In silico prediction and experimental evaluation of potential siRNAs against SARS-CoV-2 inhibition in Vero E6 cells

doi:10.1016/j.jksus.2022.102049

. 2022 Jun;34(4):102049.

doi: 10.1016/j.jksus.2022.102049. Epub 2022 Apr 26.

In silico prediction and experimental evaluation of potential siRNAs against SARS-CoV-2 inhibition in Vero E6 cells

Sayed Sartaj Sohrab^{1

2}, Sherif Aly El-Kafrawy^{1

2}, Esam Ibraheem Azhar^{1

2}

Affiliations

¹ Special Infectious Agents Unit, King Fahd Medical Research Center, King Abdulaziz University, Post Box, No-80216, Jeddah 21589, Saudi Arabia.
² Department of Medical Laboratory Sciences, Faculty of Applied Medical Sciences, King Abdulaziz University, Jeddah, Saudi Arabia.

PMID: 35493709
PMCID: PMC9040457
DOI: 10.1016/j.jksus.2022.102049

In silico prediction and experimental evaluation of potential siRNAs against SARS-CoV-2 inhibition in Vero E6 cells

Sayed Sartaj Sohrab et al. J King Saud Univ Sci. 2022 Jun.

. 2022 Jun;34(4):102049.

doi: 10.1016/j.jksus.2022.102049. Epub 2022 Apr 26.

Authors

Sayed Sartaj Sohrab^{1

2}, Sherif Aly El-Kafrawy^{1

2}, Esam Ibraheem Azhar^{1

2}

Affiliations

¹ Special Infectious Agents Unit, King Fahd Medical Research Center, King Abdulaziz University, Post Box, No-80216, Jeddah 21589, Saudi Arabia.
² Department of Medical Laboratory Sciences, Faculty of Applied Medical Sciences, King Abdulaziz University, Jeddah, Saudi Arabia.

PMID: 35493709
PMCID: PMC9040457
DOI: 10.1016/j.jksus.2022.102049

Abstract

Objective: The acute cases of pneumonia (COVID-19) were first reported from China in December 2019, and the pathogen was identified as SARS-CoV-2. Currently, many vaccines have been developed against this virus by using multiple genes, applying different platforms, and used for the vaccinations of the human population. Spike protein genes play an important role in host cell attachment and viral entry and have been extensively used for the development of vaccine and antiviral therapeutics. Short interfering RNA is also known as silencing RNA and contribute a significant role to regulate the expression of a specific gene. By using this technology, virus inhibition has been demonstrated against many viral diseases.

Methods: In this work, we have reported the Insilico prediction, designing, and experimental validation of siRNAs antiviral potency against SARS-CoV-2-S-RBD. The siDirect 2.0 was selected for siRNAs prediction, and secondary structure was predicted by RNAfold while the HNADOCK was used for molecular docking analysis and specific binding of siRNAs to the selected target. We have used and evaluated four siRNAs for cellular toxicity and determination of antiviral efficiency based on the Ct value of q-real-time PCR in Vero E6 cells.

Results: Based on the experimental evaluation and analysis of results from generated data, we observed that there is no cytotoxicity for any tested siRNAs in Vero E6 cells. Total four siRNA were filtered out from twenty-one siRNAs following the strict selection and scoring criteria. The better antiviral efficiency was observed in 3rd siRNAs based on the Ct value of q-real-time PCR. The results that emerged from this study encouraged us to validate the efficiency of these siRNAs in multiple cells by using alone and in a combination of two or more siRNAs to inhibit the SARS-CoV-2 proliferation.

Conclusion: The Insilico prediction, molecular docking analysis provided the selection of better siRNAs. Based on the experimental evaluation only 3rd siRNA was found to be more effective than others and showed better antiviral efficiency. These siRNAs should also be evaluated in other cell lines either separately or in combination against SARS-CoV-2 to determine their antiviral efficiency.

Keywords: ARDS, Acute respiratory distress syndrome; COVID-19, The new Coronavirus Disease 2019; Insilico prediction; MFE, Minimum free energy; Molecular docking; PCR, Polymerase Chain Reaction; RBD, receptor binding domain; RNAi, RNA interference; SARS-CoV-2; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus-2; Saudi Arabia; USFDA, United States Food and Drug Authority; Vero E6 cells; WHO, World Health Organization; siRNA, Short interfering RNA; siRNAs.

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Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

**Fig. 1**
The possible folding and secondary structure prediction of Insilico predicted siRNAs molecules computed using with the online web server (1–4).

**Fig. 2**
Molecular docking of predicted siRNAs (1–4-green) with SARS-CoV-2-RBD-S target gene and 3D interaction diagram of different docked complex with the target.

