RESUMO
A systematic structure-activity relationship study of 4'-thioribose containing small interfering RNAs (siRNAs) has led to the identification of highly potent and stable antisense constructs. To enable this optimization effort for both in vitro and in vivo applications, we have significantly improved the yields of 4'-thioribonucleosides by using a chirally pure (R)-sulfoxide precursor. siRNA duplexes containing strategically placed regions of 4'-thio-RNA were synthesized and evaluated for RNA interference activity and plasma stability. Stretches of 4'-thio-RNA were well tolerated in both the antisense and sense strands. However, optimization of both the number and placement of 4'-thioribonucleosides was necessary for maximal potency. These optimized siRNAs were generally equipotent or superior to native siRNAs and exhibited increased thermal and plasma stability. Furthermore, significant improvements in siRNA activity and plasma stability were achieved by judicious combination of 4'-thioribose with 2'-O-methyl and 2'-O-methoxyethyl modifications. These optimized 4'-thio-siRNAs may be valuable for developing stable siRNAs for therapeutic applications.
Assuntos
Interferência de RNA , RNA Interferente Pequeno/química , Ribonucleases/sangue , Tionucleosídeos/síntese química , Animais , Estabilidade de Medicamentos , Células HeLa , Calefação , Humanos , Técnicas In Vitro , Camundongos , Compostos Organofosforados/síntese química , Compostos Organofosforados/química , PTEN Fosfo-Hidrolase/biossíntese , PTEN Fosfo-Hidrolase/genética , Plasma , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Interferente Pequeno/metabolismo , RNA Interferente Pequeno/farmacologia , Ribose/química , Estereoisomerismo , Relação Estrutura-Atividade , Sulfóxidos/química , Tionucleosídeos/química , Tionucleosídeos/farmacologiaRESUMO
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.
Assuntos
Monoéster Fosfórico Hidrolases/genética , RNA Interferente Pequeno/farmacologia , Proteínas Supressoras de Tumor/genética , Animais , Sequência de Bases , Linhagem Celular , Mutação em Linhagem Germinativa , Células HeLa , Humanos , Mamíferos , PTEN Fosfo-Hidrolase , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Interferência de RNA/efeitos dos fármacos , RNA Antissenso/química , RNA Antissenso/farmacologia , RNA Neoplásico/efeitos dos fármacos , RNA Interferente Pequeno/química , Proteínas Supressoras de Tumor/antagonistas & inibidoresRESUMO
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/biossínteseRESUMO
In a search to identify chemical modifications to improve the properties of siRNA, we have investigated the effect of the 2 '-O-methyl-2-thiouridine modification on the biological activity of siRNA. Our results indicate that judicious placement of 2 '-O-methyl-2-thiouridine residues could lead to modified siRNA with activity in mammalian cells.
Assuntos
RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismo , Tiouridina/análogos & derivados , Células Cultivadas , Desenho de Fármacos , Células HeLa , Humanos , Oligonucleotídeos/síntese química , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , RNA Interferente Pequeno/genética , Tiouridina/síntese química , Tiouridina/química , Tiouridina/metabolismoRESUMO
A novel 2'-modification, 2'-O-[2-(methylthio)ethyl] or 2'-O-MTE, has been incorporated into oligonucleotides and evaluated for properties relevant to antisense activity. The results were compared with the previously characterized 2'-O-[2-(methoxy)ethyl] 2'-O-MOE modification. As expected, the 2'-O-MTE modified oligonucleotides exhibited improved binding to human serum albumin compared to the 2'-O-MOE modified oligonucleotides. The 2'-O-MTE oligonucleotides maintained high binding affinity to target RNA. Nuclease digestion of 2'-O-MTE oligonucleotides showed that they have limited resistance to exonuclease degradation. We analyzed the crystal structure of a decamer DNA duplex containing the 2'-O-MTE modifcation. Analysis of the crystal structure provides insight into the improved RNA binding affinity, protein binding affinity and limited resistance of 2'-O-MTE modified oligonucleotides to exonuclease degradation.