RESUMEN
Friedreich's ataxia (FRDA) is a severe multisystem disease caused by transcriptional repression induced by expanded GAA repeats located in intron 1 of the Frataxin (FXN) gene encoding frataxin. FRDA results from decreased levels of frataxin; thus, stabilization of the FXN mRNA already present in patient cells represents an attractive and unexplored therapeutic avenue. In this work, we pursued a novel approach based on oligonucleotide-mediated targeting of FXN mRNA ends to extend its half-life and availability as a template for translation. We demonstrated that oligonucleotides designed to bind to FXN 5' or 3' noncoding regions can increase FXN mRNA and protein levels. Simultaneous delivery of oligonucleotides targeting both ends increases efficacy of the treatment. The approach was confirmed in several FRDA fibroblast and induced pluripotent stem cell-derived neuronal progenitor lines. RNA sequencing and single-cell expression analyses confirmed oligonucleotide-mediated FXN mRNA upregulation. Mechanistically, a significant elongation of the FXN mRNA half-life without any changes in chromatin status at the FXN gene was observed upon treatment with end-targeting oligonucleotides, indicating that transcript stabilization is responsible for frataxin upregulation. These results identify a novel approach toward upregulation of steady-state mRNA levels via oligonucleotide-mediated end targeting that may be of significance to any condition resulting from transcription downregulation.
Asunto(s)
Ataxia de Friedreich/terapia , Terapia Genética/métodos , Proteínas de Unión a Hierro/genética , Estabilidad del ARN , ARN Mensajero/metabolismo , Regiones no Traducidas 3' , Regiones no Traducidas 5' , Células Cultivadas , Humanos , Proteínas de Unión a Hierro/metabolismo , Oligonucleótidos Antisentido/química , Oligonucleótidos Antisentido/metabolismo , ARN Mensajero/química , ARN Mensajero/genética , FrataxinaRESUMEN
Non-viral vectors offer the potential to deliver nucleic acids including mRNA and DNA into cells in vivo. However, designing materials that effectively deliver to target organs and then to desired compartments within the cell remains a challenge. Here we develop polymeric materials that can be optimized for either DNA transcription in the nucleus or mRNA translation in the cytosol. We synthesized poly(beta amino ester) terpolymers (PBAEs) with modular changes to monomer chemistry to investigate influence on nucleic acid delivery. We identified two PBAEs with a single monomer change as being effective for either DNA (D-90-C12-103) or mRNA (DD-90-C12-103) delivery to lung endothelium following intravenous injection in mice. Physical properties such as particle size or charge did not account for the difference in transfection efficacy. However, endosome co-localization studies revealed that D-90-C12-103 nanoparticles resided in late endosomes to a greater extent than DD-90-C12-103. We compared luciferase expression in vivo and observed that, even with nucleic acid optimized vectors, peak luminescence using mRNA was two orders of magnitude greater than pDNA in the lungs of mice following systemic delivery. This study indicates that different nucleic acids require tailored delivery vectors, and further support the potential of PBAEs as intracellular delivery materials.
Asunto(s)
Nanopartículas , Polímeros , Animales , ADN , Lípidos , Pulmón , Ratones , ARN Mensajero/genética , TransfecciónRESUMEN
Spinal muscular atrophy (SMA) is a neurodegenerative disease characterized by progressive motor neuron loss and caused by mutations in SMN1 (Survival Motor Neuron 1). The disease severity inversely correlates with the copy number of SMN2, a duplicated gene that is nearly identical to SMN1. We have delineated a mechanism of transcriptional regulation in the SMN2 locus. A previously uncharacterized long noncoding RNA (lncRNA), SMN-antisense 1 (SMN-AS1), represses SMN2 expression by recruiting the Polycomb Repressive Complex 2 (PRC2) to its locus. Chemically modified oligonucleotides that disrupt the interaction between SMN-AS1 and PRC2 inhibit the recruitment of PRC2 and increase SMN2 expression in primary neuronal cultures. Our approach comprises a gene-up-regulation technology that leverages interactions between lncRNA and PRC2. Our data provide proof-of-concept that this technology can be used to treat disease caused by epigenetic silencing of specific loci.
