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1.
Nucleic Acids Res ; 42(22): 13456-68, 2014 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-25398895

RESUMO

Bicyclic oxazaphospholidine monomers were used to prepare a series of phosphorothioate (PS)-modified gapmer antisense oligonucleotides (ASOs) with control of the chirality of each of the PS linkages within the 10-base gap. The stereoselectivity was determined to be 98% for each coupling. The objective of this work was to study how PS chirality influences biophysical and biological properties of the ASO including binding affinity (Tm), nuclease stability, activity in vitro and in vivo, RNase H activation and cleavage patterns (both human and E. coli) in a gapmer context. Compounds that had nine or more Sp-linkages in the gap were found to be poorly active in vitro, while compounds with uniform Rp-gaps exhibited activity very similar to that of the stereo-random parent ASOs. Conversely, when tested in vivo, the full Rp-gap compound was found to be quickly metabolized resulting in low activity. A total of 31 ASOs were prepared with control of the PS chirally of each linkage within the gap in an attempt to identify favorable Rp/Sp positions. We conclude that a mix of Rp and Sp is required to achieve a balance between good activity and nuclease stability.


Assuntos
Oligonucleotídeos Antissenso/química , Oligonucleotídeos Fosforotioatos/química , Animais , Células Cultivadas , Proteínas Fúngicas/metabolismo , Camundongos , Oligonucleotídeos Antissenso/síntese química , Oligonucleotídeos Antissenso/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Ribonuclease H/metabolismo , Endonucleases Específicas para DNA e RNA de Cadeia Simples/metabolismo , Estereoisomerismo , Temperatura Ambiente
2.
J Org Chem ; 79(18): 8877-81, 2014 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-25137618

RESUMO

We compare the duplex stabilizing properties of 2'-fluorinated nucleic acid analogues with furanose and non-furanose ring systems and dissect the relative contributions of hydration, sugar conformation, and fluorine configuration toward the overall T(m) value. We find that the stabilization imparted by fluorine substitution is additive over that obtained by restricting the conformation of the sugar ring itself. Our studies support further evaluation of fluorinated nucleic acid analogues with non-furanose sugar rings as surrogates of 2'-F RNA for therapeutic antisense applications.


Assuntos
Carboidratos/química , Ácidos Nucleicos/química , RNA/química , Ribose/química , Pareamento de Bases , Configuração de Carboidratos , Conformação Molecular , Estrutura Molecular , Conformação de Ácido Nucleico
3.
Chem Commun (Camb) ; 48(66): 8195-7, 2012 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-22614180

RESUMO

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.


Assuntos
DNA/química , Desoxirribonucleases/química , Modelos Moleculares , Nucleosídeos/química , Oligorribonucleotídeos/química
4.
Genes Dev ; 26(10): 1098-109, 2012 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-22588721

RESUMO

An established paradigm in pre-mRNA splicing is the recognition of the 5' splice site (5'ss) by canonical base-pairing to the 5' end of U1 small nuclear RNA (snRNA). We recently reported that a small subset of 5'ss base-pair to U1 in an alternate register that is shifted by 1 nucleotide. Using genetic suppression experiments in human cells, we now demonstrate that many other 5'ss are recognized via noncanonical base-pairing registers involving bulged nucleotides on either the 5'ss or U1 RNA strand, which we term "bulge registers." By combining experimental evidence with transcriptome-wide free-energy calculations of 5'ss/U1 base-pairing, we estimate that 10,248 5'ss (∼5% of human 5'ss) in 6577 genes use bulge registers. Several of these 5'ss occur in genes with mutations causing genetic diseases and are often associated with alternative splicing. These results call for a redefinition of an essential element for gene expression that incorporates these registers, with important implications for the molecular classification of splicing mutations and for alternative splicing.


