Screening of siRNAs for silencing of SARS-CoV-2 genes by bioluminescence assay
To determine the silencing activity of 15 anti-SARS-CoV-2 siRNAsin vitro , we constructed plasmids pRdRp-full, pVAX-N-IRES-LUC and pVAX-LP-IRES-LUC simultaneously expressing SARS-CoV-2 genes RdRp (RNA dependent RNA polymerase), N (nucleocapsid), LP (leader protein), respectively, and the reporter gene of firefly luciferase. Hep-2 cells were co-transfected with vectors pRdRp-full, pVAX-N-IRES-LUC and pVAX-LP-IRES-LUC and 15 different siRNA variants to evaluate their knockdown activity. siRNA against luciferase (siLuc) and GFP (siGFP) were used as a positive and negative control, respectively. Eight of the 15 designed specific siRNAs significantly reduced luciferase activity in vitro with various levels of efficacy compared to non-specific siGFP and/or cells, transfected with plasmid only (table 1, fig. 2). As indicated in table 1: siLP-1, siN-3, siN-4, siN-5, siR-6, siR-7, siR-11 and siR-13 significantly decreased the level of luminescence as follows, %: 82.43 (P<0.05), 74.53 (P<0.01), 69.21 (P<0.05), 84.03 (P<0.01), 67.95 (P<0.05), 88.28 (P<0.01), 68.77 (P=0.0519) and 65.17 (P=0.0519), respectively, compared with cells transfected with plasmid only and 84.67 (P<0.05), 76.35 (P<0.05), 64.88 (P=0.0568), 81.78 (P<0.05), 63.45 (P=0.0666), 89.12 (P<0.01), 71 (P<0.05) and 67.66 (P<0.05), respectively, compared to cells transfected with non-specific siGFP. Control siLuc significantly reduced expression of firefly luciferase as follows, %: 70.24 (P<0.001), 77.69 (P<0.05) and 53.1 (NS) compared to cells transfected with pRdRp-full, pVAX-N-IRES-LUC and pVAX-LP-IRES-LUC plasmid only, respectively; as follows, %: 72.37 (P<0.01), 74.56 (P<0.05) and 56.63 (NS) compared to cells transfected with pRdRp-full, pVAX-N-IRES-LUC and pVAX-LP-IRES-LUC plasmid, respectively, followed by transfection with siGFP. Of note, we found no significant difference in luciferase expression between cells transfected with plasmid only or after co-transfection with plasmid and non-specific siGFP (table 1, fig. 2). According to results plotted at figure 2 and listed in table 1, siN-3, siN-4, siR-7 and siR-11 significantly reduced luciferase expression in both cells treated with plasmid only and cells co-transfected with plasmid and siRNA. The latter molecules were chosen as the most potent SARS-CoV-2 silencing siRNAs and were further studied.