Asunto(s)
Atrofia Muscular Espinal/terapia , Oligonucleótidos/genética , Complejo Represivo Polycomb 2/metabolismo , ARN Largo no Codificante/metabolismo , Proteína 2 para la Supervivencia de la Neurona Motora/genética , Animales , Línea Celular , Modelos Animales de Enfermedad , Exones/genética , Fibroblastos , Dosificación de Gen , Terapia Genética/métodos , Humanos , Ratones , Terapia Molecular Dirigida/métodos , Neuronas Motoras/metabolismo , Atrofia Muscular Espinal/genética , Mutación Puntual , Complejo Represivo Polycomb 2/genética , ARN Largo no Codificante/genética , Proteína 1 para la Supervivencia de la Neurona Motora/genética , Activación Transcripcional/genética , Regulación hacia ArribaRESUMEN
The molecular origins of fibrosis affecting multiple tissue beds remain incompletely defined. Previously, we delineated the critical role of the control of extracellular matrix (ECM) stiffening by the mechanosensitive microRNA-130/301 family, as activated by the YAP/TAZ co-transcription factors, in promoting pulmonary hypertension (PH). We hypothesized that similar mechanisms may dictate fibrosis in other tissue beds beyond the pulmonary vasculature. Employing an in silico combination of microRNA target prediction, transcriptomic analysis of 137 human diseases and physiologic states, and advanced gene network modeling, we predicted the microRNA-130/301 family as a master regulator of fibrotic pathways across a cohort of seemingly disparate diseases and conditions. In two such diseases (pulmonary fibrosis and liver fibrosis), inhibition of microRNA-130/301 prevented the induction of ECM modification, YAP/TAZ, and downstream tissue fibrosis. Thus, mechanical forces act through a central feedback circuit between microRNA-130/301 and YAP/TAZ to sustain a common fibrotic phenotype across a network of human physiologic and pathophysiologic states. Such re-conceptualization of interconnections based on shared systems of disease and non-disease gene networks may have broad implications for future convergent diagnostic and therapeutic strategies.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Péptidos y Proteínas de Señalización Intracelular/metabolismo , MicroARNs/genética , Fosfoproteínas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Benzoatos/farmacología , Bencilaminas/farmacología , Modelos Animales de Enfermedad , Matriz Extracelular , Fibrosis , Humanos , Hipertensión Pulmonar/genética , Hipertensión Pulmonar/metabolismo , Hipertensión Pulmonar/patología , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Relacionadas con Receptor de LDL/genética , Proteínas Relacionadas con Receptor de LDL/metabolismo , Cirrosis Hepática/genética , Cirrosis Hepática/metabolismo , Cirrosis Hepática/patología , Ratones , Fosfoproteínas/genética , Fibrosis Pulmonar/genética , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/patología , Transactivadores , Factores de Transcripción , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ , Proteínas Señalizadoras YAPRESUMEN
Pulmonary hypertension (PH) is a deadly vascular disease with enigmatic molecular origins. We found that vascular extracellular matrix (ECM) remodeling and stiffening are early and pervasive processes that promote PH. In multiple pulmonary vascular cell types, such ECM stiffening induced the microRNA-130/301 family via activation of the co-transcription factors YAP and TAZ. MicroRNA-130/301 controlled a PPAR?-APOE-LRP8 axis, promoting collagen deposition and LOX-dependent remodeling and further upregulating YAP/TAZ via a mechanoactive feedback loop. In turn, ECM remodeling controlled pulmonary vascular cell crosstalk via such mechanotransduction, modulation of secreted vasoactive effectors, and regulation of associated microRNA pathways. In vivo, pharmacologic inhibition of microRNA-130/301, APOE, or LOX activity ameliorated ECM remodeling and PH. Thus, ECM remodeling, as controlled by the YAP/TAZ-miR-130/301 feedback circuit, is an early PH trigger and offers combinatorial therapeutic targets for this devastating disease.
