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.