RESUMEN
RIG-I is a cytosolic receptor of viral RNA essential for the immune response to numerous RNA viruses. Accordingly, RIG-I must sensitively detect viral RNA yet tolerate abundant self-RNA species. The basic binding cleft and an aromatic amino acid of the RIG-I C-terminal domain(CTD) mediate high-affinity recognition of 5'triphosphorylated and 5'base-paired RNA(dsRNA). Here, we found that, while 5'unmodified hydroxyl(OH)-dsRNA demonstrated residual activation potential, 5'-monophosphate(5'p)-termini, present on most cellular RNAs, prevented RIG-I activation. Determination of CTD/dsRNA co-crystal structures and mutant activation studies revealed that the evolutionarily conserved I875 within the CTD sterically inhibits 5'p-dsRNA binding. RIG-I(I875A) was activated by both synthetic 5'p-dsRNA and endogenous long dsRNA within the polyA-rich fraction of total cellular RNA. RIG-I(I875A) specifically interacted with long, polyA-bearing, mitochondrial(mt) RNA, and depletion of mtRNA from total RNA abolished its activation. Altogether, our study demonstrates that avoidance of 5'p-RNA recognition is crucial to prevent mtRNA-triggered RIG-I-mediated autoinflammation.
Asunto(s)
Proteína 58 DEAD Box , Isoleucina , Receptores Inmunológicos , Proteína 58 DEAD Box/química , Proteína 58 DEAD Box/genética , Proteína 58 DEAD Box/metabolismo , Tolerancia Inmunológica , Isoleucina/genética , ARN Bicatenario/genética , ARN Mitocondrial/genética , ARN Mitocondrial/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , Humanos , Receptores Inmunológicos/química , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismoRESUMEN
The presence of the cap structure on the 5'-end of in vitro-transcribed (IVT) mRNA determines its translation and stability, underpinning its use in therapeutics. Both enzymatic and co-transcriptional capping may lead to incomplete positioning of the cap on newly synthesized RNA molecules. IVT mRNAs are rapidly emerging as novel biologics, including recent vaccines against COVID-19 and vaccine candidates against other infectious diseases, as well as for cancer immunotherapies and protein replacement therapies. Quality control methods necessary for the preclinical and clinical stages of development of these therapeutics are under ongoing development. Here, we described a method to assess the presence of the cap structure of IVT mRNAs. We designed a set of ribozyme assays to specifically cleave IVT mRNAs at a unique position and release 5'-end capped or uncapped cleavage products up to 30 nt long. We purified these products using silica-based columns and visualized/quantified them using denaturing polyacrylamide gel electrophoresis (PAGE) or liquid chromatography and mass spectrometry (LC-MS). Using this technology, we determined the capping efficiencies of IVT mRNAs with different features, which include: Different cap structures, diverse 5' untranslated regions, different nucleoside modifications, and diverse lengths. Taken together, the ribozyme cleavage assays we developed are fast and reliable for the analysis of capping efficiency for research and development purposes, as well as a general quality control for mRNA-based therapeutics.
RESUMEN
While viral replication processes are largely understood, comparably little is known on cellular mechanisms degrading viral RNA. Some viral RNAs bear a 5'-triphosphate (PPP-) group that impairs degradation by the canonical 5'-3' degradation pathway. Here we show that the Nudix hydrolase 2 (NUDT2) trims viral PPP-RNA into monophosphorylated (P)-RNA, which serves as a substrate for the 5'-3' exonuclease XRN1. NUDT2 removes 5'-phosphates from PPP-RNA in an RNA sequence- and overhang-independent manner and its ablation in cells increases growth of PPP-RNA viruses, suggesting an involvement in antiviral immunity. NUDT2 is highly homologous to bacterial RNA pyrophosphatase H (RppH), a protein involved in the metabolism of bacterial mRNA, which is 5'-tri- or diphosphorylated. Our results show a conserved function between bacterial RppH and mammalian NUDT2, indicating that the function may have adapted from a protein responsible for RNA turnover in bacteria into a protein involved in the immune defense in mammals.