Asunto(s)
Matriz Extracelular/metabolismo , Retroalimentación Fisiológica , Hipertensión Pulmonar/metabolismo , Mecanotransducción Celular , MicroARNs/genética , Factores de Transcripción/metabolismo , Animales , Apolipoproteínas E/metabolismo , Matriz Extracelular/patología , Humanos , Concentración de Iones de Hidrógeno , Hipertensión Pulmonar/patología , Proteínas Relacionadas con Receptor de LDL/metabolismo , Ratones , Ratones Endogámicos C57BL , PPAR gamma/metabolismo , Ratas , Ratas Sprague-Dawley , Factores de Transcripción/genéticaRESUMEN
The ss-siRNA activity in vivo requires a metabolically stable 5'-phosphate analog. In this report we used crystal structure of the 5'-phosphate binding pocket of Ago-2 bound with guide strand to design and synthesize ss-siRNAs containing various 5'-phosphate analogs. Our results indicate that the electronic and spatial orientation of the 5'-phosphate analog was critical for ss-siRNA activity. Chemically modified ss-siRNA targeting human apoC III mRNA demonstrated good potency for inhibiting ApoC III mRNA and protein in transgenic mice. Moreover, ApoC III ss-siRNAs were able to reduce the triglyceride and LDL cholesterol in transgenic mice demonstrating pharmacological effect of ss-siRNA. Our study provides guidance to develop surrogate phosphate analog for ss-siRNA and demonstrates that ss-siRNA provides an alternative strategy for therapeutic gene silencing.
Asunto(s)
ARN Interferente Pequeño/química , ARN Interferente Pequeño/metabolismo , Animales , Apolipoproteína C-III/genética , Apolipoproteína C-III/metabolismo , Proteínas Argonautas/química , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Secuencia de Bases , Sitios de Unión , LDL-Colesterol/sangre , Células HeLa , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Modelos Moleculares , Estructura Molecular , Conformación de Ácido Nucleico , Fosfatos/química , Dominios y Motivos de Interacción de Proteínas , Interferencia de ARN , Estabilidad del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Triglicéridos/sangreRESUMEN
Pulmonary hypertension (PH) is a complex disorder, spanning several known vascular cell types. Recently, we identified the microRNA-130/301 (miR-130/301) family as a regulator of multiple pro-proliferative pathways in PH, but the true breadth of influence of the miR-130/301 family across cell types in PH may be even more extensive. Here, we employed targeted network theory to identify additional pathogenic pathways regulated by miR-130/301, including those involving vasomotor tone. Guided by these predictions, we demonstrated, via gain- and loss-of-function experimentation in vitro and in vivo, that miR-130/301-specific control of the peroxisome proliferator-activated receptor γ regulates a panel of vasoactive factors communicating between diseased pulmonary vascular endothelial and smooth muscle cells. Of these, the vasoconstrictive factor endothelin-1 serves as an integral point of communication between the miR-130/301-peroxisome proliferator-activated receptor γ axis in endothelial cells and contractile function in smooth muscle cells. Thus, resulting from an in silico analysis of the architecture of the PH disease gene network coupled with molecular experimentation in vivo, these findings clarify the expanded role of the miR-130/301 family in the global regulation of PH. They further emphasize the importance of molecular cross-talk among the diverse cellular populations involved in PH.
Asunto(s)
Regulación de la Expresión Génica , Hipertensión Pulmonar/metabolismo , MicroARNs/metabolismo , Vasoconstricción , Algoritmos , Animales , Células Cultivadas , Endotelina-1/metabolismo , Endotelinas/metabolismo , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/metabolismo , Oligonucleótidos/química , PPAR gamma/metabolismo , Arteria Pulmonar/metabolismo , Transducción de Señal , Biología de SistemasRESUMEN
MicroRNAs (miRNAs), small RNA molecules that post-transcriptionally regulate mRNA expression, are crucial in diverse developmental and physiological programs and their misregulation can lead to disease. Chemically modified oligonucleotides have been developed to modulate miRNA activity for therapeutic intervention in disease settings, but their mechanism of action has not been fully elucidated. Here we show that the miRNA inhibitors (anti-miRs) physically associate with Argonaute proteins in the context of the cognate target miRNA in vitro and in vivo. The association is mediated by the seed region of the miRNA and is sensitive to the placement of chemical modifications. Furthermore, the targeted miRNAs are stable and continue to be associated with Argonaute. Our results suggest that anti-miRs specifically associate with Argonaute-bound miRNAs, preventing association with target mRNAs, which leads to subsequent stabilization and thus increased expression of the targeted mRNAs.