**Fig. 3**
Cytotoxicity of different siRNAs (1–4) at various concentrations (0.1–50 nM) in Vero E6 cells.

**Fig. 4**
Graphical representation of Ct value of RT-PCR result for the inhibition of SARS-CoV-2 in Vero E6 cells.

See this image and copyright information in PMC

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1. Azhar E.I., Hindawi S.I., El‐Kafrawy S.A., Hassan A.M., Tolah A.M., Alandijany T.A., Bajrai L.H., Damanhouri G.A. Amotosalen and ultraviolet A light treatment efficiently inactivates severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in human plasma. Vox Sanguinis (December) 2020;116(6):673–681. - PMC - PubMed
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[1] Alnylam Pharmaceuticals., 2020. Vir and Alnylam Expand Collaboration to Advance Rnai Therapeutics for the Treatment of Coronavirus Infection, Including Covid-19. Available online at: https://investors.alnylam.com/press-release?id= 24656.

[2] Alnylam Pharmaceuticals., 2020. Vir and Alnylam Expand Collaboration to Advance Rnai Therapeutics for the Treatment of Coronavirus Infection, Including Covid-19. Available online at: https://investors.alnylam.com/press-release?id= 24656.

[3] Alshukairi A.N., Tolah A.M., Dada A., Al-Tawfiq J.A., Almagharbi R.S., Saeedi M.F., Al-Hamzi M.A., El-Kafrawy S.A., Bahaudden H.A., El-Saed A., Al-Mozaini M.A., Khalid I., Hefni L.K., Hassan A.M., Alandijany T.A., Bajrai L.H., Bayumi D.T., Albishi G.E., Althawadi S.I., Zabani N.A., Perlman S., Azhar E.I. Test-based de-isolation in COVID-19 immunocompromised patients: Cycle threshold value versus SARS-CoV-2 viral culture. Internat. J. Infect. Diseases IJID. 2021;108:112–115. - PMC - PubMed

[4] Alshukairi A.N., Tolah A.M., Dada A., Al-Tawfiq J.A., Almagharbi R.S., Saeedi M.F., Al-Hamzi M.A., El-Kafrawy S.A., Bahaudden H.A., El-Saed A., Al-Mozaini M.A., Khalid I., Hefni L.K., Hassan A.M., Alandijany T.A., Bajrai L.H., Bayumi D.T., Albishi G.E., Althawadi S.I., Zabani N.A., Perlman S., Azhar E.I. Test-based de-isolation in COVID-19 immunocompromised patients: Cycle threshold value versus SARS-CoV-2 viral culture. Internat. J. Infect. Diseases IJID. 2021;108:112–115. - PMC - PubMed

[5] Azhar E.I., Hindawi S.I., El‐Kafrawy S.A., Hassan A.M., Tolah A.M., Alandijany T.A., Bajrai L.H., Damanhouri G.A. Amotosalen and ultraviolet A light treatment efficiently inactivates severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in human plasma. Vox Sanguinis (December) 2020;116(6):673–681. - PMC - PubMed

[6] Azhar E.I., Hindawi S.I., El‐Kafrawy S.A., Hassan A.M., Tolah A.M., Alandijany T.A., Bajrai L.H., Damanhouri G.A. Amotosalen and ultraviolet A light treatment efficiently inactivates severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in human plasma. Vox Sanguinis (December) 2020;116(6):673–681. - PMC - PubMed

[7] Azoulay E., Fartoukh M., Darmon M., Géri G., Voiriot G., Dupont T., Zafrani L., Girodias L., Labbé V., Dres M., Beurton A., Vieillard-Baron A., Demoule A. Increased mortality in patients with severe SARS-CoV-2 infection admitted within seven days of disease onset. Intensive Care Med. 2020;46(9):1714–1722. - PMC - PubMed

[8] Azoulay E., Fartoukh M., Darmon M., Géri G., Voiriot G., Dupont T., Zafrani L., Girodias L., Labbé V., Dres M., Beurton A., Vieillard-Baron A., Demoule A. Increased mortality in patients with severe SARS-CoV-2 infection admitted within seven days of disease onset. Intensive Care Med. 2020;46(9):1714–1722. - PMC - PubMed

[9] Bappy S.S., Shibly A.Z., Sultana S., Mohiuddin A.K.M., Kabir Y. Designing potential siRNA molecule for the nucleocapsid(N) gene silencing of different SARS-CoV-2 strains of Bangladesh: Computational approach. Comput. Biol. Chem. 2021;92:107486. - PMC - PubMed

[10] Bappy S.S., Shibly A.Z., Sultana S., Mohiuddin A.K.M., Kabir Y. Designing potential siRNA molecule for the nucleocapsid(N) gene silencing of different SARS-CoV-2 strains of Bangladesh: Computational approach. Comput. Biol. Chem. 2021;92:107486. - PMC - PubMed

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In silico prediction and experimental evaluation of potential siRNAs against SARS-CoV-2 inhibition in Vero E6 cells

Affiliations

In silico prediction and experimental evaluation of potential siRNAs against SARS-CoV-2 inhibition in Vero E6 cells

Authors

Affiliations

Abstract

Conflict of interest statement

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Abstract

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