RESUMEN
Locked nucleic acid (LNA) analogues with 2',4'-bridged sugars show promise in antisense applications. S-5'-Me-LNA has high RNA affinity, and modified oligonucleotides show weakened immune stimulation in vivo. Conversely, an R-5'-methyl group dramatically lowers RNA affinity. To test the effects of S- and R-6'-methyl groups on 3'-fluoro hexitol nucleic acid (FHNA) stability, we synthesized S- and R-6'-Me-FHNA thymidine and incorporated them into oligo-2'-deoxynucleotides. As with LNA, S-6'-Me is stabilizing whereas R-6'-Me is destabilizing. Crystal structures of 6'-Me-FHNA-modified DNAs explain the divergent consequences for stability and suggest convergent origins of these effects by S- and R-6'-Me (FHNA) [-5'-Me (LNA and RNA)] substituents.
Asunto(s)
Conformación de Ácido Nucleico , Ácidos Nucleicos Heterodúplex/química , ARN/química , Alcoholes del Azúcar/química , Cristalografía por Rayos X , Hexosadifosfatos/química , Oligonucleótidos Antisentido/química , Estereoisomerismo , Relación Estructura-Actividad , TermodinámicaRESUMEN
We report the design and synthesis of 2'-fluoro cyclohexenyl nucleic acid (F-CeNA) pyrimidine phosphoramidites and the synthesis and biophysical, structural, and biological evaluation of modified oligonucleotides. The synthesis of the nucleoside phosphoramidites was accomplished in multigram quantities starting from commercially available methyl-D-mannose pyranoside. Installation of the fluorine atom was accomplished using nonafluorobutanesulfonyl fluoride, and the cyclohexenyl ring system was assembled by means of a palladium-catalyzed Ferrier rearrangement. Installation of the nucleobase was carried out under Mitsunobu conditions followed by standard protecting group manipulations to provide the desired pyrimidine phosphoramidites. Biophysical evaluation indicated that F-CeNA shows behavior similar to that of a 2'-modified nucleotide, and duplexes with RNA showed slightly lower duplex thermostability as compared to that of the more rigid 3'-fluoro hexitol nucleic acid (FHNA). However, F-CeNA modified oligonucleotides were significantly more stable against digestion by snake venom phosphodiesterases (SVPD) as compared to unmodified DNA, 2'-fluoro RNA (FRNA), 2'-methoxyethyl RNA (MOE), and FHNA modified oligonucleotides. Examination of crystal structures of a modified DNA heptamer duplex d(GCG)-T*-d(GCG):d(CGCACGC) by X-ray crystallography indicated that the cyclohexenyl ring system exhibits both the (3)H(2) and (2)H(3) conformations, similar to the C3'-endo/C2'-endo conformation equilibrium seen in natural furanose nucleosides. In the (2)H(3) conformation, the equatorial fluorine engages in a relatively close contact with C8 (2.94 Å) of the 3'-adjacent dG nucleotide that may represent a pseudo hydrogen bond. In contrast, the cyclohexenyl ring of F-CeNA was found to exist exclusively in the (3)H(2) (C3'-endo like) conformation in the crystal structure of the modified A-form DNA decamer duplex [d(GCGTA)-T*-d(ACGC)](2.) In an animal experiment, a 16-mer F-CeNA gapmer ASO showed similar RNA affinity but significantly improved activity compared to that of a sequence matched MOE ASO, thus establishing F-CeNA as a useful modification for antisense applications.