Asunto(s)
Proteínas Argonautas/metabolismo , MicroARNs/antagonistas & inhibidores , MicroARNs/metabolismo , Oligonucleótidos Antisentido/farmacología , Animales , Femenino , Humanos , Masculino , Ratones Endogámicos C57BL , Unión Proteica/efectos de los fármacosRESUMEN
Development of the vascular disease pulmonary hypertension (PH) involves disparate molecular pathways that span multiple cell types. MicroRNAs (miRNAs) may coordinately regulate PH progression, but the integrative functions of miRNAs in this process have been challenging to define with conventional approaches. Here, analysis of the molecular network architecture specific to PH predicted that the miR-130/301 family is a master regulator of cellular proliferation in PH via regulation of subordinate miRNA pathways with unexpected connections to one another. In validation of this model, diseased pulmonary vessels and plasma from mammalian models and human PH subjects exhibited upregulation of miR-130/301 expression. Evaluation of pulmonary arterial endothelial cells and smooth muscle cells revealed that miR-130/301 targeted PPARγ with distinct consequences. In endothelial cells, miR-130/301 modulated apelin-miR-424/503-FGF2 signaling, while in smooth muscle cells, miR-130/301 modulated STAT3-miR-204 signaling to promote PH-associated phenotypes. In murine models, induction of miR-130/301 promoted pathogenic PH-associated effects, while miR-130/301 inhibition prevented PH pathogenesis. Together, these results provide insight into the systems-level regulation of miRNA-disease gene networks in PH with broad implications for miRNA-based therapeutics in this disease. Furthermore, these findings provide critical validation for the evolving application of network theory to the discovery of the miRNA-based origins of PH and other diseases.
Asunto(s)
Hipertensión Pulmonar/etiología , Hipertensión Pulmonar/genética , MicroARNs/genética , Animales , Apelina , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proliferación Celular , Simulación por Computador , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Células Endoteliales/patología , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Redes Reguladoras de Genes , Humanos , Hipertensión Pulmonar/patología , Hipoxia/complicaciones , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Ratones , Ratones Endogámicos C57BL , MicroARNs/metabolismo , Modelos Biológicos , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , PPAR gamma/metabolismo , Arteria Pulmonar/metabolismo , Arteria Pulmonar/patología , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Teoría de Sistemas , Regulación hacia ArribaRESUMEN
Acute myelogenous leukemia (AML) subtypes that result from oncogenic activation of homeobox (HOX) transcription factors are associated with poor prognosis. The HOXA9 transcription activator and growth factor independent 1 (GFI1) transcriptional repressor compete for occupancy at DNA-binding sites for the regulation of common target genes. We exploited this HOXA9 versus GFI1 antagonism to identify the genes encoding microRNA-21 and microRNA-196b as transcriptional targets of HOX-based leukemia oncoproteins. Therapeutic inhibition of microRNA-21 and microRNA-196b inhibited in vitro leukemic colony forming activity and depleted in vivo leukemia-initiating cell activity of HOX-based leukemias, which led to leukemia-free survival in a murine AML model and delayed disease onset in xenograft models. These data establish microRNA as functional effectors of endogenous HOXA9 and HOX-based leukemia oncoproteins, provide a concise in vivo platform to test RNA therapeutics, and suggest therapeutic value for microRNA antagonists in AML.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Leucemia Mieloide Aguda/metabolismo , MicroARNs/genética , Células Madre Neoplásicas/fisiología , Animales , Secuencia de Bases , Sitios de Unión , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Terapia Combinada , Citarabina/administración & dosificación , Proteínas de Unión al ADN/metabolismo , Doxorrubicina/administración & dosificación , Regulación Leucémica de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Humanos , Quimioterapia de Inducción , Leucemia Mieloide Aguda/patología , Leucemia Mieloide Aguda/terapia , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones SCID , MicroARNs/metabolismo , Proteína 1 del Sitio de Integración Viral Ecotrópica Mieloide , Proteínas de Neoplasias/metabolismo , Oligonucleótidos Fosforotioatos/genética , Factor de Transcripción 1 de la Leucemia de Células Pre-B , Unión Proteica , Proteínas Proto-Oncogénicas/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Factores de Transcripción/metabolismo , Transcriptoma , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
MicroRNAs (miRNAs) bind to mRNAs and fine-tune protein output by affecting mRNA stability and/or translation. miR-21 is a ubiquitous, highly abundant, and stress-responsive miRNA linked to several diseases, including cancer, fibrosis, and inflammation. Although the RNA silencing activity of miR-21 in diseased cells has been well documented, the roles of miR-21 under healthy cellular conditions are not well understood. Here, we show that pharmacological inhibition or genetic deletion of miR-21 in healthy mouse liver has little impact on regulation of canonical seed-matched mRNAs and only a limited number of genes enriched in stress response pathways. These surprisingly weak and selective regulatory effects on known and predicted target mRNAs contrast with those of other abundant liver miRNAs such as miR-122 and let-7. Moreover, miR-21 shows greatly reduced binding to polysome-associated target mRNAs compared to miR-122 and let-7. Bioinformatic analysis suggests that reduced thermodynamic stability of seed pairing and target binding may contribute to this deficiency of miR-21. Significantly, these trends are reversed in human cervical carcinoma (HeLa) cells, where miRNAs including miR-21 show enhanced target binding within polysomes and where miR-21 triggers strong degradative activity toward target mRNAs. Taken together, our results suggest that, under normal cellular conditions in liver, miR-21 activity is maintained below a threshold required for binding and silencing most of its targets. Consequently, enhanced association with polysome-associated mRNA is likely to explain in part the gain of miR-21 function often found in diseased or stressed cells.
