natural compounds and small RNAs, the design and application of nanocarriers, and the latest research progress in co-delivery systems are introduced in detail in this paper. The application prospects of the co-delivery of natural compounds and small RNAs in cancer therapy are also discussed. Full article

Nuclease P1 (NP1) is critical for producing 5′-nucleotides, which are essential flavor enhancers in the food industry. Traditional use of free NP1 is hampered by poor reusability, high costs, and potential residual enzyme protein contamination, compromising product quality. This study introduces a novel immobilization technique using a cross-linking approach with food-grade ion exchange resin AER1 to create NP1@AER1-GA. This method achieves an enzyme activity of 51,015 U/g, with a notable immobilization yield of 67.7%. The immobilized NP1@AER1-GA exhibits significantly enhanced stability and catalytic efficiency while ensuring industrial compatibility and maintaining stringent safety standards. Under optimized conditions, NP1@AER1-GA demonstrates exceptional performance in 5′-nucleotide production, retaining approximately 85% of its initial activity after 10 cycles of reuse. This breakthrough not only boosts the efficiency and sustainability of nucleotide synthesis but also offers a scalable solution for industrial applications, promoting sustainable manufacturing practices within the food industry. By addressing key challenges associated with traditional enzymatic methods, this immobilization technique sets a new benchmark for biocatalyst design in the food processing industry. Full article

The association between microRNAs and various diseases, especially cancer, has been established in recent years, indicating that miRNAs can potentially serve as biomarkers for these diseases. Determining miRNA concentrations in biological samples is crucial for disease diagnosis. Nevertheless, the stem-loop reverse transcription quantitative PCR method, the gold standard for detecting miRNA, has great challenges in terms of high costs and enzyme limitations when applied to clinical biological samples. In this study, an isothermal signal amplification method based on a duplex-specific nuclease (DSN) enzyme-driven DNA walker and an improved catalytic hairpin assembly (CHA) was designed for miRNA detection. First, biotin–triethylene glycol-modified trigger-releasable DNA probes were conjugated to the streptavidin-coated magnetic beads for recognizing the target miRNA. The DSN enzyme specifically hydrolyzes DNA strands when the DNA probe hybridizes with the targeted miRNA. This recycling process converts the input miRNA into short trigger fragments (catalysts). Finally, three hairpins of improved CHA are driven by this catalyst, resulting in the three-armed CHA products and a fluorescence signal as the output. This dual-cycle biosensor shows a good linear relationship in the detection of miR-21 and miR-141 over the final concentration range of 250 fM to 50 nM, presenting an excellent limit of detection (2.95 amol). This system was used to detect miR-21 and miR-141 in MCF-7 and 22RV1 cells, as well as in 1% human serum. This system can be used to evaluate the expression levels of miRNAs in different biological matrices for the clinical diagnosis and prognosis of different cancers. Full article

Synthetic cytosine-phosphate-guanine oligodeoxynucleotides (CpG ODNs) are promising candidates for vaccine adjuvants, because they activate immune responses through the Toll-like receptor 9 (TLR9) pathway. However, unmodified CpG ODNs are quickly degraded by serum nucleases, and their negative charge hinders cellular uptake, limiting their clinical application. Our group previously reported that guanine-quadruplex (G4)-forming CpG ODNs exhibit enhanced stability and cellular uptake. G4 structures can form in parallel, anti-parallel, or hybrid topologies, depending on strand orientation, but the effects of these topologies on CpG ODNs have not yet been explored. In this study, we designed three distinct G4 topologies as scaffolds for CpG ODNs. Among the three topology, the parallel G4 CpG ODN demonstrated the highest serum stability and cellular uptake, resulting in the strongest immune response from macrophage cells. Additionally, we investigated the binding affinities of the different G4 topologies to macrophage scavenger receptor-1 and TLR9, both of which are key to immune activation. These findings provide valuable insights into the development of CpG ODN-based vaccine adjuvants. Full article