Assuntos
Processamento Alternativo , Pareamento de Bases , Sítios de Splice de RNA/genética , RNA Nuclear Pequeno/química , RNA Nuclear Pequeno/genética , Ribonucleotídeos/química , Células HeLa , Humanos , Conformação de Ácido Nucleico , Transcriptoma
5.
Nucleic Acids Res ; 40(13): 6135-43, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22467214

RESUMO

We report the evaluation of 20-, 18-, 16- and 14-mer phosphorothioate (PS)-modified tricycloDNA (tcDNA) gapmer antisense oligonucleotides (ASOs) in T(m), cell culture and animal experiments and compare them to their gap-matched 20-mer 2'-O-methoxyethyl (MOE) and 14-mer 2',4'-constrained ethyl (cEt) counterparts. The sequence-matched 20-mer tcDNA and MOE ASOs showed similar T(m) and activity in cell culture under free-uptake and cationic lipid-mediated transfection conditions, while the 18-, 16- and 14-mer tcDNA ASOs were moderate to significantly less active. These observations were recapitulated in the animal experiments where the 20-mer tcDNA ASO formulated in saline showed excellent activity (ED(50) 3.9 mg/kg) for reducing SR-B1 mRNA in liver. The tcDNA 20-mer ASO also showed better activity than the MOE 20-mer in several extra-hepatic tissues such as kidney, heart, diaphragm, lung, fat, gastrocnemius and quadriceps. Interestingly, the 14-mer cEt ASO showed the best activity in the animal experiments despite significantly lower T(m) and 5-fold reduced activity in cell culture relative to the 20-mer tcDNA and MOE-modified ASOs. Our experiments establish tcDNA as a useful modification for antisense therapeutics and highlight the role of chemical modifications in influencing ASO pharmacology and pharmacokinetic properties in animals.


Assuntos
Oligodesoxirribonucleotídeos Antissenso/química , Receptores Depuradores Classe B/genética , Animais , Linhagem Celular , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Oligodesoxirribonucleotídeos Antissenso/administração & dosagem , Oligodesoxirribonucleotídeos Antissenso/farmacocinética , RNA Mensageiro/metabolismo , Receptores Depuradores Classe B/metabolismo , Distribuição Tecidual
6.
Arthritis Res Ther ; 14(1): R38, 2012 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-22353730

RESUMO

INTRODUCTION: The c-Jun N-terminal kinase (JNK) is a key regulator of matrix metalloproteinase (MMP) and cytokine production in rheumatoid arthritis (RA) and JNK deficiency markedly protects mice in animal models of arthritis. Cytokine-induced JNK activation is strictly dependent on the mitogen-activated protein kinase kinase 7 (MKK7) in fibroblast-like synoviocytes (FLS). Therefore, we evaluated whether targeting MKK7 using anti-sense oligonucleotides (ASO) would decrease JNK activation and severity in K/BxN serum transfer arthritis. METHODS: Three 2'-O-methoxyethyl chimeric ASOs for MKK7 and control ASO were injected intravenously in normal C57BL/6 mice. PBS, control ASO or MKK7 ASO was injected from Day -8 to Day 10 in the passive K/BxN model. Ankle histology was evaluated using a semi-quantitative scoring system. Expression of MKK7 and JNK pathways was evaluated by quantitative PCR and Western blot analysis. RESULTS: MKK7 ASO decreased MKK7 mRNA and protein levels in ankles by about 40% in normal mice within three days. There was no effect of control ASO on MKK7 expression and MKK7 ASO did not affect MKK3, MKK4 or MKK6. Mice injected with MKK7 ASO had significantly less severe arthritis compared with control ASO (P < 0.01). Histologic evidence of synovial inflammation, bone erosion and cartilage damage was reduced in MKK7 ASO-treated mice (P < 0.01). MKK7 deficiency decreased phospho-JNK and phospho-c-Jun in ankle extracts (P < 0.05), but not phospho-MKK4. Interleukin-1beta (IL-1ß), MMP3 and MMP13 gene expression in ankle joints were decreased by MKK7 ASO (P < 0.01). CONCLUSIONS: MKK7 plays a critical regulatory role in the JNK pathway in a murine model of arthritis. Targeting MKK7 rather than JNK could provide site and event specificity when treating synovitis.