Asunto(s)
Ciclohexenos/síntesis química , Ácidos Nucleicos/síntesis química , Oligonucleótidos/química , ARN/química , Alcoholes del Azúcar/química , Catálisis , Cristalografía por Rayos X , Ciclohexenos/química , Modelos Moleculares , Estructura Molecular , Conformación de Ácido Nucleico , Ácidos Nucleicos/químicaRESUMEN
The synthesis and biophysical evaluation of R and S-5'-Me-α-l-LNA nucleoside phosphoramidites and modified oligo-2'-deoxyribonucleotides is reported. Synthesis of the nucleoside phosphoramidites was accomplished in multi-gram quantities starting from diacetone glucose. The 5'-methyl group in the S configuration was introduced by reacting the sugar 5'-aldehyde with MeMgBr. Synthesis of the R-5'-Me isomer was accomplished from the S-5'-Me nucleoside by a late stage inversion using Mitsunobu conditions. Evaluation of the modified oligonucleotides in thermal denaturation experiments revealed that R-5'-Me-α-l-LNA showed similar RNA affinity as α-l-LNA while the S-5'-Me analog was less stabilizing. This result is in contrast to the ß-d-series where the S-5'-Me isomer showed LNA-like affinity for RNA while the R-5'-Me group completely reversed the stabilization effect on duplex thermostability.
Asunto(s)
Oligonucleótidos/química , Animales , Bioquímica/métodos , Biofisica/métodos , Cristalografía por Rayos X/métodos , Diseño de Fármacos , Glucosa/química , Ratones , Modelos Químicos , Conformación Molecular , Conformación de Ácido Nucleico , Hibridación de Ácido Nucleico , Nucleósidos/química , Compuestos Organofosforados/química , Estereoisomerismo , TemperaturaRESUMEN
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
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
The synthesis and hybridization properties of an α-L-LNA analog where the 2'-oxygen atom is replaced with an exocyclic methylene group is reported. Contrary to the ß-D series where the exocyclic methylene group is extremely well tolerated, this group was very poorly tolerated in the α-L-series and lead to duplex destabilization. Modeling studies showed that the exocyclic methylene group results in a steric clash with the nucleobase 3' to the modified residue. Based on this structural model one can anticipate that replacing the 2'-oxygen atom of α-L-LNA with larger groups is likely to be detrimental to duplex stability. The model also provides insights into what type of 2',4'-bridges are most likely to be tolerated in α-L-LNA modified oligonucleotide duplexes.
Asunto(s)
Oligonucleótidos/química , Oxígeno/química , Conformación de Ácido Nucleico , Temperatura de TransiciónRESUMEN
The synthesis and biophysical evaluation of 3'-Me-α-L-LNA is reported. The synthesis of the nucleoside building block phosphoramidite was accomplished starting from diacetone glucose. The 3'-Me group was introduced in the desired configuration by hydride mediated opening of an exocyclic epoxide. Inversion of the 2'-hydroxyl group was achieved by means of an oxidation/reduction sequence followed by cyclization onto a 5'-leaving group to assemble the [2.2.1] ring system. Biophysical evaluation of 3'-Me-α-L-LNA modified oligonucleotides showed good duplex thermal stabilizing properties which were similar to α-L-LNA. Mismatch discrimination experiments revealed that 3'-Me-α-L-LNA possess slightly enhanced discrimination properties for the GU wobble base-pair as compared to related nucleic acid analogs.
Asunto(s)
Oligonucleótidos/química , Modelos Moleculares , Conformación Molecular , Oligonucleótidos/síntesis química , Estereoisomerismo , TemperaturaRESUMEN
The synthesis and biophysical properties of R-6'-Me-α-L-LNA, which has a methyl group in the (R) configuration on the 2',4'-bridging substituent of α-L-LNA, is reported. The synthesis of the uracil nucleobase phosphoramidite was efficiently accomplished in 14 steps and 8 chromatographic purifications starting from a known sugar intermediate. Biophysical evaluation revealed that substitution along the edge of the major groove does not impair the high affinity duplex forming ability of α-L-LNA modified oligonucleotides.