Asunto(s)
Hígado/metabolismo , MicroARNs/fisiología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Animales , Western Blotting , Perfilación de la Expresión Génica , Células HeLa , Humanos , Hígado/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Análisis de Secuencia por Matrices de Oligonucleótidos , Polirribosomas/metabolismo , Biosíntesis de Proteínas , Estabilidad del ARN/genética , ARN Mensajero/antagonistas & inhibidoresRESUMEN
We report the structure activity relationships of short 14-mer phosphorothioate gapmer antisense oligonucleotides (ASOs) modified with α-L-locked nucleic acid (LNA) and related modifications targeting phosphatase and tensin homologue (PTEN) messenger RNA in mice. α-L-LNA represents the α-anomer of enantio-LNA and modified oligonucleotides show LNA like binding affinity for complementary RNA. In contrast to sequence matched LNA gapmer ASOs which showed elevations in plasma alanine aminotransferase (ALT) levels indicative of hepatotoxicity, gapmer ASOs modified with α-L-LNA and related analogs in the flanks showed potent downregulation of PTEN messenger RNA in liver tissue without producing elevations in plasma ALT levels. However, the α-L-LNA ASO showed a moderate dose-dependent increase in liver and spleen weights suggesting a higher propensity for immune stimulation. Interestingly, replacing α-L-LNA nucleotides in the 3'- and 5'-flanks with R-5'-Me-α-L-LNA but not R-6'-Me- or 3'-Me-α-L-LNA nucleotides, reversed the drug induced increase in organ weights. Examination of structural models of dinucleotide units suggested that the 5'-Me group increases steric bulk in close proximity to the phosphorothioate backbone or produces subtle changes in the backbone conformation which could interfere with recognition of the ASO by putative immune receptors. Our data suggests that introducing steric bulk at the 5'-position of the sugar-phosphate backbone could be a general strategy to mitigate the immunostimulatory profile of oligonucleotide drugs. In a clinical setting, proinflammatory effects manifest themselves as injection site reactions and flu-like symptoms. Thus, a mitigation of these effects could increase patient comfort and compliance when treated with ASOs.Molecular Therapy - Nucleic Acids (2012) 1, e47; doi:10.1038/mtna.2012.34; published online 18 September 2012.
RESUMEN
The synthesis, biophysical, structural, and biological properties of both isomers of 3'-fluoro hexitol nucleic acid (FHNA and Ara-FHNA) modified oligonucleotides are reported. Synthesis of the FHNA and Ara-FHNA thymine phosphoramidites was efficiently accomplished starting from known sugar precursors. Optimal RNA affinities were observed with a 3'-fluorine atom and nucleobase in a trans-diaxial orientation. The Ara-FHNA analog with an equatorial fluorine was found to be destabilizing. However, the magnitude of destabilization was sequence-dependent. Thus, the loss of stability is sharply reduced when Ara-FHNA residues were inserted at pyrimidine-purine (Py-Pu) steps compared to placement within a stretch of pyrimidines (Py-Py). Crystal structures of A-type DNA duplexes modified with either monomer provide a rationalization for the opposing stability effects and point to a steric origin of the destabilization caused by the Ara-FHNA analog. The sequence dependent effect can be explained by the formation of an internucleotide C-F···H-C pseudo hydrogen bond between F3' of Ara-FHNA and C8-H of the nucleobase from the 3'-adjacent adenosine that is absent at Py-Py steps. In animal experiments, FHNA-modified antisense oligonucleotides formulated in saline showed a potent downregulation of gene expression in liver tissue without producing hepatotoxicity. Our data establish FHNA as a useful modification for antisense therapeutics and also confirm the stabilizing influence of F(Py)···H-C(Pu) pseudo hydrogen bonds in nucleic acid structures.
