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The remarkable success of messenger RNA (mRNA)-based vaccines has underscored their potential as a novel biotechnology platform for vaccine development and therapeutic protein delivery. However, the single-subunit RNA polymerase from bacteriophage T7 widely used for in vitro transcription is well known to generate double-stranded RNA (dsRNA) by-products that strongly stimulate the mammalian innate immune response. The dsRNA was reported to be originated from self-templated RNA extension or promoter-independent transcription. Here, we identified that the primary source of the full-length dsRNA during in vitro transcription is the DNA-terminus-initiated transcription by T7 RNA polymerase. Guanosines or cytosines at the end of DNA templates enhance the DNA-terminus-initiated transcription. Moreover, we found that aromatic residues located at position 47 in the C-helix lead to a significant reduction in the production of full-length dsRNA. As a result, the mRNA synthesized using the T7 RNA polymerase G47W mutant exhibits higher expression efficiency and lower immunogenicity compared to the mRNA produced using the wild-type T7 RNA polymerase.
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ARN Polimerasas Dirigidas por ADN , Transcripción Genética , Proteínas Virales , ARN Polimerasas Dirigidas por ADN/metabolismo , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/química , Proteínas Virales/metabolismo , Proteínas Virales/genética , Proteínas Virales/química , Mutación , ARN Bicatenario/genética , ARN Bicatenario/metabolismo , Animales , ADN/metabolismo , ADN/genética , ADN/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , Bacteriófago T7/genética , Bacteriófago T7/enzimología , RatonesRESUMEN
Peripheral nerve injury (PNI) poses a significant public health issue, often leading to muscle atrophy and persistent neuropathic pain, which can drastically impact the quality of life for patients. Electrical stimulation represents an effective and non-pharmacological treatment to promote nerve regeneration. Yet, the postoperative application of electrical stimulation remains a challenge. Here, we propose a fully biodegradable, self-powered nerve guidance conduit (NGC) based on dissolvable zinc-molybdenum batteries. The conduit can offer topographic guidance for nerve regeneration and deliver sustained electrical cues between both ends of a transected nerve stump, extending beyond the surgical window. Schwann cell proliferation and adenosine triphosphate (ATP) production are enhanced by the introduction of the zinc-molybdenum batteries. In rodent models with 10-mm sciatic nerve damage, the device effectively enhances nerve regeneration and motor function recovery. This study offers innovative strategies for creating biodegradable and electroactive devices that hold important promise to optimize therapeutic outcomes for nerve regeneration.
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Regeneración Nerviosa , Traumatismos de los Nervios Periféricos , Nervio Ciático , Zinc , Animales , Traumatismos de los Nervios Periféricos/terapia , Zinc/química , Nervio Ciático/fisiología , Nervio Ciático/lesiones , Ratas , Suministros de Energía Eléctrica , Molibdeno/química , Células de Schwann , Ratas Sprague-Dawley , Humanos , Regeneración Tisular Dirigida/instrumentación , Regeneración Tisular Dirigida/métodos , Técnicas Biosensibles , Implantes AbsorbiblesRESUMEN
The main challenge for immune checkpoint blockade (ICB) therapy lies in immunosuppressive tumor microenvironment (TME). Repolarizing M2-like tumor-associated macrophages (TAMs) into inflammatory M1 phenotype is a promising strategy for cancer immunotherapy. Here, this study shows that the tumor suppressive protein SHISA3 regulates the antitumor functions of TAMs. Local delivery of mRNA encoding Shisa3 enables cancer immunotherapy by reprogramming TAMs toward an antitumoral phenotype, thus enhancing the efficacy of programmed cell death 1 (PD-1) antibody. Enforced expression of Shisa3 in TAMs increases their phagocytosis and antigen presentation abilities and promotes CD8+ T cell-mediated antitumor immunity. The expression of SHISA3 is induced by damage/pathogen-associated molecular patterns (DAMPs/PAMPs) in macrophages via nuclear factor-κB (NF-κB) transcription factors. Reciprocally, SHISA3 forms a complex with heat shock protein family A member 8 (HSPA8) to activate NF-κB signaling thus maintaining M1 polarization of macrophages. Knockout Shisa3 largely abolishes the antitumor efficacy of combination immunotherapy with Toll-like receptor 4 (TLR4) agonist monophosphoryl lipid A (MPLA) and PD-1 antibody. It further found that higher expression of SHISA3 in antitumoral TAMs is associated with better overall survival in lung cancer patients. Taken together, the findings describe the role of SHISA3 in reprogramming TAMs that ameliorate cancer immunotherapy.