Asunto(s)
Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/metabolismo , Estabilidad del ARN , ARN Viral/metabolismo , Adaptación Fisiológica , Animales , Antivirales , Células de la Médula Ósea , Sistemas CRISPR-Cas , Exonucleasas , Exorribonucleasas , Femenino , Técnicas de Inactivación de Genes , Células HEK293 , Células HeLa , Humanos , Inmunidad Innata , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Asociadas a Microtúbulos , Polifosfatos , ARN Bacteriano , ARN Mensajero , Replicación ViralRESUMEN
Influenza A virus infection causes substantial morbidity and mortality in seasonal epidemic outbreaks, and more efficient treatments are urgently needed. Innate immune sensing of viral nucleic acids stimulates antiviral immunity, including cell-autonomous antiviral defense mechanisms that restrict viral replication. RNA oligonucleotide ligands that potently activate the cytoplasmic helicase retinoic-acid-inducible gene I (RIG-I) are promising candidates for the development of new antiviral therapies. Here, we demonstrate in an Mx1-expressing mouse model of influenza A virus infection that a single intravenous injection of low-dose RIG-I ligand 5'-triphosphate RNA (3pRNA) completely protected mice from a lethal challenge with influenza A virus for at least 7 days. Furthermore, systemic administration of 3pRNA rescued mice with pre-established fulminant influenza infection and prevented the fatal effects of a streptococcal superinfection. Type I interferon, but not interferon-λ, was required for the therapeutic effect. Our results suggest that the use of RIG-I activating oligonucleotide ligands has the clinical potential to confine influenza epidemics when a strain-specific vaccine is not yet available and to reduce lethality of influenza in severely infected patients.
Asunto(s)
Infecciones Bacterianas , Virus de la Influenza A , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Infecciones por Orthomyxoviridae/metabolismo , Infecciones por Orthomyxoviridae/virología , Sobreinfección , Animales , Quimiocina CXCL10/metabolismo , Virus de la Influenza A/inmunología , Interferón Tipo I/metabolismo , Ligandos , Pulmón/inmunología , Pulmón/metabolismo , Pulmón/patología , Pulmón/virología , Proteínas de la Membrana/agonistas , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/agonistas , Oligonucleótidos/administración & dosificación , Oligonucleótidos/genética , Infecciones por Orthomyxoviridae/mortalidad , Sustancias Protectoras/administración & dosificación , ARN/administración & dosificación , ARN/genética , Receptores de Superficie Celular , Análisis de Supervivencia , Receptores Toll-Like/metabolismoRESUMEN
A hypoxic tumor microenvironment is linked to poor prognosis. It promotes tumor cell dedifferentiation and metastasis and desensitizes tumor cells to type-I IFN, chemotherapy, and irradiation. The cytoplasmic immunoreceptor retinoic acid-inducible gene-I (RIG-I) is ubiquitously expressed in tumor cells and upon activation by 5'-triphosphate RNA (3pRNA) drives the induction of type I IFN and immunogenic cell death. Here, we analyzed the impact of hypoxia on the expression of RIG-I in various human and murine tumor and nonmalignant cell types and further investigated its function in hypoxic murine melanoma. 3pRNA-inducible RIG-I-expression was reduced in hypoxic melanoma cells compared with normoxic controls, a phenomenon that depended on the hypoxia-associated transcription factor HIF1α. Still, RIG-I functionality was conserved in hypoxic melanoma cells, whereas responsiveness to recombinant type-I IFN was abolished, due to hypoxia-induced loss of type I IFN receptor expression. Likewise, RIG-I activation in hypoxic melanoma cells, but not exposure to recombinant IFNα, provoked melanocyte antigen-specific CD8+ T-cell and NK-cell attack. Scavenging of hypoxia-induced reactive oxygen species by vitamin C restored the inducible expression of RIG-I under hypoxia in vitro, boosted in vitro anti-melanoma NK- and CD8+ T-cell attack, and augmented 3pRNA antitumor efficacy in vivo These results demonstrate that RIG-I remains operational under hypoxia and that RIG-I function is largely insensitive to lower cell surface expression of the IFNα receptor. RIG-I function could be fortified under hypoxia by the combined use of 3pRNA with antioxidants. Cancer Immunol Res; 5(6); 455-67. ©2017 AACR.