Assuntos
Artrite Reumatoide/genética , Proteínas Quinases JNK Ativadas por Mitógeno/genética , MAP Quinase Quinase 7/genética , Sistema de Sinalização das MAP Quinases/genética , Animais , Articulação do Tornozelo/metabolismo , Articulação do Tornozelo/patologia , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Western Blotting , Expressão Gênica , Técnicas de Silenciamento de Genes , Injeções Intravenosas , Interleucina-1beta/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , MAP Quinase Quinase 7/metabolismo , Metaloproteinase 13 da Matriz/genética , Camundongos , Camundongos Endogâmicos C57BL , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/genética , Fosforilação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Índice de Gravidade de Doença , Membrana Sinovial/metabolismo , Membrana Sinovial/patologia , Sinovite/genética , Sinovite/metabolismo , Fatores de Tempo
7.
J Am Chem Soc ; 133(41): 16642-9, 2011 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-21919455

RESUMO

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.


Assuntos
Ácidos Nucleicos/química , Oligonucleotídeos/química , RNA/química , Álcoois Açúcares/química , Modelos Moleculares , Conformação Molecular , Estereoisomerismo
8.
Bioorg Med Chem Lett ; 21(4): 1122-5, 2011 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-21256012

RESUMO

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.


Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/síntese química , Oligonucleotídeos/síntese química , Sequência de Bases , Compostos Bicíclicos Heterocíclicos com Pontes/química , Conformação de Ácido Nucleico , Oligonucleotídeos/química , Estereoisomerismo , Temperatura de Transição
9.
Bioorg Med Chem Lett ; 21(1): 588-91, 2011 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-21075634

RESUMO

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.


Assuntos
Oligonucleotídeos/química , Oxigênio/química , Conformação de Ácido Nucleico , Temperatura de Transição
10.
J Med Chem ; 53(23): 8309-18, 2010 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-21058707

RESUMO

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.


Assuntos
Oligonucleotídeos/farmacologia , Biofísica , Ressonância Magnética Nuclear Biomolecular , Oligonucleotídeos/química , Relação Estrutura-Atividade
11.
J Am Chem Soc ; 132(42): 14942-50, 2010 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-20886816

RESUMO

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.


Assuntos
Alcenos/química , Ácidos Nucleicos/química , Oxigênio/química , Animais , Cristalografia por Raios X , Camundongos , Modelos Moleculares , Hibridização de Ácido Nucleico , PTEN Fosfo-Hidrolase/genética , RNA Mensageiro/genética , Relação Estrutura-Atividade
12.
J Med Chem ; 53(10): 3919-26, 2010 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-20420385

RESUMO

A peptide nucleic acid (PNA) targeting a splice junction of the murine PTEN primary transcript was covalently conjugated to various basic peptides. When systemically administered to healthy mice, the conjugates displayed sequence-specific alteration of PTEN mRNA splicing as well as inhibition of full length PTEN protein expression. Correlating activity with drug concentration in various tissues indicated strong tissue-dependence, with highest levels of activity observed in adipose tissue. While the presence of a peptide carrier was found to be crucial for efficient delivery to tissue, little difference was observed between the various peptides evaluated. A second PNA-conjugate targeting the murine insulin receptor primary transcript showed a similar activity profile, suggesting that short basic peptides can generally be used to effectively deliver peptide nucleic acids to adipose tissue.


