Materials and Methods
Plasmid constructs
and design of siRNA
The primary screening of siRNAs was performed in vitro using
vectors expressing viral genes fused with the firefly luciferase
reporter gene. Three vectors expressing full-size genes and gene
fragments of SARS-CoV-2 (NCBI
RefSeq NC_045512.2) leader protein NSP1 (540 bp), RdRp RNA-dependent
RNA polymerase (2796 bp) and nucleocapsid protein N (1260 bp) were
designed (pVAX-LP-IRES-LUC, pRdRp-full and pVAX-N-IRES-LUC,
respectively) (Fig.1). Plasmid pVAX-1 (Thermo Fisher Scientific,
Waltham, Massachusetts, USA) ensuring stable protein expression in
eukaryotic cells, was used as expression vector. The expression plasmids
containing virus-specific inserts were synthesized as three bicistronic
expression plasmids containing IRES fragment (Takara Bio USA, Mountain
View, California, USA) (Fig. 1a-c). The vectors allow mRNA transcription
of target (RdRp or N or NSP1) and reporter (Luc) genes under a single
CMV promoter followed by translation of the target and reporter
proteins. A single RNA-transcript assures comparable expression of both
genes.
In total 15 siRNA duplexes targeting NSP1, RdRp and N proteins of
SARS-CoV-2 (Fig. 1d) were designed in silico and synthesized by
“DNA-technology” company (Moscow, Russia) (sequences are described in
a patent application (27)). siRNA against
firefly luciferase (siLuc)(28) and GFP
(sense strand: 5’- CAAGCUGACCCUGAAGUUCtt-3’) were used as controls.