Asunto(s)
Hipoxia/metabolismo , Tolerancia Inmunológica , Melanoma/metabolismo , Receptores de Ácido Retinoico/metabolismo , Animales , Antioxidantes/farmacología , Ácido Ascórbico/farmacología , Línea Celular , Línea Celular Tumoral , Femenino , Técnicas de Inactivación de Genes , Humanos , Ratones Endogámicos C57BL , ARN/farmacología , Especies Reactivas de Oxígeno/metabolismo , Receptores de Ácido Retinoico/genética , Bazo/citologíaRESUMEN
Cytosolic DNA that emerges during infection with a retrovirus or DNA virus triggers antiviral type I interferon responses. So far, only double-stranded DNA (dsDNA) over 40 base pairs (bp) in length has been considered immunostimulatory. Here we found that unpaired DNA nucleotides flanking short base-paired DNA stretches, as in stem-loop structures of single-stranded DNA (ssDNA) derived from human immunodeficiency virus type 1 (HIV-1), activated the type I interferon-inducing DNA sensor cGAS in a sequence-dependent manner. DNA structures containing unpaired guanosines flanking short (12- to 20-bp) dsDNA (Y-form DNA) were highly stimulatory and specifically enhanced the enzymatic activity of cGAS. Furthermore, we found that primary HIV-1 reverse transcripts represented the predominant viral cytosolic DNA species during early infection of macrophages and that these ssDNAs were highly immunostimulatory. Collectively, our study identifies unpaired guanosines in Y-form DNA as a highly active, minimal cGAS recognition motif that enables detection of HIV-1 ssDNA.
Asunto(s)
ADN Complementario/química , ADN Viral/química , ADN Viral/inmunología , VIH-1/genética , VIH-1/inmunología , Interferón-alfa/inmunología , Nucleotidiltransferasas/genética , Animales , Línea Celular , Células Cultivadas , ADN Complementario/genética , ADN Complementario/inmunología , ADN Viral/genética , Células HEK293 , Humanos , Inmunización , RatonesRESUMEN
The cytosolic helicase retinoic acid-inducible gene-I (RIG-I) initiates immune responses to most RNA viruses by detecting viral 5'-triphosphorylated RNA (pppRNA). Although endogenous mRNA is also 5'-triphosphorylated, backbone modifications and the 5'-ppp-linked methylguanosine ((m7)G) cap prevent immunorecognition. Here we show that the methylation status of endogenous capped mRNA at the 5'-terminal nucleotide (N1) was crucial to prevent RIG-I activation. Moreover, we identified a single conserved amino acid (H830) in the RIG-I RNA binding pocket as the mediator of steric exclusion of N1-2'O-methylated RNA. H830A alteration (RIG-I(H830A)) restored binding of N1-2'O-methylated pppRNA. Consequently, endogenous mRNA activated the RIG-I(H830A) mutant but not wild-type RIG-I. Similarly, knockdown of the endogenous N1-2'O-methyltransferase led to considerable RIG-I stimulation in the absence of exogenous stimuli. Studies involving yellow-fever-virus-encoded 2'O-methyltransferase and RIG-I(H830A) revealed that viruses exploit this mechanism to escape RIG-I. Our data reveal a new role for cap N1-2'O-methylation in RIG-I tolerance of self-RNA.
Asunto(s)
ARN Helicasas DEAD-box/genética , Tolerancia Inmunológica/genética , Procesamiento Postranscripcional del ARN/genética , ARN/genética , Virus de la Fiebre Amarilla/enzimología , Secuencia de Aminoácidos , Animales , Células Cultivadas , Proteína 58 DEAD Box , Activación Enzimática/genética , Activación Enzimática/inmunología , Histidina/genética , Humanos , Metilación , Metiltransferasas/genética , Ratones , Estructura Terciaria de Proteína , ARN/química , ARN/inmunología , ARN Viral/inmunología , Receptores Inmunológicos , Virus de la Fiebre Amarilla/genéticaRESUMEN
Mammalian cells possess mechanisms to detect and defend themselves from invading viruses. In the cytosol, the RIG-I-like receptors (RLRs), RIG-I (retinoic acid-inducible gene I; encoded by DDX58) and MDA5 (melanoma differentiation-associated gene 5; encoded by IFIH1) sense atypical RNAs associated with virus infection. Detection triggers a signalling cascade via the adaptor MAVS that culminates in the production of type I interferons (IFN-α and ß; hereafter IFN), which are key antiviral cytokines. RIG-I and MDA5 are activated by distinct viral RNA structures and much evidence indicates that RIG-I responds to RNAs bearing a triphosphate (ppp) moiety in conjunction with a blunt-ended, base-paired region at the 5'-end (reviewed in refs 1, 2, 3). Here we show that RIG-I also mediates antiviral responses to RNAs bearing 5'-diphosphates (5'pp). Genomes from mammalian reoviruses with 5'pp termini, 5'pp-RNA isolated from yeast L-A virus, and base-paired 5'pp-RNAs made by in vitro transcription or chemical synthesis, all bind to RIG-I and serve as RIG-I agonists. Furthermore, a RIG-I-dependent response to 5'pp-RNA is essential for controlling reovirus infection in cultured cells and in mice. Thus, the minimal determinant for RIG-I recognition is a base-paired RNA with 5'pp. Such RNAs are found in some viruses but not in uninfected cells, indicating that recognition of 5'pp-RNA, like that of 5'ppp-RNA, acts as a powerful means of self/non-self discrimination by the innate immune system.