Assuntos
Tecido Adiposo/metabolismo , Oligopeptídeos/química , PTEN Fosfo-Hidrolase/biossíntese , Ácidos Nucleicos Peptídicos/farmacologia , RNA Antissenso/farmacologia , Receptor de Insulina/biossíntese , Animais , Linhagem Celular , Portadores de Fármacos , Rim/metabolismo , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , PTEN Fosfo-Hidrolase/genética , Ácidos Nucleicos Peptídicos/administração & dosagem , Ácidos Nucleicos Peptídicos/química , Ácidos Nucleicos Peptídicos/farmacocinética , Sítios de Splice de RNA , Processamento de RNA , RNA Antissenso/administração & dosagem , RNA Antissenso/química , RNA Antissenso/farmacocinética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptor de Insulina/genética , Relação Estrutura-Atividade , Distribuição Tecidual
13.
J Org Chem ; 75(5): 1569-81, 2010 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-20136157

RESUMO

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.


Assuntos
Cristalinas/síntese química , Ácidos Nucleicos/síntese química , Nucleosídeos/síntese química , Oligonucleotídeos/química , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Fenômenos Biofísicos , Catálise , Cristalinas/química , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Estrutura Molecular , Ácidos Nucleicos/química , Nucleosídeos/química , Estereoisomerismo
14.
J Biol Chem ; 284(4): 2535-48, 2009 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-19017633

RESUMO

Human Dicer is an integral component of the RNA interference pathway. Dicer processes premicro-RNA and double-strand RNA to, respectively, mature micro-RNA and short interfering RNA (siRNA) and transfers the processed products to the RNA-induced silencing complex. To better understand the factors that are important for the binding, translocation, and selective recognition of the siRNA strands, we determined the binding affinities of human Dicer for processed products (siRNA) and short single-strand RNAs (ssRNA). siRNAs and ssRNAs competitively inhibited human Dicer activity, suggesting that they are interacting with the active site of the enzyme. The dissociation constants (Kd) for unmodified siRNAs were 5-11-fold weaker compared with a 27-nucleotide double-strand RNA substrate. Chemically modified siRNAs exhibited binding affinities for Dicer comparable with the substrate. 3'-dinucleotide overhangs in the siRNA affected the binding affinity of human Dicer for the siRNA and biased strand loading into RNA-induced silencing complex. The Kd values for the ssRNAs ranged from 3- to 40-fold weaker than the Kd for the substrate. Sequence composition of the 3'-terminal nucleotides of the ssRNAs exhibited the greatest effect on Dicer binding. Dicer cleaved substrates containing short siRNA-like double-strand regions and extended 3' or 5' ssRNA overhangs in the adjacent ssRNA regions. Remarkably, cleavage sites were observed consistent with the enzyme entering the substrate from the extended 3' ssRNA terminus. These data suggest that the siRNAs and ssRNAs interact predominantly with the PAZ domain of the enzyme. Finally, the tightest binding siRNAs were also more potent inhibitors of gene expression.


Assuntos
Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Ribonuclease III/metabolismo , Sequência de Bases , Cromatografia Líquida de Alta Pressão , Inativação Gênica , Células HeLa , Humanos , Modelos Genéticos , RNA/química , Proteínas de Ligação a RNA/genética , Ribonuclease III/genética , Espectrometria de Massas por Ionização por Electrospray , Especificidade por Substrato
15.
J Med Chem ; 48(4): 901-4, 2005 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-15715458

RESUMO

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.


Assuntos
Oligonucleotídeos/síntese química , Monoéster Fosfórico Hidrolases/genética , RNA Mensageiro/química , RNA Interferente Pequeno/química , Proteínas Supressoras de Tumor/genética , Animais , Portadores de Fármacos , Estabilidade de Medicamentos , Células HeLa , Humanos , Camundongos , Oligonucleotídeos/química , Oligonucleotídeos/farmacologia , PTEN Fosfo-Hidrolase , Fosfatidiletanolaminas , Monoéster Fosfórico Hidrolases/biossíntese , RNA Mensageiro/biossíntese , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Relação Estrutura-Atividade , Transfecção , Proteínas Supressoras de Tumor/biossíntese
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