Knockdown of Severe Acute Respiratory Syndrome Corona Virus (SARS-CoV) Genes by Small Interfering RNA (siRNA) Using siRNA-expression Vectors and Synthetic Double-stranded RNA (dsRNA) as a Model for siRNA Design

摘要

References(31) While offering the promise of therapeutic use in the future, RNA interference (RNAi) technology is useful to knock down genes posttranscriptionally. We attempted to knock down severe acute respiratory syndrome (SARS)-corona virus (Cov) genes using small interfering RNA (siRNA) employing siRNA-expression vectors and synthetic double-stranded RNA (dsRNA) as a model for effective siRNA design. First, we selected three target sites without mutations among 15 SARS-Cov strains using a prediction algorithm and constructed three siRNA-expression vectors. Using a pGL3 vector, we constructed three corresponding model target vectors to the firefly luciferase gene (Fluc), to which the model targets were connected. Using Renilla luciferase gene (Rluc) as the internal control, the three siRNA vectors knocked down the targets, providing effective target sequences. Almost identical results were obtained when Rluc was integrated into the pGL3-Fluc target vector. Next, effective structures of synthetic double-stranded RNA (dsRNA) were investigated using two targets. In all, six RNAs per target were synthesized: complementary sense and antisense 19-mer core RNAs; sense 21-mer RNAs having a 2-nucleotide (nt) match or unmatch overhang at the 3'-end; and antisense 21-mer RNAs having a 2-nt match or unmatch overhang at the 3'-end. The six RNAs provided nine species of dsRNAs (a blunt 19-mer duplex, a total of 4 19-mer/21-mer duplexes with a match or unmatch 2-nt overhang at the 3'- end of the sense or antisense strand, 4 21-mer/21-mer duplexes with match or unmatch 2-nt overhang at both ends) in combination. Targets were sense or antisense sequences. Generally, 19-mer/21-mer dsRNA with a match 2-nt overhang at the 3'- end of the antisense strand showed the highest activity, irrespective of the thermodynamic stabilities at terminal ends, suggesting that the 2-nt overhang is more critical than thermodynamic stabilities to select the antisense strand to the RNA-induced silencing complex (RISC).