Asunto(s)
ARN Helicasas DEAD-box/metabolismo , Difosfatos/metabolismo , Inmunidad Innata , ARN Viral/química , ARN Viral/metabolismo , Reoviridae/genética , Reoviridae/inmunología , Animales , Emparejamiento Base , Secuencia de Bases , Proteína 58 DEAD Box , Femenino , Genoma Viral/genética , Masculino , Ratones , ARN Viral/genética , Reoviridae/fisiologíaRESUMEN
Detection of cytoplasmic DNA represents one of the most fundamental mechanisms of the innate immune system to sense the presence of microbial pathogens. Moreover, erroneous detection of endogenous DNA by the same sensing mechanisms has an important pathophysiological role in certain sterile inflammatory conditions. The endoplasmic-reticulum-resident protein STING is critically required for the initiation of type I interferon signalling upon detection of cytosolic DNA of both exogenous and endogenous origin. Next to its pivotal role in DNA sensing, STING also serves as a direct receptor for the detection of cyclic dinucleotides, which function as second messenger molecules in bacteria. DNA recognition, however, is triggered in an indirect fashion that depends on a recently characterized cytoplasmic nucleotidyl transferase, termed cGAMP synthase (cGAS), which upon interaction with DNA synthesizes a dinucleotide molecule that in turn binds to and activates STING. We here show in vivo and in vitro that the cGAS-catalysed reaction product is distinct from previously characterized cyclic dinucleotides. Using a combinatorial approach based on mass spectrometry, enzymatic digestion, NMR analysis and chemical synthesis we demonstrate that cGAS produces a cyclic GMP-AMP dinucleotide, which comprises a 2'-5' and a 3'-5' phosphodiester linkage >Gp(2'-5')Ap(3'-5')>. We found that the presence of this 2'-5' linkage was required to exert potent activation of human STING. Moreover, we show that cGAS first catalyses the synthesis of a linear 2'-5'-linked dinucleotide, which is then subject to cGAS-dependent cyclization in a second step through a 3'-5' phosphodiester linkage. This 13-membered ring structure defines a novel class of second messenger molecules, extending the family of 2'-5'-linked antiviral biomolecules.
Asunto(s)
Proteínas de la Membrana/metabolismo , Nucleotidiltransferasas/metabolismo , Oligorribonucleótidos/metabolismo , Sistemas de Mensajero Secundario/fisiología , Adenosina Monofosfato/química , Animales , Biocatálisis , Línea Celular , GMP Cíclico/química , Ciclización , Células HEK293 , Humanos , Espectroscopía de Resonancia Magnética , Ratones , Modelos Moleculares , Estructura Molecular , Nucleotidiltransferasas/genética , Oligorribonucleótidos/biosíntesis , Oligorribonucleótidos/químicaRESUMEN
In vitro-transcribed mRNA has great therapeutic potential to transiently express the encoded protein without the adverse effects of viral and DNA-based constructs. Mammalian cells, however, contain RNA sensors of the innate immune system that must be considered in the generation of therapeutic RNA. Incorporation of modified nucleosides both reduces innate immune activation and increases translation of mRNA, but residual induction of type I interferons (IFNs) and proinflammatory cytokines remains. We identify that contaminants, including double-stranded RNA, in nucleoside-modified in vitro-transcribed RNA are responsible for innate immune activation and their removal by high performance liquid chromatography (HPLC) results in mRNA that does not induce IFNs and inflammatory cytokines and is translated at 10- to 1000-fold greater levels in primary cells. Although unmodified mRNAs were translated significantly better following purification, they still induced high levels of cytokine secretion. HPLC purified nucleoside-modified mRNA is a powerful vector for applications ranging from ex vivo stem cell generation to in vivo gene therapy.
