In Silico and In Vitro development of novel small interfering RNAs (siRNAs) to inhibit SARS-CoV-2

SARS-CoV-2 is causing severe to moderate respiratory tract infections, posing global health, social life, and economic threats. Our design strategy for siRNAs differs from existing studies through a step-by-step filtration process utilizing integrative bioinformatics protocols and web tools. Stage one: Multiple Sequence Alignment was employed to identify the most conserved areas. Stage two involves using various online tools, among the most reputable tools for building siRNA. The first filtration step of siRNA uses the Huesken dataset, estimating a 90 % experimental inhibition. The second filtration stage involves choosing the most suitable and targeted siRNA by utilizing thermodynamics and Target Accessibility of siRNAs. The final filtration step is off-target filtration using BLAST with specific parameters. Four of the 258 siRNAs were chosen for their potency and specificity, targeting conserved regions (NSP8, NSP12, and NSP14) with minimal human transcripts off-targets. We conducted in-vitro experiments, including cytotoxicity, TCID50, and RT-PCR assays. When tested on the SARS-CoV-2 strain hCoV-19/Egypt/NRC-03/2020 at 100 nM, none showed cellular toxicity. The TCID50 assay confirmed viral replication reduction at 12 h.p.i; the efficacy of the four siRNAs and their P value were highly significant. siRNA2 maintaining efficacy at 24, 36, and 48 h.p.i, while siRNA4 had a significant P value (≤0.0001) at 48 h.p.i. At 24 h.p.i, siRNA2 and siRNA4 showed statistical significance in viral knockdown of the virus's S gene and ORF1b gene by 95 %, 89 %, and 96 %, 97 %, respectively. Our computational method and experimental assessment of specific siRNAs have led us to conclude that siRNA2 and siRNA4 could be promising new therapies for SARS-CoV-2 that need further development.