RNase III-prepared short interfering RNAs induce degradation of SARS-coronavirus mRNAs in human cells / 生物工程学报
Chinese Journal of Biotechnology
;
(12): 484-489, 2004.
Artigo
em Chinês
| WPRIM
| ID: wpr-270100
ABSTRACT
SARS-associated coronavirus has been identified for the cause of Severe Acute Respiratory Syndrome, for which there is no efficacious drugs or vaccines. RNA interference (RNAi) is a process in cell to degradation specific target mRNA by double-stranded RNA. In mammalian cells, RNAi can be triggered by short interfering RNA (siRNA). RNA interference of virus-specific genes has emerged as a potential antiviral mechanism. This work evaluated if RNase III-prepared short interfering RNAs can induce specific degradation of SARS-coronavirus mRNAs in human cells. Three of SARS genes, RNA dependent RNA polymerase (RdRp), spike and nucleocapsid, were amplified with T7 promoter-flanked primers. Long length double-stranded RNA of these genes were transcribed in vitro and then were cleaved to <30bp length short interfering RNA with E. coli RNase III. These siRNAs were termed esiRNA-R, esiRNA-S and esiRNA-N respectively. RdRp, spike and nucleocapsid DNA fragments were inserted into the plasmid pGL3-Control, obtained plasmids pGL-R, pGL-S and pGL-N can express hybrid mRNAs luciferase-RdRp, spike and -nucleocapsid in cells. Above plasmids and esiRNAs were co-transfected to HEK293F cells with reference plasmid pRL-TK. Firefly luciferase and Renilla luciferase activity were measured. Hybrid mRNAs' abundance was measured using reverse transcription real-time PCR. Firefly luciferase expression of pGL-R was reduced to 13% by esiRNA-R. Expression of pGLS was reduced to 11% by esiRNA-S. Expression of pGL-N was reduced to 40% by esiRNA-N. Control esiRNAs didn't affect luciferase expression; Hybrid mRNAs' abundance was dramatically reduced by corresponding esiRNAs. RNase III-prepared short interfering RNAs induce robust and specific degradation of SARS-coronavirus mRNAs in HEK293F cells. These siRNAs could be used to inhibit SARS-coronavirus in future research.
Texto completo:
DisponíveL
Índice:
WPRIM (Pacífico Ocidental)
Assunto principal:
Fisiologia
/
Plasmídeos
/
RNA Mensageiro
/
RNA Viral
/
Células Cultivadas
/
RNA Interferente Pequeno
/
Interferência de RNA
/
Coronavírus Relacionado à Síndrome Respiratória Aguda Grave
/
Ribonuclease III
/
Genética
Limite:
Humanos
Idioma:
Chinês
Revista:
Chinese Journal of Biotechnology
Ano de publicação:
2004
Tipo de documento:
Artigo
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