Asunto(s)
Terapia Genética , Biosíntesis de Proteínas , ARN Mensajero/inmunología , Animales , Células Cultivadas , Cromatografía Líquida de Alta Presión , Citidina/análogos & derivados , Citidina/química , Células HEK293 , Humanos , Ratones , Seudouridina/química , ARN Bicatenario/inmunología , ARN Mensajero/química , ARN Mensajero/aislamiento & purificación , Transcripción Genética , TransfecciónRESUMEN
Sequencing of small RNA cDNA libraries is an important tool for the discovery of new RNAs and the analysis of their mutational status as well as expression changes across samples. It requires multiple enzyme-catalyzed steps, including sequential oligonucleotide adapter ligations to the 3' and 5' ends of the small RNAs, reverse transcription (RT), and PCR. We assessed biases in representation of miRNAs relative to their input concentration, using a pool of 770 synthetic miRNAs and 45 calibrator oligoribonucleotides, and tested the influence of Rnl1 and two variants of Rnl2, Rnl2(1-249) and Rnl2(1-249)K227Q, for 3'-adapter ligation. The use of the Rnl2 variants for adapter ligations yielded substantially fewer side products compared with Rnl1; however, the benefits of using Rnl2 remained largely obscured by additional biases in the 5'-adapter ligation step; RT and PCR steps did not have a significant impact on read frequencies. Intramolecular secondary structures of miRNA and/or miRNA/3'-adapter products contributed to these biases, which were highly reproducible under defined experimental conditions. We used the synthetic miRNA cocktail to derive correction factors for approximation of the absolute levels of individual miRNAs in biological samples. Finally, we evaluated the influence of 5'-terminal 5-nt barcode extensions for a set of 20 barcoded 3' adapters and observed similar biases in miRNA read distribution, thereby enabling cost-saving multiplex analysis for large-scale miRNA profiling.
Asunto(s)
Biblioteca de Genes , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , MicroARNs/análisis , ARN Ligasa (ATP)/genética , Cartilla de ADN , Perfilación de la Expresión Génica/métodos , Familia de Multigenes , Oligonucleótidos/genética , Reacción en Cadena de la Polimerasa , ARN Ligasa (ATP)/análisis , Análisis de Secuencia de ARNRESUMEN
RIG-I is a cytosolic helicase that senses 5'-ppp RNA contained in negative-strand RNA viruses and triggers innate antiviral immune responses. Calorimetric binding studies established that the RIG-I C-terminal regulatory domain (CTD) binds to blunt-end double-stranded 5'-ppp RNA a factor of 17 more tightly than to its single-stranded counterpart. Here we report on the crystal structure of RIG-I CTD bound to both blunt ends of a self-complementary 5'-ppp dsRNA 12-mer, with interactions involving 5'-pp clearly visible in the complex. The structure, supported by mutation studies, defines how a lysine-rich basic cleft within the RIG-I CTD sequesters the observable 5'-pp of the bound RNA, with a stacked phenylalanine capping the terminal base pair. Key intermolecular interactions observed in the crystalline state are retained in the complex of 5'-ppp dsRNA 24-mer and full-length RIG-I under in vivo conditions, as evaluated from the impact of binding pocket RIG-I mutations and 2'-OCH(3) RNA modifications on the interferon response.