Asunto(s)
Compuestos Bicíclicos Heterocíclicos con Puentes/síntesis química , Oligonucleótidos/síntesis química , Secuencia de Bases , Compuestos Bicíclicos Heterocíclicos con Puentes/química , Conformación de Ácido Nucleico , Oligonucleótidos/química , Estereoisomerismo , Temperatura de TransiciónRESUMEN
We show for the first time that it is possible to obtain LNA-like (Locked Nucleic Acid 1) binding affinity and biological activity with carbocyclic LNA (cLNA) analogs by replacing the 2'-oxygen atom in LNA with an exocyclic methylene group. Synthesis of the methylene-cLNA nucleoside was accomplished by an intramolecular cyclization reaction between a radical at the 2'-position and a propynyl group at the C-4' position. Only methylene-cLNA modified oligonucleotides showed similar thermal stability and mismatch discrimination properties for complementary nucleic acids as LNA. In contrast, the close structurally related methyl-cLNA analogs showed diminished hybridization properties. Analysis of crystal structures of cLNA modified self-complementary DNA decamer duplexes revealed that the methylene group participates in a tight interaction with a 2'-deoxyribose residue of the 5'-terminal G of a neighboring duplex, resulting in the formation of a CH...O type hydrogen bond. This indicates that the methylene group retains a negative polarization at the edge of the minor groove in the absence of a hydrophilic 2'-substituent and provides a rationale for the superior thermal stability of this modification. In animal experiments, methylene-cLNA antisense oligonucleotides (ASOs) showed similar in vivo activity but reduced toxicity as compared to LNA ASOs. Our work highlights the interchangeable role of oxygen and unsaturated moieties in nucleic acid structure and emphasizes greater use of this bioisostere to improve the properties of nucleic acids for therapeutic and diagnostic applications.
Asunto(s)
Alquenos/química , Ácidos Nucleicos/química , Oxígeno/química , Animales , Cristalografía por Rayos X , Ratones , Modelos Moleculares , Hibridación de Ácido Nucleico , Fosfohidrolasa PTEN/genética , ARN Mensajero/genética , Relación Estructura-ActividadRESUMEN
We have recently shown that combining the structural elements of 2'O-methoxyethyl (MOE) and locked nucleic acid (LNA) nucleosides yielded a series of nucleoside modifications (cMOE, 2',4'-constrained MOE; cEt, 2',4'-constrained ethyl) that display improved potency over MOE and an improved therapeutic index relative to that of LNA antisense oligonucleotides. In this report we present details regarding the synthesis of the cMOE and cEt nucleoside phosphoramidites and the biophysical evaluation of oligonucleotides containing these nucleoside modifications. The synthesis of the cMOE and cEt nucleoside phosphoramidites was efficiently accomplished starting from inexpensive commercially available diacetone allofuranose. The synthesis features the use of a seldom used 2-naphthylmethyl protecting group that provides crystalline intermediates during the synthesis and can be cleanly deprotected under mild conditions. The synthesis was greatly facilitated by the crystallinity of a key mono-TBDPS-protected diol intermediate. In the case of the cEt nucleosides, the introduction of the methyl group in either configuration was accomplished in a stereoselective manner. Ring closure of the 2'-hydroxyl group onto a secondary mesylate leaving group with clean inversion of stereochemistry was achieved under surprisingly mild conditions. For the S-cEt modification, the synthesis of all four (thymine, 5-methylcytosine, adenine, and guanine) nucleobase-modified phosphoramidites was accomplished on a multigram scale. Biophysical evaluation of the cMOE- and cEt-containing oligonucleotides revealed that they possess hybridization and mismatch discrimination attributes similar to those of LNA but greatly improved resistance to exonuclease digestion.