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The cyclic-oligonucleotide-based anti-phage signalling system (CBASS) is a type of innate prokaryotic immune system. Composed of a cyclic GMP-AMP synthase (cGAS) and CBASS-associated proteins, CBASS uses cyclic oligonucleotides to activate antiviral immunity. One major class of CBASS contains a homologue of eukaryotic ubiquitin-conjugating enzymes, which is either an E1-E2 fusion or a single E2. However, the functions of single E2s in CBASS remain elusive. Here, using biochemical, genetic, cryo-electron microscopy and mass spectrometry investigations, we discover that the E2 enzyme from Serratia marcescens regulates cGAS by imitating the ubiquitination cascade. This includes the processing of the cGAS C terminus, conjugation of cGAS to a cysteine residue, ligation of cGAS to a lysine residue, cleavage of the isopeptide bond and poly-cGASylation. The poly-cGASylation activates cGAS to produce cGAMP, which acts as an antiviral signal and leads to cell death. Thus, our findings reveal a unique regulatory role of E2 in CBASS.
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Nucleotidiltransferasas , Enzimas Ubiquitina-Conjugadoras , Ubiquitinación , Enzimas Ubiquitina-Conjugadoras/metabolismo , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/química , Nucleotidiltransferasas/metabolismo , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/química , Transducción de Señal , Nucleótidos Cíclicos/metabolismo , Bacteriófagos/genética , Bacteriófagos/enzimología , Ubiquitina/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Humanos , Microscopía por Crioelectrón , Inmunidad InnataRESUMEN
The underlying mechanisms of information processing for two basic motion types, rotation and flicker, are not fully understood. Rotational and flickering animations at four speeds - 7 frames per second (fps), 8 fps, 11 fps, and 12 fps, respectively - are presented as visual stimuli. The motion-onset visual evoked potentials (VEPs) and steady-state VEPs (SSVEP) elicited by these motion stimuli were compared between the rotation and flicker motion types at time windows of 0-500 ms and 1000-5000 ms post-stimulus, respectively. The standardized low-resolution electromagnetic tomography (sLORETA) source localization was investigated as well. Four motion speeds had no effect on the whole VEP waveform in either the rotation or the flicker groups. Significant differences in motion-onset VEPs and sLORETA source localization were found between the rotation and the flicker motion types at time windows of 200-500 ms post-stimulus. For the time windows of 1000-5000 ms post-stimulus, both the rotation and flicker groups all demonstrated the characteristics of SSVEP, with the peak spectral topographies showing at the four different frequencies, which correspond to the four motion speeds. Additionally, a higher power of spectral topography at each of the four motion speeds was found in the rotation relative to the flicker stimulation. The perceptual and cognitive processes are distinct for two types of motion: rotation and flicker. In terms of motion-onset VEPs and the characteristics of SSVEP, rotating visual stimulation is superior to flicker stimulation and may be more appropriate for clinical and engineering applications.