Asunto(s)
ARN Helicasas DEAD-box/química , ARN Helicasas DEAD-box/metabolismo , ARN Bicatenario/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Proteína 58 DEAD Box , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/inmunología , Humanos , Inmunidad Innata , Helicasa Inducida por Interferón IFIH1 , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Mutación Puntual , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , ARN Helicasas/química , ARN Helicasas/metabolismo , ARN Bicatenario/química , Receptores Inmunológicos , Alineación de SecuenciaRESUMEN
TLR9 detects DNA in endolysosomal compartments of human B cells and PDC. Recently, the concept of the CpG motif specificity of TLR9-mediated detection, specifically of natural phosphodiester DNA, has been challenged. Unlike in human B cells, CpG specificity of natural phosphodiester DNA recognition in human PDC has not been analyzed in the literature. Here, we found that the induction of IFN-alpha and TNF-alpha in human PDC by phosphodiester ODNs containing one or two CG dinucleotides was reduced to a lower level when the CG dinucleotides were methylated and was abolished if the CGs were switched to GCs. Consistent with a high frequency of unmethylated CG dinucleotides, bacterial DNA induced high levels of IFN-alpha in PDC; IFN-alpha was reduced but not abolished upon methylation of bacterial DNA. Mammalian DNA containing low numbers of CG dinucleotides, which are frequently methylated, induced IFN-alpha in PDC consistently but on a much lower level than bacterial DNA. For activation of PDC, phosphodiester ODNs and genomic DNA strictly required complexation with cationic molecules such as the keratinocyte-derived antimicrobial peptide LL37 or a scrambled derivative. In conclusion, we demonstrate that self-DNA complexed to cationic molecules activate PDC and thus, indeed, may function as DAMPs; nevertheless, the preference of PDC for CpG containing DNA provides the basis for the discrimination of microbial from self-DNA even if DNA is presented in the condensed form of a complex.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/inmunología , ADN Bacteriano/inmunología , Células Dendríticas/inmunología , Fosfatos de Dinucleósidos/inmunología , Oligonucleótidos Fosforotioatos/inmunología , Receptor Toll-Like 9/inmunología , Catelicidinas , Línea Celular , Células Cultivadas , Islas de CpG/genética , Islas de CpG/inmunología , ADN/genética , ADN/inmunología , Metilación de ADN , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , Humanos , Riñón/citología , Leucocitos Mononucleares/inmunología , Activación de Linfocitos/inmunologíaRESUMEN
Antiviral immunity is triggered by immunorecognition of viral nucleic acids. The cytosolic helicase RIG-I is a key sensor of viral infections and is activated by RNA containing a triphosphate at the 5' end. The exact structure of RNA activating RIG-I remains controversial. Here, we established a chemical approach for 5' triphosphate oligoribonucleotide synthesis and found that synthetic single-stranded 5' triphosphate oligoribonucleotides were unable to bind and activate RIG-I. Conversely, the addition of the synthetic complementary strand resulted in optimal binding and activation of RIG-I. Short double-strand conformation with base pairing of the nucleoside carrying the 5' triphosphate was required. RIG-I activation was impaired by a 3' overhang at the 5' triphosphate end. These results define the structure of RNA for full RIG-I activation and explain how RIG-I detects negative-strand RNA viruses that lack long double-stranded RNA but do contain blunt short double-stranded 5' triphosphate RNA in the panhandle region of their single-stranded genome.
Asunto(s)
ARN Helicasas DEAD-box/inmunología , Polifosfatos/inmunología , Virus ARN/inmunología , ARN Bicatenario/inmunología , ARN Viral/inmunología , Animales , Células Cultivadas , Proteína 58 DEAD Box , ARN Helicasas DEAD-box/genética , Humanos , Interferón-alfa/biosíntesis , Interferón-alfa/inmunología , Ratones , Ratones Mutantes , Monocitos/inmunología , Monocitos/metabolismo , Oligorribonucleótidos/síntesis química , Oligorribonucleótidos/inmunología , Polifosfatos/metabolismo , ARN Bicatenario/metabolismo , ARN Viral/metabolismo , Receptores InmunológicosRESUMEN
MicroRNAs are small regulatory RNAs with many biological functions and disease associations. We showed that in situ hybridization (ISH) using conventional formaldehyde fixation results in substantial microRNA loss from mouse tissue sections, which can be prevented by fixation with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide that irreversibly immobilizes the microRNA at its 5' phosphate. We determined optimal hybridization parameters for 130 locked nucleic acid probes by recording nucleic acid melting temperature during ISH.