Asunto(s)
Cristalinas/síntesis química , Ácidos Nucleicos/síntesis química , Nucleósidos/síntesis química , Oligonucleótidos/química , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Fenómenos Biofísicos , Catálisis , Cristalinas/química , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Estructura Molecular , Ácidos Nucleicos/química , Nucleósidos/química , EstereoisomerismoRESUMEN
Autosomal dominant polycystic kidney disease (ADPKD), caused by mutations in either PKD1 or PKD2 genes, is one of the most common human monogenetic disorders and the leading genetic cause of end-stage renal disease. Unfortunately, treatment options for ADPKD are limited. Here we report the discovery and characterization of RGLS4326, a first-in-class, short oligonucleotide inhibitor of microRNA-17 (miR-17), as a potential treatment for ADPKD. RGLS4326 is discovered by screening a chemically diverse and rationally designed library of anti-miR-17 oligonucleotides for optimal pharmaceutical properties. RGLS4326 preferentially distributes to kidney and collecting duct-derived cysts, displaces miR-17 from translationally active polysomes, and de-represses multiple miR-17 mRNA targets including Pkd1 and Pkd2. Importantly, RGLS4326 demonstrates a favorable preclinical safety profile and attenuates cyst growth in human in vitro ADPKD models and multiple PKD mouse models after subcutaneous administration. The preclinical characteristics of RGLS4326 support its clinical development as a disease-modifying treatment for ADPKD.
Asunto(s)
MicroARNs/antagonistas & inhibidores , Oligonucleótidos/uso terapéutico , Enfermedades Renales Poliquísticas/tratamiento farmacológico , Enfermedades Renales Poliquísticas/genética , Animales , Secuencia de Bases , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Redes Reguladoras de Genes/efectos de los fármacos , Células HeLa , Hematopoyesis/efectos de los fármacos , Humanos , Túbulos Renales/patología , Macaca fascicularis , Masculino , Ratones Endogámicos C57BL , MicroARNs/genética , Oligonucleótidos/farmacocinética , Oligonucleótidos/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Distribución Tisular/efectos de los fármacosRESUMEN
Short interfering RNAs (siRNA) guide degradation of target RNA by the RNA-induced silencing complex (RISC). The use of siRNA in animals is limited partially due to the short half-life of siRNAs in tissues. Chemically modified siRNAs are necessary that maintain mRNA degradation activity, but are more stable to nucleases. In this study, we utilized alternating 2'-O-methyl and 2'-deoxy-2'-fluoro (OMe/F) chemically modified siRNA targeting PTEN and Eg5. OMe/F-modified siRNA consistently reduced mRNA and protein levels with equal or greater potency and efficacy than unmodified siRNA. We showed that modified siRNAs use the RISC mechanism and lead to cleavage of target mRNA at the same position as unmodified siRNA. We further demonstrated that siRNAs can compete with each other, where highly potent siRNAs can compete with less potent siRNAs, thus limiting the ability of siRNAs with lower potency to mediate mRNA degradation. In contrast, a siRNA with low potency cannot compete with a highly efficient siRNA. We established a correlation between siRNA potency and ability to compete with other siRNAs. Thus, siRNAs that are more potent inhibitors for mRNA destruction have the potential to out-compete less potent siRNAs indicating that the amount of a cellular component, perhaps RISC, limits siRNA activity.
Asunto(s)
Interferencia de ARN , ARN Interferente Pequeño/química , Complejo Silenciador Inducido por ARN/metabolismo , Unión Competitiva , Línea Celular Tumoral , Humanos , Cinesinas/genética , Fosfohidrolasa PTEN/genética , ARN Mensajero/metabolismo , Ribonucleasas/metabolismoRESUMEN
A systematic study on the effect of 2'-sugar modifications (2'-F (2'-F-2'-deoxy-nucleoside residues), 2'-O-Me (2'-O-methyl-nucleoside residues), and 2'-O-MOE [2'-O-(2-methoxyethyl)]-nucleoside residues) in the antisense and sense strands of short interference RNA (siRNA) was performed in HeLa cells. The study of the antisense strand of siRNAs demonstrated that activity depends on the position of the modifications in the sequence. The siRNAs with modified ribonucleotides at the 5'-end of the antisense strand were less active relative to the 3'-modified ones. The 2'-F sugar was generally well-tolerated on the antisense strand, whereas the 2'-O-Me showed significant shift in activity depending on the position of modification. The 2'-O-MOE modification in the antisense strand resulted in less active siRNA constructs regardless of placement position in the construct. The incorporation of the modified residues, e.g., 2'-O-Me and 2'-O-MOE, in the sense strand of siRNA did not show a strong positional preference. These results may provide guidelines to design effective and stable siRNAs for RNA interference mediated therapeutic applications.