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Cognición , Potenciales Evocados Visuales , Rotación , Estimulación Luminosa/métodos , Examen Neurológico , Electroencefalografía/métodosRESUMEN
Peripheral nerve injuries (PNI) can lead to mitochondrial dysfunction and energy depletion within the affected microenvironment. The objective is to investigate the potential of transplanting mitochondria to reshape the neural regeneration microenvironment. High-purity functional mitochondria with an intact structure are extracted from human umbilical cord-derived mesenchymal stem cells (hUCMSCs) using the Dounce homogenization combined with ultracentrifugation. Results show that when hUCMSC-derived mitochondria (hUCMSC-Mitos) are cocultured with Schwann cells (SCs), they promote the proliferation, migration, and respiratory capacity of SCs. Acellular nerve allografts (ANAs) have shown promise in nerve regeneration, however, their therapeutic effect is not satisfactory enough. The incorporation of hUCMSC-Mitos within ANAs has the potential to remodel the regenerative microenvironment. This approach demonstrates satisfactory outcomes in terms of tissue regeneration and functional recovery. Particularly, the use of metabolomics and bioenergetic profiling is used for the first time to analyze the energy metabolism microenvironment after PNI. This remodeling occurs through the enhancement of the tricarboxylic acid cycle and the regulation of associated metabolites, resulting in increased energy synthesis. Overall, the hUCMSC-Mito-loaded ANAs exhibit high functionality to promote nerve regeneration, providing a novel regenerative strategy based on improving energy metabolism for neural repair.
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Células Madre Mesenquimatosas , Tejido Nervioso , Traumatismos de los Nervios Periféricos , Humanos , Nervio Ciático , Células de Schwann , Traumatismos de los Nervios Periféricos/terapia , Matriz Extracelular , Regeneración Nerviosa/fisiologíaRESUMEN
Spinal cord injury (SCI) is a serious traumatic disease with no effective treatment. This study aimed to explore the therapeutic effect of syringaresinol on SCI. First, the potential targets and associated signaling pathways of syringaresinol were predicted by bioinformatics analysis and molecular docking. Second, MTT was employed to evaluate cell proliferation rate, Western blot was performed to detect protein expression, RT-qPCR was conducted to detect mRNA expression levels, flow cytometry and 5-ethynyl-2'-deoxyuridine (EDU) staining were used to determine cell apoptosis, and immunofluorescence and immunohistochemistry were used to estimate the expression of RNA binding fox-1 homolog 3 and clipped caspase 3. Basso-Beattie-Bresnahan scores and inclined plate tests were conducted to analyze hindlimb locomotor function. Results showed that syringaresinol could inhibit the apoptosis of glutamate-treated SHSY5Y cells by upregulating the expression of ubiquitination factor E4B (UBE4B) and activating the AKT serine/threonine kinase (AKT) signaling pathway. This effect can be rescued by UBE4B knockdown or AKT pathway inhibition. Syringaresinol remarkably improved locomotor function and increased neuronal survival in SCI rats. Our results suggested that syringaresinol could promote locomotor functional recovery by reducing neuronal apoptosis by activating the UBE4B/AKT signaling pathway.
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Proteínas Proto-Oncogénicas c-akt , Traumatismos de la Médula Espinal , Ratas , Animales , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Médula Espinal/metabolismo , Ratas Sprague-Dawley , Simulación del Acoplamiento Molecular , Transducción de Señal , Traumatismos de la Médula Espinal/metabolismo , Apoptosis , Neuronas/metabolismo , Ubiquitinación , Serina/metabolismo , Recuperación de la FunciónRESUMEN
The Gabija complex is a prokaryotic antiviral system consisting of the GajA and GajB proteins. GajA was identified as a DNA nicking endonuclease but the functions of GajB and the complex remain unknown. Here, we show that synergy between GajA-mediated DNA cleavage and nucleotide hydrolysis by GajB initiates efficient abortive infection defense against virulent bacteriophages. The antiviral activity of GajA requires GajB, which senses DNA termini produced by GajA to hydrolyze (d)A/(d)GTP, depleting essential nucleotides. This ATPase activity of Gabija complex is only activated upon DNA binding. GajA binds to GajB to form stable complexes in vivo and in vitro. However, a functional Gabija complex requires a molecular ratio between GajB and GajA below 1:1, indicating stoichiometric regulation of the DNA/nucleotide processing complex. Thus, the Gabija system exhibits distinct and efficient antiviral defense through sequential sensing and activation of nucleotide depletion and DNA cleavage, causing a cascade suicide effect.