Asunto(s)
Carbodiimidas/química , Formaldehído/química , Hibridación in Situ/métodos , MicroARNs/análisis , Fijación del Tejido/métodos , Animales , Encéfalo/citología , Encéfalo/metabolismo , Química Encefálica , Ratones , MicroARNs/genética , Microscopía Fluorescente/métodos , Neuronas/citología , Neuronas/metabolismoRESUMEN
In vitro-transcribed mRNAs encoding physiologically important proteins have considerable potential for therapeutic applications. However, in its present form, mRNA is unfeasible for clinical use because of its labile and immunogenic nature. Here, we investigated whether incorporation of naturally modified nucleotides into transcripts would confer enhanced biological properties to mRNA. We found that mRNAs containing pseudouridines have a higher translational capacity than unmodified mRNAs when tested in mammalian cells and lysates or administered intravenously into mice at 0.015-0.15 mg/kg doses. The delivered mRNA and the encoded protein could be detected in the spleen at 1, 4, and 24 hours after the injection, where both products were at significantly higher levels when pseudouridine-containing mRNA was administered. Even at higher doses, only the unmodified mRNA was immunogenic, inducing high serum levels of interferon-alpha (IFN-alpha). These findings indicate that nucleoside modification is an effective approach to enhance stability and translational capacity of mRNA while diminishing its immunogenicity in vivo. Improved properties conferred by pseudouridine make such mRNA a promising tool for both gene replacement and vaccination.
Asunto(s)
Biosíntesis de Proteínas , Seudouridina/química , ARN Mensajero/química , Animales , Línea Celular , Vectores Genéticos , Humanos , Luciferasas/biosíntesis , Luciferasas/genética , Ratones , ARN Mensajero/inmunología , Transcripción GenéticaRESUMEN
Distinct classes of small RNAs, 20-32 nucleotides long, play important regulatory roles for diverse cellular processes. It is therefore important to identify and quantify small RNAs as a function of development, tissue and cell type, in normal and disease states. Here we describe methods to prepare cDNA libraries from pools of small RNAs isolated from organisms, tissues or cells. These methods enable the identification of new members or new classes of small RNAs, and they are also suitable to obtain miRNA expression profiles based on clone count frequencies. This protocol includes the use of new deep sequencing methods (454/Roche and Solexa) to facilitate the characterization of diverse sequence pools of small RNAs.
Asunto(s)
Biblioteca de Genes , MicroARNs/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Secuencias Reguladoras de Ácido Ribonucleico/genética , Animales , Humanos , MicroARNs/aislamiento & purificaciónRESUMEN
BACKGROUND/AIMS: Four different ribozymes (Rz) targeting the hepatitis C virus (HCV) 5'-non-coding region (NCR) at nucleotide (nt) positions GUA 165 (Rz1), GUC 270 (Rz2), GUA 330 (Rz3) and GCA 348 (Rz1293) were compared for in vitro cleavage using a 455 nt HCV RNA substrate. The GUA 330 (Rz3) and GCA 348 (Rz1293) ribozymes, both targeting the HCV loop IV region, were found to be the most efficient, and were further analyzed in an in vitro translation system. METHODS: For this purpose RNA transcribed from a construct encoding a HCV-5'-NCR-luciferase fusion protein was used. Cleavage-inactive (Rz1426), mismatch (Rz1293m) or unrelated ribozymes (Rz1437) were synthesized as controls for Rz-1293. HCV specificity was analysed by competition experiments using sense and mismatch oligodeoxynucleotides HCVrzCI and HCVrzMM, respectively. RESULTS: A chemically modified nuclease-resistant variant of the GCA 348 cleaving ribozyme was selected for cell culture experiments using recombinant HepG2 or CCL13 cell lines stably transfected with a HCV-5'-NCR-luciferase target construct. CONCLUSIONS: This ribozyme (Rz1293) showed an inhibitory activity of translation of more than 70% thus verifying that the GCA 348 cleavage site in the HCV loop IV is an accessible target site in vivo and may be suitable for the development of novel optimized hammerhead structures.
Asunto(s)
Antivirales/farmacología , Hepacivirus/efectos de los fármacos , ARN Catalítico/farmacología , ARN Viral/efectos de los fármacos , Regiones no Traducidas/efectos de los fármacos , Células Cultivadas , Humanos , Conformación de Ácido Nucleico , Biosíntesis de Proteínas/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
This report describes a one-pot synthesis of alpha-P-borano-, alpha-P-thio-, and alpha-P-seleno-modified nucleoside diphosphate analogues that are otherwise difficult to obtain. The key step involves the intramolecular nucleophilic attack by an amino group in 5 to remove the gamma-phosphate. The absolute configurations of P-diastereomers were confirmed by analysis of their 1H NMR. Affinity studies revealed that the nucleoside boranodiphosphates are potentially useful in antiviral research.