Asunto(s)
Monoéster Fosfórico Hidrolasas/genética , ARN Interferente Pequeño/farmacología , Proteínas Supresoras de Tumor/genética , Animales , Secuencia de Bases , Línea Celular , Mutación de Línea Germinal , Células HeLa , Humanos , Mamíferos , Fosfohidrolasa PTEN , Monoéster Fosfórico Hidrolasas/antagonistas & inhibidores , Interferencia de ARN/efectos de los fármacos , ARN sin Sentido/química , ARN sin Sentido/farmacología , ARN Neoplásico/efectos de los fármacos , ARN Interferente Pequeño/química , Proteínas Supresoras de Tumor/antagonistas & inhibidoresRESUMEN
We have identified a small interfering RNA (siRNA) motif, consisting entirely of 2'-O-methyl and 2'-fluoro nucleotides, that displays enhanced plasma stability and increased in vitro potency. At one site, this motif showed remarkable >500-fold improvement in potency over the unmodified siRNA. This marks the first report of such a potent fully modified motif, which may represent a useful design for therapeutic oligonucleotides.
Asunto(s)
Oligonucleótidos/síntesis química , Monoéster Fosfórico Hidrolasas/genética , ARN Mensajero/química , ARN Interferente Pequeño/química , Proteínas Supresoras de Tumor/genética , Animales , Portadores de Fármacos , Estabilidad de Medicamentos , Células HeLa , Humanos , Ratones , Oligonucleótidos/química , Oligonucleótidos/farmacología , Fosfohidrolasa PTEN , Fosfatidiletanolaminas , Monoéster Fosfórico Hidrolasas/biosíntesis , ARN Mensajero/biosíntesis , ARN Interferente Pequeño/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Relación Estructura-Actividad , Transfección , Proteínas Supresoras de Tumor/biosíntesisRESUMEN
Substitution at the C(7) position of purine nucleotides by a potent electron-withdrawing nitro group facilitates the cleavage of glycosidic bonds under alkaline conditions. This property is useful for sequence-specific cleavage of DNA containing these analogues. Here we describe the preparation of 7-deaza-7-NO(2)-dA and 7-deaza-7-NO(2)-dG using two different approaches, starting from 2'-deoxy-adenosine and 6-chloro-7-deaza-guanine, respectively. These modified nucleosides were converted to nucleotide triphosphates, each of which can replace the corresponding, naturally occurring triphosphate to support PCR amplification. [structure: see text]
Asunto(s)
Nucleótidos de Desoxiadenina/síntesis química , Nucleótidos de Desoxiguanina/síntesis química , ADN/química , Estructura Molecular , Nucleósidos de Purina/síntesis química , Nucleósidos de Purina/química , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
UNLABELLED: Hepatocellular carcinoma (HCC) remains a significant clinical challenge with few therapeutic options available to cancer patients. MicroRNA 21-5p (miR-21) has been shown to be upregulated in HCC, but the contribution of this oncomiR to the maintenance of tumorigenic phenotype in liver cancer remains poorly understood. We have developed potent and specific single-stranded oligonucleotide inhibitors of miR-21 (anti-miRNAs) and used them to interrogate dependency on miR-21 in a panel of liver cancer cell lines. Treatment with anti-miR-21, but not with a mismatch control anti-miRNA, resulted in significant derepression of direct targets of miR-21 and led to loss of viability in the majority of HCC cell lines tested. Robust induction of caspase activity, apoptosis, and necrosis was noted in anti-miR-21-treated HCC cells. Furthermore, ablation of miR-21 activity resulted in inhibition of HCC cell migration and suppression of clonogenic growth. To better understand the consequences of miR-21 suppression, global gene expression profiling was performed on anti-miR-21-treated liver cancer cells, which revealed striking enrichment in miR-21 target genes and deregulation of multiple growth-promoting pathways. Finally, in vivo dependency on miR-21 was observed in two separate HCC tumor xenograft models. In summary, these data establish a clear role for miR-21 in the maintenance of tumorigenic phenotype in HCC in vitro and in vivo. IMPLICATIONS: miR-21 is important for the maintenance of the tumorigenic phenotype of HCC and represents a target for pharmacologic intervention.