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Antivirales , Nucleótidos , Humanos , Nucleótidos/metabolismo , División del ADN , ADN/metabolismo , HidrólisisRESUMEN
Grapefruit peel polysaccharide has antioxidant, antitumor, hypoglycemic and other biological activities, and chemical modification can further improve the properties of the polysaccharide. Acetylation modification of polysaccharides has the advantages of simple operation, low cost and little pollution, and is widely used at present. Different degrees of acetylation modification have different effects on the properties of polysaccharides, so it is necessary to optimize the preparation technology of acetylated grapefruit peel polysaccharides. In this article, acetylated grapefruit peel polysaccharide was prepared by acetic anhydride method. With the degree of acetyl substitution as the evaluation index, combined with the analysis of sugar content and protein content in the polysaccharide before and after modification, the effects of three feeding ratios of 1:0.6, 1 : 1.2 and 1 : 1.8 (polysaccharide: acetic anhydride, mass/volume) on acetylation modification were explored through single factor experiments. The results showed that the optimum ratio of material to liquid for acetylation modification of grapefruit peel polysaccharide was 1:0.6. Under these conditions, the degree of substitution of acetylated grapefruit peel polysaccharide was 0.323, the sugar content was 59.50 % and the protein content was 1.038 %. The results provide some reference for the study of acetylated grapefruit peel polysaccharide.
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Citrus paradisi , Anhídridos Acéticos , Polisacáridos/farmacología , Polisacáridos/química , Azúcares , AcetilaciónRESUMEN
Given the advantages of high energy density and easy deployment, biodegradable primary battery systems remain as a promising power source to achieve bioresorbable electronic medicine, eliminating secondary surgeries for device retrieval. However, currently available biobatteries are constrained by operational lifetime, biocompatibility, and biodegradability, limiting potential therapeutic outcomes as temporary implants. Herein, we propose a fully biodegradable primary zinc-molybdenum (Zn-Mo) battery with a prolonged functional lifetime of up to 19 days and desirable energy capacity and output voltage compared with reported primary Zn biobatteries. The Zn-Mo battery system is shown to have excellent biocompatibility and biodegradability and can significantly promote Schwann cell proliferation and the axonal growth of dorsal root ganglia. The biodegradable battery module with 4 Zn-Mo cells in series using gelatin electrolyte accomplishes electrochemical generation of signaling molecules (nitric oxide, NO) that can modulate the behavior of the cellular network, with efficacy comparable with that of conventional power sources. This work sheds light on materials strategies and fabrication schemes to develop high-performance biodegradable primary batteries to achieve a fully bioresorbable electronic platform for innovative medical treatments that could be beneficial for health care.
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Suministros de Energía Eléctrica , Zinc , Electrónica , Gelatina , Proliferación Celular , Molibdeno , Óxido NítricoRESUMEN
Background: Although Andrographis paniculata (AP) exhibits various biological functions such as anticancer, anti-inflammatory, antimalarial, antimicrobial, antioxidant, cardioprotective and immunomodulatory, its role in estrogen deficiency-related osteoporosis remains unclear. Methods: Ovariectomy (OVX)-induced estrogen deficiency-related osteoporotic mouse models and sham mouse models were established using 8-week-old female C57BL/6J mice. Micro-computed tomography (µCT) scanning was performed to assess the skeletal phenotype. The differentiation potential of bone marrow mesenchymal stem cells (BMSCs) from the OVX and sham groups was assessed by osteogenic or adipogenic induction medium in vitro. To verify the effects of AP, alizarin red S (ARS) staining, alkaline phosphatase (ALP) staining and oil red O (ORO) staining, reverse transcription assay and quantitative real-time polymerase chain reaction were applied to detect the lineage differentiation ability of BMSCs. Results: µCT scanning showed that AP treatment attenuated the osteoporotic phenotype in OVX-induced estrogen deficiency-related osteoporotic mice. The results of ARS staining, ALP staining, ORO staining and quantitative real-time polymerase chain reaction indicated that BMSCs from OVX-induced osteoporotic mice displayed a significant reduction in osteogenic differentiation and an increase in adipogenic differentiation, which could be reversed by AP treatment. Conclusions: Our findings suggested that AP regulated the differentiation potential of BMSCs and ameliorated the development of estrogen deficiency-related osteoporosis, which might be an effective therapeutic method for estrogen deficiency-related osteoporosis.