The design of controllable and precise RNA-targeted CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) systems is an important problem of modern molecular biology and genetic technology. Herein, we have designed a series of photocleavable guide CRISPR RNAs (crRNA) and their 2′-modified (2′-fluoro and locked nucleic acid) analogs containing one or two 1-(2-nitrophenyl)-1,2-ethanediol photolabile linkers (PL). We have demonstrated that these crRNAs can be destroyed by relatively mild UVA irradiation with the rate constants 0.24–0.77 min⁻¹ and that the photocleavage markedly slows down the action of Cas9 nuclease in the model in vitro system. Two PLs provide more rapid crRNA destruction than a single linker. PLs in the crRNA structure improve the specificity of DNA cleavage by Cas9 nuclease for the fully complementary target. The application of photocleavable crRNA in CRISPR/Cas9 genome editing permits the system to be switched off in a spatiotemporally controlled manner, thus alleviating its off-target effects. Full article

Fluorescent biosensor, which has the characteristics of high sensitivity, specificity, and low cost, can be directly detected in physiological fluids such as blood and serum. Therefore, the development of fluorescence sensor platforms for miRNA detection has a positive effect on the prevention and treatment of various diseases. In this paper, miR-34a was selected as a biological indicator of Alzheimer’s disease (AD). We designed a circular single-stranded DNA (CSSD) biosensor, which uses two unmodified single-stranded DNA (ssDNA) with complementary ends, DNAa and DNAb, to form CSSD by DNA sequence pairing to improve thermal stability and achieve signal amplification. At the same time, CSSD can react with miR-34a, and then the DNA of the DNA–RNA chain is hydrolyzed by duplex-specific nuclease (DSN enzyme). Finally, miR-34a is released to partake in the subsequent step, thus realizing cycle amplification. By evaluating the change in fluorescence signal under the optimized conditions, we discovered that this approach exhibits impressive sensitivity, with a detection threshold reaching as low as 0.36 nM. This surpasses the performance of numerous preceding miRNA detection biosensors. Furthermore, the system displays excellent detection capabilities even in intricate settings like serum, showcasing a strong ability to differentiate and choose effectively. In summary, this is a signal-off fluorescent biosensor, which realizes the purpose of double amplification of biosensor signal by using CSSD and enzyme assistance so that it can be used as a valuable tool for early diagnosis of diseases. Full article

Cationic vaccines of nanometric sizes can directly perform the delivery of antigen(s) and immunomodulator(s) to dendritic cells in the lymph nodes. The positively charged nanovaccines are taken up by antigen-presenting cells (APCs) of the lymphatic system often originating the cellular immunological defense required to fight intracellular microbial infections and the proliferation of cancers. Cationic molecules imparting the positive charges to nanovaccines exhibit a dose-dependent toxicity which needs to be systematically addressed. Against the coronavirus, mRNA cationic nanovaccines evolved rapidly. Nowadays cationic nanovaccines have been formulated against several infections with the advantage of cationic compounds granting protection of nucleic acids in vivo against biodegradation by nucleases. Up to the threshold concentration of cationic molecules for nanovaccine delivery, cationic nanovaccines perform well eliciting the desired Th 1 improved immune response in the absence of cytotoxicity. A second strategy in the literature involves dilution of cationic components in biocompatible polymeric matrixes. Polymeric nanoparticles incorporating cationic molecules at reduced concentrations for the cationic component often result in an absence of toxic effects. The progress in vaccinology against cancer involves in situ designs for cationic nanovaccines. The lysis of transformed cancer cells releases several tumoral antigens, which in the presence of cationic nanoadjuvants can be systemically presented for the prevention of metastatic cancer. In addition, these local cationic nanovaccines allow immunotherapeutic tumor treatment. Full article

Overexpression and aberrant activation of signal transducer and activator of transcription 3 (STAT3) contribute to tumorigenesis, drug resistance, and tumor-immune evasion, making it a potential cancer therapeutic target. BP1003 is a neutral liposome incorporated with a nuclease-resistant P-ethoxy antisense oligodeoxynucleotide (ASO) targeting the STAT3 mRNA. Its unique design enhances BP1003 stability, cellular uptake, and target affinity. BP1003 efficiently reduces STAT3 expression and enhances the sensitivity of breast cancer cells (HER2⁺, triple negative) and ovarian cancer cells (late stage, invasive ovarian cancer) to paclitaxel and 5-fluorouracil (5-FU) in both 2D and 3D cell cultures. Similarly, ex vivo and in vivo patient-derived models of pancreatic ductal adenocarcinoma (PDAC) show reduced tissue viability and tumor volume with BP1003 and gemcitabine combination treatments. In addition to directly affecting tumor cells, BP1003 can modulate the tumor microenvironment. Unlike M1 differentiation, monocyte differentiation into anti-inflammatory M2 macrophages is suppressed by BP1003, indicating its potential contribution to immunotherapy. The broad anti-tumor effect of BP1003 in numerous preclinical solid tumor models, such as breast, ovarian, and pancreatic cancer models shown in this work, makes it a promising cancer therapeutic. Full article