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Proliferación Celular/efectos de los fármacos , Redes Reguladoras de Genes/efectos de los fármacos , Neoplasias Hepáticas/metabolismo , MicroARNs/metabolismo , Oligorribonucleótidos Antisentido/farmacología , Animales , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Femenino , Xenoinjertos , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/patología , Ratones Desnudos , MicroARNs/antagonistas & inhibidores , MicroARNs/química , Invasividad Neoplásica , Oligorribonucleótidos Antisentido/uso terapéuticoRESUMEN
As part of a continued effort to identify inhibitors of hepatitis C viral (HCV) replication, we report here the synthesis and evaluation of a series of nucleoside analogues and their corresponding triphosphates. Nucleosides were evaluated for their ability to inhibit HCV RNA replication in a cell-based, subgenomic replicon system, while nucleoside triphosphates were evaluated for their ability to inhibit in vitro RNA synthesis mediated by the HCV RNA-dependent RNA polymerase, NS5B. 2'-C-Methyladenosine and 2'-C-methylguanosine were identified as potent inhibitors of HCV RNA replication, and the corresponding triphosphates were found to be potent inhibitors of HCV NS5B-mediated RNA synthesis. The data generated in the cell-based assay demonstrated a fairly stringent structure-activity relationship around the active nucleosides. Increase in steric bulk beyond methyl on C2, change in the stereo- or regiochemistry of the methyl substituent, or change of identity of the heterobase beyond that of the endogenous adenine or guanine was found to lead to loss of inhibitory activity. The results highlight the importance of the ribo configuration 2'- and 3'-hydroxy pharmacophores for inhibition of HCV RNA replication in the cell-based assay and demonstrate that inclusion of the 2'-C-methylribonucleoside pharmacophore leads to increased resistance to adenosine deaminase and purine nucleoside phosphorylase mediated metabolism.
Asunto(s)
Hepacivirus/química , Nucleósidos de Purina/síntesis química , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , Ribonucleósidos/síntesis química , Proteínas no Estructurales Virales/antagonistas & inhibidores , Adenosina Desaminasa/química , Enlace de Hidrógeno , Metilación , Conformación Molecular , Nucleósidos de Purina/química , Purina-Nucleósido Fosforilasa/química , Purinas/química , ARN Polimerasa Dependiente del ARN/química , Ribonucleósidos/química , Ribosa/química , Relación Estructura-Actividad , Proteínas no Estructurales Virales/químicaRESUMEN
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
Combining the structural elements of the second generation 2'-O-methoxyethyl (MOE) and locked nucleic acid (LNA) antisense oligonucleotide (AON) modifications yielded the highly nuclease resistant 2',4'-constrained MOE and ethyl bicyclic nucleic acids (cMOE and cEt BNA, respectively). Crystal structures of DNAs with cMOE or cEt BNA residues reveal their conformational preferences. Comparisons with MOE and LNA structures allow insights into their favourable properties for AON applications.
Asunto(s)
ADN/química , Desoxirribonucleasas/química , Modelos Moleculares , Nucleósidos/química , Oligorribonucleótidos/químicaRESUMEN
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.