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RNA research and applications are underpinned by in vitro transcription (IVT), but RNA impurities resulting from the enzymatic reagents severely impede downstream applications. To improve the stability and purity of synthesized RNA, we have characterized a novel single-subunit RNA polymerase (RNAP) encoded by the psychrophilic phage VSW-3 from a plateau lake. The VSW-3 RNAP is capable of carrying out in vitro RNA synthesis at low temperatures (4-25°C). Compared to routinely used T7 RNAP, VSW-3 RNAP provides a similar yield of transcripts but is insensitive to class II transcription terminators and synthesizes RNA without redundant 3'-cis extensions. More importantly, through dot-blot detection with the J2 monoclonal antibody, we found that the RNA products synthesized by VSW-3 RNAP contained a much lower amount of double-stranded RNA byproducts (dsRNA), which are produced by transcription from both directions and are significant in T7 RNAP IVT products. Taken together, the VSW-3 RNAP almost eliminates both terminal loop-back dsRNA and full-length dsRNA in IVT and thus is especially advantageous for producing RNA for in vivo use.
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Bacteriófagos , ARN Bicatenario , ARN Bicatenario/genética , Bacteriófagos/genética , Transcripción Genética , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Anticuerpos Monoclonales/genética , Bacteriófago T7/genética , Bacteriófago T7/metabolismoRESUMEN
In the treatment of patients with locally advanced prostate cancer (PCa), androgen deprivation therapy (ADT) significantly enhances the efficacy of radiotherapy by weakening the DNA damage response (DDR) pathway. Recently, several studies have suggested that androgen receptor splicing variants (ARvs) may mediate a compensatory DDR pathway when canonical androgen receptor (AR) signaling is inhibited, thus contributing to the resistance of some patients to this combinational treatment. However, the specific roles of certain ARvs as well as the detailed mechanism of how ARvs regulate the DDR are not well understood. Here, we demonstrated that AR splicing variant 7 (ARv7), which is the most abundant form of ARvs, significantly promotes the DDR of PCa cells under severe DNA damage independent of its parental AR by using the ionizing radiation (IR) and doxorubicin (Dox)-treated cell models. Mechanistically, ARv7 is sufficient to upregulate both the homologous recombination (HR) and the nonhomologous end joining (NHEJ) pathways by forming a positive regulatory loop with poly ADP-ribose polymerase 1 (PARP1). Moreover, the presence of ARv7 impairs the synergistic effect between AR antagonists and poly ADP-ribose polymerase (PARP) inhibitor, which has been recently shown to be a promising future treatment strategy for metastatic castration resistant prostate cancer (mCRPC). Combined, our data indicate that constitutively active ARv7 not only contributes to radioresistance after ADT, but may also serve as a potential predictive biomarker for assessing the efficacy of novel PARP inhibitor-based therapy in PCa.
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Neoplasias de la Próstata Resistentes a la Castración , Receptores Androgénicos , Adenosina Difosfato Ribosa , Empalme Alternativo , Antagonistas de Andrógenos , Línea Celular Tumoral , Daño del ADN , Humanos , Masculino , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Neoplasias de la Próstata Resistentes a la Castración/genética , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismoRESUMEN
Osteosarcoma (OS) is the commonest malignant bone tumor in adolescent patients, and patients face amputation, tumor metastasis, chemotherapy resistance, and even death. We investigated the potential connection between abnormal methylation differentially expressed genes and the survival rate of osteosarcoma patients. GSE36002 and GSE12865 datasets of GEO database were utilized for abnormal methylation differentially expressed genes, followed by function and pathway enrichment analyses, the protein-protein interaction network in the STRING database, and cluster analysis in the MCODE app of Cytoscape. The RNA-seq and clinical data from the TARGET-OS project of TCGA were used for univariate and least absolute shrinkage and selection operator (LASSO) Cox regression analyses to predict the risk genes of osteosarcoma. 1191 hypermethylation-downregulated genes might function through plasma membrane, negative regulation of transcription from the RNA polymerase II promoter, and pathways, including transcriptional misregulation in cancer. 127 hypomethylation-upregulated genes were enriched in proteolysis, negative regulation of the canonical Wnt signaling pathway, and metabolic signaling pathways. The univariate Cox analysis revealed 638 genes (P < 0.01), including 50 hypermethylation-downregulated genes and 4 hypomethylation-upregulated genes, subsequently based on LASSO Cox regression analysis for 54 aberrant methylation-driven genes, and three genes (COL13A1, MXI1, and TBRG1) were selected to construct the risk score model. The three genes (COL13A1, MXI1, and TBRG1) regulated by DNA methylation were identified to relate with the outcomes of OS patients, which might provide a new insight to the pathological mechanism of osteosarcoma.