Synthetic antisense oligonucleotides (ASOs) are emerging as an attractive platform to treat various diseases. By specifically binding to a target mRNA transcript through Watson–Crick base pairing, ASOs can alter gene expression in a desirable fashion to either rescue loss of function or downregulate pathogenic protein expression. To be clinically relevant, ASOs are generally synthesized using modified analogs to enhance resistance to enzymatic degradation and pharmacokinetic and dynamic properties. Phosphorothioate (PS) belongs to the first generation of modified analogs and has played a vital role in the majority of approved ASO drugs, mainly based on the RNase H mechanism. In contrast to RNase H-dependent ASOs that bind and cleave target mature mRNA, splice-switching oligonucleotides (SSOs) mainly bind and alter precursor mRNA splicing in the cell nucleus. To date, only one approved SSO (Nusinersen) possesses a PS backbone. Typically, the synthesis of PS oligonucleotides generates two types of stereoisomers that could potentially impact the ASO’s pharmaco-properties. This can be limited by introducing the naturally occurring phosphodiester (PO) linkage to the ASO sequence. In this study, towards fine-tuning the current strategy in designing SSOs, we reported the design, synthesis, and evaluation of several stereo-random SSOs on a mixed PO–PS backbone for their binding affinity, biological potency, and nuclease stability. Based on the results, we propose that a combination of PO and PS linkages could represent a promising approach toward limiting undesirable stereoisomers while not largely compromising the efficacy of SSOs. Full article

Salmonella is a common foodborne pathogen that can cause food poisoning, posing a serious threat to human health. Therefore, quickly, sensitively, and accurately detecting Salmonella is crucial to ensuring food safety. For the Salmonella hilA gene, we designed Recombinase-aided amplification (RAA) primers and dsDNA-specific nuclease (DNase) probes. The ideal primer and probe combination was found when conditions were optimized. Under UV light, a visual Salmonella detection technique (RAA-dsDNase) was developed. Additionally, the RAA-dsDNase was modified to further reduce pollution hazards and simplify operations. One-pot RAA-dsDNase-UV or one-pot RAA-dsDNase-LFD was developed as a Salmonella detection method, using UV or a lateral flow dipstick (LFD) for result observation. Among them, one-pot RAA-dsDNase and one-pot RAA-dsDNase-LFD had detection times of 50 min and 60 min, respectively, for detecting Salmonella genomic DNA. One-pot RAA-dsDNase-UV had a detection limit of 10¹ copies/μL and 10¹ CFU/mL, while one-pot RAA-dsDNase-LFD had a sensitivity of 10² copies/μL and 10² CFU/mL. One-pot RAA-dsDNase-UV and one-pot RAA-dsDNase-LFD assays may identify 17 specific Salmonella serovars witho ut causing a cross-reaction with the remaining 8 bacteria, which include E. coli. Furthermore, Salmonella in tissue and milk samples has been reliably detected using both approaches. Overall, the detection method developed in this study can quickly, sensitively, and accurately detect Salmonella, and it is expected to become an important detection tool for the prevention and control of Salmonella in the future. Full article

The monogenetic disease epidermolysis bullosa (EB) is characterised by the formation of extended blisters and lesions on the patient’s skin upon minimal mechanical stress. Causal for this severe condition are genetic mutations in genes, leading to the functional impairment, reduction, or absence of the encoded protein within the skin’s basement membrane zone connecting the epidermis to the underlying dermis. The major burden of affected families justifies the development of long-lasting and curative therapies operating at the genomic level. The landscape of causal therapies for EB is steadily expanding due to recent breakthroughs in the gene therapy field, providing promising outcomes for patients suffering from this severe disease. Currently, two gene therapeutic approaches show promise for EB. The clinically more advanced gene replacement strategy was successfully applied in severe EB forms, leading to a ground-breaking in vivo gene therapy product named beremagene geperpavec (B-VEC) recently approved from the US Food and Drug Administration (FDA). In addition, the continuous innovations in both designer nucleases and gene editing technologies enable the efficient and potentially safe repair of mutations in EB in a potentially permanent manner, inspiring researchers in the field to define and reach new milestones in the therapy of EB. Full article