Asunto(s)
Ácidos Nucleicos/química , Oligonucleótidos/química , ARN/química , Alcoholes del Azúcar/química , Modelos Moleculares , Conformación Molecular , EstereoisomerismoRESUMEN
As part of a program aimed at exploring the structure- activity relationships of 2',4'-bridged nucleic acid (BNA) containing antisense oligonucleotides (ASOs), we report the synthesis and biophysical and biological properties of R- and S-5'-Me LNA modified oligonucleotides. We show that introduction of a methyl group in the (S) configuration at the 5'-position is compatible with the high affinity recognition of complementary nucleic acids observed with LNA. In contrast, introduction of a methyl group in the (R) configuration reversed the stabilization effect of LNA. NMR studies indicated that the R-5'-Me group changes the orientation around torsion angle γ from the +sc to the ap range at the nucleoside level, and this may in part be responsible for the poor hybridization behavior exhibited by this modification. In animal experiments, S-5'-Me-LNA modified gapmer antisense olignucleotides showed slightly reduced potency relative to the sequence matched LNA ASOs while improving the therapeutic profile.
Asunto(s)
Oligonucleótidos/farmacología , Biofisica , Resonancia Magnética Nuclear Biomolecular , Oligonucleótidos/química , Relación Estructura-ActividadRESUMEN
A high-resolution solution structure of a stable 42-nt RNA dimeric construct has been derived based on a high number of NMR observables including nuclear overhauser effects (NOEs), J-coupling constants and residual dipolar couplings (RDCs), which were all obtained with isotopically unlabeled molecules. Two 21-nt siRNA that efficiently hybridize consist of ribose units that were alternately substituted by 2'-fluoro or 2'-methoxy groups. Structure calculations utilized a set of H-F RDC values for all 21 2'-fluoro modified nucleotides under conditions of weak alignment achieved by Pf1 phages. A completely 2'-F/2'-OMe modified dimeric RNA construct adopts an antiparallel double-helical structure consisting of 19 Watson-Crick base pairs with additional 3' UU overhangs and a 5' phosphate group on the antisense strand. NMR data suggest that the stability of individual base pairs is not uniform throughout the construct. While most of the double helical segment exhibits well dispersed imino resonances, the last three base pairs either display uncharacteristic chemical shifts of imino protons or absence of imino resonances even at lower temperatures. Accessibility of imino protons to solvent exchange suggests a difference in stability of duplex ends, which might be of importance for incorporation of the guide siRNA strand into a RISC.
Asunto(s)
ARN Interferente Pequeño/química , Dimerización , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Conformación de Ácido Nucleico , Protones , SolucionesRESUMEN
MicroRNAs (miRNAs) are increasingly implicated in the regulation of metastasis. Despite their potential as targets for anti-metastatic therapy, miRNAs have only been silenced in normal tissues of rodents and nonhuman primates. Therefore, the development of effective approaches for sequence-specific inhibition of miRNAs in tumors remains a scientific and clinical challenge. Here we show that systemic treatment of tumor-bearing mice with miR-10b antagomirs-a class of chemically modified anti-miRNA oligonucleotide-suppresses breast cancer metastasis. Both in vitro and in vivo, silencing of miR-10b with antagomirs significantly decreases miR-10b levels and increases the levels of a functionally important miR-10b target, Hoxd10. Administration of miR-10b antagomirs to mice bearing highly metastatic cells does not reduce primary mammary tumor growth but markedly suppresses formation of lung metastases in a sequence-specific manner. The miR-10b antagomir, which is well tolerated by normal animals, appears to be a promising candidate for the development of new anti-metastasis agents.