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With the increasing urbanization in China, the mental health problems of migrant children have attracted widespread attention. From the perspective of social cognition, psychological capital, self-esteem, and other factors are closely related to the social and cultural adaptation of the group. In order to explore the relationship among the psychological capital, self-esteem and socio-cultural adaptations of migrant children, a total of 245 Chinese migrant children were investigated with the psychological capital scale, self-esteem scale, and socio-cultural adaptation scale. At the same time, 24 months group longitudinal intervention exercises was designed scientifically, the participants were provided with it continuously to enhance their psychological capital. According to the results, there were significant differences in t-test in experiment group and control group of migrant children. At the same time, there was a significant linear correlation between predictive variables' psychological capital and self-esteem and outcome variable's socio-cultural adaptation. In the next step of mediating effect analysis, psychological capital and self-esteem were adopted for predicting socio-cultural adaptation. The psychological capital could not only directly affect the social and cultural adaptation of migrant children, but also indirectly affect the degree of social and cultural adaptation of migrant children through changing self-esteem. The results showed that the self-esteem mediation model fitted well with the data. Furthermore, the mediation effect accounted for 17.3% in the direct effect. Longitudinal group intervention could improve the psychological capital and social and cultural adaptation of migrant children. In a word, the research was helpful to explore the influence mechanism of migrant children's psychological capital on social and cultural adaptation, and had certain practical value in preventing and reducing the crisis caused by the social and cultural adaptation of migrant children.
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The purpose of this research was to develop an efficient and non-destructive method for decolorizing of polysaccharides extracted from Isaria cicadae Miquel by magnetic chitosan microspheres (MCM). The optimum decolorization parameters were achieved by response surface methodology as follows: the MCM amount was 8.0%, the adsorption temperature was 48 °C, the adsorption time was 82 min and the pH was 7. Under these optimal conditions, the D r%, R r%, and K c were 90.31 ± 0.12%, 95.40 ± 0.11% and 19.66 ± 0.49, respectively. MCM adsorption of pigment molecules was a spontaneous and endothermic process that could be fitted with the pseudo-second-order equation and the Freundlich equation. Besides, the adsorption mechanism could be controlled by multiple-diffusion steps, including film diffusion and intra-particle diffusion. Furthermore, MCM is a recyclable material. Adsorption with MCM is a promising method to remove pigment molecules of polysaccharide, it may replace the traditional decolorization method.
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Fungal infections cause ~1.5 million deaths each year worldwide, and the mortality rate of disseminated candidiasis currently exceeds that of breast cancer and malaria. The major reasons for the high mortality of candidiasis are the limited number of antifungal drugs and the emergence of drug-resistant species. Therefore, a better understanding of antifungal host defense mechanisms is crucial for the development of effective preventive and therapeutic strategies. Here, we report that DOCK2 (dedicator of cytokinesis 2) promotes indispensable antifungal innate immune signaling and proinflammatory gene expression in macrophages. DOCK2-deficient macrophages exhibit decreased RAC GTPase (Rac family small GTPase) activation and ROS (reactive oxygen species) production, which in turn attenuates the killing of intracellular fungi and the activation of downstream signaling pathways. Mechanistically, after fungal stimulation, activated SYK (spleen-associated tyrosine kinase) phosphorylates DOCK2 at tyrosine 985 and 1405, which promotes the recruitment and activation of RAC GTPases and then increases ROS production and downstream signaling activation. Importantly, nanoparticle-mediated delivery of in vitro transcribed (IVT) Rac1 mRNA promotes the activity of Rac1 and helps to eliminate fungal infection in vivo. Taken together, this study not only identifies a critical role of DOCK2 in antifungal immunity via regulation of RAC GTPase activity but also provides proof of concept for the treatment of invasive fungal infections by using IVT mRNA.