Over the past decade, genetic engineering has witnessed a revolution with the emergence of a relatively new genetic editing tool based on RNA-guided nucleases: the CRISPR/Cas9 system. Since the first report in 1987 and characterization in 2007 as a bacterial defense mechanism, this system has garnered immense interest and research attention. CRISPR systems provide immunity to bacteria against invading genetic material; however, with specific modifications in sequence and structure, it becomes a precise editing system capable of modifying the genomes of a wide range of organisms. The refinement of these modifications encompasses diverse approaches, including the development of more accurate nucleases, understanding of the cellular context and epigenetic conditions, and the re-designing guide RNAs (gRNAs). Considering the critical importance of the correct performance of CRISPR/Cas9 systems, our scope will emphasize the latter approach. Hence, we present an overview of the past and the most recent guide RNA web-based design tools, highlighting the evolution of their computational architecture and gRNA characteristics over the years. Our study explains computational approaches that use machine learning techniques, neural networks, and gRNA/target interactions data to enable predictions and classifications. This review could open the door to a dynamic community that uses up-to-date algorithms to optimize and create promising gRNAs, suitable for modern CRISPR/Cas9 engineering. Full article

Thrombin-binding aptamer (TBA) is one of the best-known G-quadruplex (G4)-forming aptamers. By adopting its peculiar chair-like G4 structure, TBA can efficiently bind to thrombin, thus producing an anticoagulant effect. The major limit to its therapeutic application is represented by its poor thermal and biological resistance. Therefore, numerous research studies have been focused on the design of TBA analogues with chemical modifications to improve its pharmacokinetic and pharmacodynamic properties. To maintain the functional recognition to protein surface on which TBA anticoagulant activity depends, it is essential to preserve the canonical antiparallel topology of the TBA quadruplex core. In this paper, we have designed three TBA variants with modified G-tetrads to evaluate the effects of nucleobase and sugar moiety chemical modifications on biological properties of TBA, preserving its chair-like G-quadruplex structure. All derivatives contain 8-bromo-2′-deoxyguanosine (G^Br) in syn positions, while in the anti-positions, locked nucleic acid guanosine (G^LNA) in the analogue TBABL, 2’-O-methylguanosine (G^OMe) in TBABM, and 2’-F-riboguanosine (G^F) in TBABF is present. CD (Circular Dichroism), CD melting, 1H-NMR (Nuclear Magnetic Resonance), and non-denaturing PAGE (Polyacrylamide Gel Electrophoresis), nuclease stability, prothrombin time (PT) and fibrinogen-clotting assays have been performed to investigate the structural and biological properties of these TBA analogues. The most interesting results have been obtained with TBABF, which revealed extraordinary thermal stability (T_m approximately 40 °C higher than that of TBA), anticoagulant activity almost doubled compared to the original aptamer, and, above all, a never-observed resistance to nucleases, as 50% of its G4 species was still present in 50% FBS at 24 h. These data indicate TBABF as one of the best TBA analogue ever designed and investigated, to the best of our knowledge, overcoming the main limitations to therapeutic applications of this aptamer. Full article

Tuberculosis, caused by Mycobacterium tuberculosis, is a lethal infectious disease of significant public health concern. The rise of multidrug-resistant and drug-tolerant strains has necessitated novel approaches to combat the disease. Toxin–antitoxin (TA) systems, key players in bacterial adaptive responses, are prevalent in prokaryotic genomes and have been linked to tuberculosis. The genome of M. tuberculosis strains harbors an unusually high number of TA systems, prompting questions about their biological roles. The VapBC family, a representative type II TA system, is characterized by the VapC toxin, featuring a PilT N-terminal domain with nuclease activity. Its counterpart, VapB, functions as an antitoxin, inhibiting VapC’s activity. Additionally, we explore peptide mimics designed to replicate protein helical structures in this review. Investigating these synthetic peptides offers fresh insights into molecular interactions, potentially leading to therapeutic applications. These synthetic peptides show promise as versatile tools for modulating cellular processes and protein–protein interactions. We examine the rational design strategies employed to mimic helical motifs, their biophysical properties, and potential applications in drug development and bioengineering. This review aims to provide an in-depth understanding of TA systems by introducing known complex structures, with a focus on both structural aspects and functional and molecular details associated with each system. Full article