RESUMO
RNase L is activated by 2',5'-oligoadenylates (2-5A) at subnanomolar levels to cleave single-stranded RNA. We previously reported the hypothesis that the introduction of an 8-methyladenosine residue at the 2'-terminus of the 2-5A tetramer shifts the 2-5A binding site of RNase L. In this study, we synthesized various 5'-modified 2-5A analogs with 8-methyladenosine at the 2'-terminus. The doxifluridine-conjugated 8-methyladenosine-substituted 2-5A analog was significantly more effective as an activator of RNase L than the parent 5'-monophophorylated 2-5A tetramer and showed a tumor suppressive effect against human cervical cancer cells.
Assuntos
Antineoplásicos/farmacologia , Endorribonucleases/metabolismo , Floxuridina/farmacologia , Antineoplásicos/química , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Ativação Enzimática , Feminino , Floxuridina/química , Células HeLa , Humanos , Espectrometria de Massas , Espectroscopia de Prótons por Ressonância MagnéticaRESUMO
We developed a practical and reliable method for synthesizing an abasic deoxyribonucleoside, 1,2-dideoxy-d-ribofuranose (dR(H)) via elimination of nucleobase from thymidine. To synthesize oligonucleotides bearing dR(H) by the standard phosphoramidite solid-phase method, dR(H) was converted to the corresponding phosphoramidite derivative and linked to a solid support (controlled pore glass resin). Chemically modified small interfering RNAs (siRNAs) possessing dR(H) at their 3'-overhang regions were synthesized. Introducing dR(H) to the 3'-end of the antisense strand of siRNA reduced its knockdown effect.
Assuntos
Desoxirribose/análogos & derivados , Interferência de RNA , RNA Interferente Pequeno/genética , Desoxirribose/síntese química , Desoxirribose/química , Expressão Gênica , Inativação Gênica , Genes Reporter , Células HeLa , Humanos , Oligonucleotídeos/síntese química , Oligonucleotídeos/químicaRESUMO
Gene silencing and RNA interference are major cellular processes that control gene expression via the cleavage of target mRNA. Eukaryotic translation initiation factor 2C2 (EIF2C2, Argonaute protein 2, Ago2) is considered to be the major player of RNAi as it is the core component of RISC complexes. While a considerable amount of research has focused on RNA interference and its associated mechanisms, the nature and mechanisms of nucleotide recognition by the PAZ domain of EIF2C2/Ago2 have not yet been characterized. Here, we demonstrate that the EIF2C2/Ago2 PAZ domain has an inherent lack of binding to adenine nucleotides, a feature that highlights the poor binding of 3'-adenylated RNAs with the PAZ domain as well as the selective high trimming of the 3'-ends of miRNA containing adenine nucleotides. We further show that the PAZ domain selectively binds all ribonucleotides (except adenosine), whereas it poorly recognizes deoxyribonucleotides. In this context, the modification of dTMP to its ribonucleotide analogue gave a drastic improvement of binding enthalpy and, hence, binding affinity. Additionally, higher in vivo gene silencing efficacy was correlated with the stronger PAZ domain binders. These findings provide new insights into the nature of the interactions of the EIF2C2/Ago2 PAZ domain.
Assuntos
Proteínas Argonautas/química , Proteínas Argonautas/metabolismo , Metabolismo Energético , Nucleotídeos/metabolismo , Interferência de RNA , Proteínas Argonautas/deficiência , Proteínas Argonautas/genética , Sequência de Bases , Entropia , Células HeLa , Temperatura Alta , Humanos , Modelos Moleculares , Ligação Proteica , Estrutura Terciária de Proteína , RNA Interferente Pequeno/genética , Especificidade por SubstratoRESUMO
Positron emission tomography (PET) is a highly sensitive quantitative imaging technique for studying molecular pathways and interactions in vivo. This imaging technique plays a key role in drug discovery, pharmacokinetics, pharmacodynamics, and assessing in vivo distribution. In this study, we designed an ethynylbenzene-substituted glycol (M(E)) as a versatile probe for PET labeling of oligonucleotides through a click reaction.
Assuntos
Acetileno/análogos & derivados , Glicóis/química , Sondas Moleculares/síntese química , Oligonucleotídeos/química , Acetileno/química , Química Click , Glicóis/síntese química , Sondas Moleculares/química , Oligonucleotídeos/síntese química , Tomografia por Emissão de Pósitrons , Estereoisomerismo , TemperaturaRESUMO
We have developed chemically modified siRNAs and miRNAs bearing urea/thiourea-bridged aromatic compounds at their 3'-end for RNAi therapy. Chemically modified RNAs possessing urea/thiourea-bridged aromatic compounds instead of naturally occurring dinucleotides at the 3'-overhang region were easily prepared in good yields and were more resistant to nucleolytic hydrolysis than unmodified RNA. siRNAs containing urea or thiourea derivatives showed the desired knockdown effect. Furthermore, modified miR-143 duplexes carrying the urea/thiourea compounds in the 3'-end of each strand were able to inhibit the growth of human bladder cancer T24 cells.
Assuntos
Terapia Genética/métodos , MicroRNAs/química , Interferência de RNA , RNA Interferente Pequeno/química , Tioureia/química , Ureia/química , Hidrocarbonetos Aromáticos com Pontes , Linhagem Celular Tumoral , Células HeLa , Humanos , MicroRNAs/genética , MicroRNAs/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , TransfecçãoRESUMO
Short interfering RNA (siRNA) has been proven to be an utilizable tool for post-transcriptional gene silencing research. In this study, we designed and synthesized two glucosamine analogues and tried to modify the siRNA using these two glucosamine analogues at the 3'-overhang region of siRNAs to improve the nuclease resistance and to overcome some other weak points. The siRNAs modified with glucosamine analogues had almost no effect of the thermal stability and showed strong resistance to nuclease degradation. Some of them kept the same gene silencing activity level as unmodified siRNA.
Assuntos
Glucosamina/química , Oligonucleotídeos/síntese química , RNA Interferente Pequeno/síntese química , Animais , Exonucleases/metabolismo , Células HeLa , Humanos , Luciferases de Renilla/genética , Luciferases de Renilla/metabolismo , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Renilla/enzimologia , Venenos de Serpentes/enzimologia , Temperatura de TransiçãoRESUMO
To elucidate the role of the sugar moiety in the two natural nucleotides of the 3'-overhang region of small interfering RNA (siRNA), we synthesized siRNAs that incorporated two abasic nucleosides, 1-deoxy-D-ribofuranose (R(H)). We improved the method for preparing an O-protected abasic nucleoside, 1-deoxy-2,3,5-tri-O-benzoyl-ß-D-ribofuranose, via the reductive cleavage of the anomeric position of 1-O-acetyl-2,3,5-tri-O-benzoyl-ß-D-ribofuranose. To incorporate R(H) into oligonucleotides by the standard phosphoramidite solid phase method, R(H) was converted into its phosphoramidite derivative and the solid support linked to a controlled pore glass resin. Chemically modified RNAs possessing R(H) at the 3'-overhang region were easily prepared in good yields. siRNAs containing R(H) showed moderate nuclease-resistance and a desirable knockdown effect.