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Candidiasis , Proteínas Activadoras de GTPasa , Factores de Intercambio de Guanina Nucleótido , Inmunidad Innata , Proteínas de Unión al GTP rac , Animales , Candidiasis/inmunología , Proteínas Activadoras de GTPasa/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , ARN Mensajero , Especies Reactivas de Oxígeno/metabolismo , Proteínas de Unión al GTP rac/metabolismoRESUMEN
In vitro preparation of mRNA is a key step for mRNA therapeutics. The widely used T7 RNA polymerase (RNAP) was shown to have many by-products during in vitro transcription (IVT) process, among which double-stranded RNA (dsRNA) is the major by-product to activate the intracellular immune response. Here, we describe the use of a new VSW-3 RNAP that reduced dsRNA production during IVT and the resulting mRNA exhibited low inflammatory stimulation in cells. Compared to T7 RNAP transcripts, these mRNA exhibited superior protein expression levels, with an average of 14-fold increase in Hela cells and 5-fold increase in mice. In addition, we found that VSW-3 RNAP did not require modified nucleotides to improve protein production of IVT products. Our data suggest that VSW-3 RNAP could be a useful tool for mRNA therapeutics.
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Transcription termination is one of the least understood processes of gene expression. As the prototype model for transcription studies, the single-subunit T7 RNA polymerase (RNAP) is known to respond to two types of termination signals, but the mechanism underlying such termination, especially the specific elements of the polymerase involved, is still unclear, due to a lack of knowledge with respect to the structure of the termination complex. Here we applied phage-assisted continuous evolution to obtain variants of T7 RNAP that can bypass the typical class I T7 terminator with stem-loop structure. Through in vivo selection and in vitro characterization, we discovered a single mutation (S43Y) that significantly decreased the termination efficiency of T7 RNAP at all transcription terminators tested. Coincidently, the S43Y mutation almost eliminates the RNA-dependent RNAP (RdRp) activity of T7 RNAP without impeding the major DNA-dependent RNAP (DdRp) activity of the enzyme. S43 is located in a hinge region and regulates the transformation between transcription initiation and elongation of T7 RNAP. Steady-state kinetics analysis and an RNA binding assay indicate that the S43Y mutation increases the transcription efficiency while weakening RNA binding of the enzyme. As an enzymatic reagent for in vitro transcription, the T7 RNAP S43Y mutant reduces the undesired termination in run-off RNA synthesis and produces RNA with higher terminal homogeneity.
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Bacteriófago T7/enzimología , ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/metabolismo , Mutación , ARN Polimerasa Dependiente del ARN/metabolismo , Terminación de la Transcripción Genética , Transcripción Genética , Proteínas Virales/metabolismo , ARN Polimerasas Dirigidas por ADN/genética , Escherichia coli/genética , Escherichia coli/virología , Regiones Promotoras Genéticas , ARN Polimerasa Dependiente del ARN/genética , Proteínas Virales/genéticaRESUMEN
The arms race between bacteria and phages has led to the development of exquisite bacterial defense systems including a number of uncharacterized systems distinct from the well-known restriction-modification and CRISPR/Cas systems. Here, we report functional analyses of the GajA protein from the newly predicted Gabija system. The GajA protein is revealed as a sequence-specific DNA nicking endonuclease unique in that its activity is strictly regulated by nucleotide concentration. NTP and dNTP at physiological concentrations can fully inhibit the robust DNA cleavage activity of GajA. Interestingly, the nucleotide inhibition is mediated by an ATPase-like domain, which usually hydrolyzes ATP to stimulate the DNA cleavage when associated with other nucleases. These features suggest a mechanism of the Gabija defense in which an endonuclease activity is suppressed under normal conditions, while it is activated by the depletion of NTP and dNTP upon the replication and transcription of invading phages. This work highlights a concise strategy to utilize a DNA nicking endonuclease for phage resistance via nucleotide regulation.