Design of siRNA molecules for silencing of membrane glycoprotein, nucleocapsid phosphoprotein, and surface glycoprotein genes of SARS-CoV2

Abstract

The global COVID-19 pandemic caused by SARS-CoV2 infected millions of people and resulted in more than 4 million deaths worldwide. Apart from vaccines and drugs, RNA silencing is a novel approach for treating COVID-19. In the present study, siRNAs were designed for the conserved regions targeting three structural genes, M, N, and S, from forty whole-genome sequences of SARS-CoV2 using four different software, RNAxs, siDirect, i-Score Designer, and OligoWalk. Only siRNAs which were predicted in common by all the four servers were considered for further shortlisting. A multistep filtering approach has been adopted in the present study for the final selection of siRNAs by the usage of different online tools, viz., siRNA scales, MaxExpect, DuplexFold, and SMEpred. All these web-based tools consider several important parameters for designing functional siRNAs, e.g., target-site accessibility, duplex stability, position-specific nucleotide preference, inhibitory score, thermodynamic parameters, GC content, and efficacy in cleaving the target. In addition, a few parameters like GC content and dG value of the entire siRNA were also considered for shortlisting of the siRNAs. Antisense strands were subjected to check for any off-target similarities using BLAST. Molecular docking was carried out to study the interactions of guide strands with AGO2 protein. A total of six functional siRNAs (two for each gene) have been finally selected for targeting M, N, and S genes of SARS-CoV2. The siRNAs have not shown any off-target effects, interacted with the domain(s) of AGO2 protein, and were efficacious in cleaving the target mRNA. However, the siRNAs designed in the present study need to be tested in vitro and in vivo in the future.