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Computational Design and Experimental Evaluation of MERS-CoV siRNAs in Selected Cell Lines.

ABSTRACT: Middle East respiratory syndrome coronavirus (MERS-CoV) is caused by a well-known coronavirus first identified in a hospitalized patient in the Kingdom of Saudi Arabia. MERS-CoV is a serious pathogen affecting both human and camel health globally, with camels being known carriers of viruses that spread to humans. In this work, MERS-CoV genomic sequences were retrieved and analyzed by multiple sequence alignment to design and predict siRNAs with online software. The siRNAs were designed from the orf1ab region of the virus genome because of its high sequence conservation and vital role in virus replication. The designed siRNAs were used for experimental evaluation in selected cell lines: Vero cells, HEK-293-T, and Huh-7. Virus inhibition was assessed according to the cycle threshold value during a quantitative real-time polymerase chain reaction. Out of 462 potential siRNAs, we filtered out 21 based on specific selection criteria without off-target effect. The selected siRNAs did not show any cellular toxicity in the tested cell lines at various concentrations. Based on our results, it was obvious that the combined use of siRNAs exhibited a reduction in MERS-CoV replication in the Vero, HEK-293-T, and Huh-7 cell lines, with the highest efficacy displayed in the Vero cells.

SUBMITTER: Sohrab SS

PROVIDER: S-EPMC9818142 | biostudies-literature | 2023 Jan

REPOSITORIES: biostudies-literature

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Computational Design and Experimental Evaluation of MERS-CoV siRNAs in Selected Cell Lines.

Sohrab Sayed S SS El-Kafrawy Sherif A SA Mirza Zeenat Z Hassan Ahmed M AM Alsaqaf Fatima F Azhar Esam I EI

Diagnostics (Basel, Switzerland) 20230102 1

Middle East respiratory syndrome coronavirus (MERS-CoV) is caused by a well-known coronavirus first identified in a hospitalized patient in the Kingdom of Saudi Arabia. MERS-CoV is a serious pathogen affecting both human and camel health globally, with camels being known carriers of viruses that spread to humans. In this work, MERS-CoV genomic sequences were retrieved and analyzed by multiple sequence alignment to design and predict siRNAs with online software. The siRNAs were designed from the ...[more]

PMID: 36611443

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Project description:Oncogenic KRAS mutations drive cancer progression in lung, colon, breast, and pancreatic ductal adenocarcinomas. Apart from the current strategies, such as KRAS upstream inhibitors, downstream effector inhibitors, interaction inhibitors, cell cycle inhibitors, and direct KRAS inhibitors, against KRAS-mutated cancers, the therapeutic small interfering RNAs (siRNAs) represent a promising alternative strategy that directly binds with the target mRNA and inhibits protein translation via mRNA degradation. Here, in the present study, we utilized various in silico approaches to design potential siRNA candidates against KRAS mRNA. We have predicted nearly 17 siRNAs against the KRAS mRNA, and further through various criteria, such as U, R, and A rules, GC%, secondary structure formation, mRNA-siRNA duplex stability, Tm (Cp), Tm (Conc), and inhibition efficiency, they have been filtered into 4 potential siRNAs namely siRNA8, siRNA11, siRNA12, and siRNA17. Further, the molecular docking analysis revealed that the siRNA8, siRNA11, siRNA12, and siRNA17 showed higher negative binding energies, such as - 379.13 kcal/mol, - 360.19 kcal/mol, - 288.47 kcal/mol, and - 329.76 kcal/mol, toward the human Argonaute2 protein (hAgo2) respectively. In addition, the normal mode analysis of the hAgo2-siRNAs complexes indicates the structural changes and deformation of the hAgo2 protein upon the binding of siRNA molecules in the dynamic environment which suggests that these siRNAs could be effective. Finally, we conclude that these 4 siRNAs have therapeutic potential against KRAS mRNA and also have to be studied in vitro and in vivo to evaluate their specificity toward mutant KRAS (not degrading wild-type KRAS). Also, the current challenges in the use of siRNA therapeutics could be overcome by the emerging siRNA delivery methods, such as Antibody-siRNA conjugates (ARCs) and Gelatin-Antibody Delivery System (GADS), in the near future and these siRNAs could be employed as potential therapeutic agents against KRAS-mutated cancers.Supplementary informationThe online version contains supplementary material available at 10.1007/s13205-023-03767-w.

Project description:SARS-CoV-2 is a highly transmissible and pathogenic coronavirus that first emerged in late 2019 and has since triggered a pandemic of acute respiratory disease named COVID-19 which poses a significant threat to all public health institutions in the absence of specific antiviral treatment. Since the outbreak began in March 2020, India has reported 4.77 lakh Coronavirus deaths, according to the World Health Organization (WHO). The innate RNA interference (RNAi) pathway, on the other hand, allows for the development of nucleic acid-based antiviral drugs in which complementary small interfering RNAs (siRNAs) mediate the post-transcriptional gene silencing (PTGS) of target mRNA. Therefore, in this current study, the potential of RNAi was harnessed to construct siRNA molecules that target the consensus regions of specific structural proteins associated genes of SARS-CoV-2, such as the envelope protein gene (E), membrane protein gene (M), nucleocapsid phosphoprotein gene (N), and surface glycoprotein gene (S) which are important for the viral pathogenesis. Conserved sequences of 811 SARS-CoV-2 strains from around India were collected to design 21 nucleotides long siRNA duplex based on various computational algorithms and parameters targeting E, M, N and S genes. The proposed siRNA molecules possessed sufficient nucleotide-based and other features for effective gene silencing and BLAST results revealed that siRNAs' targets have no significant matches across the whole human genome. Hence, siRNAs were found to have no off-target effects on the genome, ruling out the possibility of off-target silencing. Finally, out of 157 computationally identified siRNAs, only 4 effective siRNA molecules were selected for each target gene which is proposed to exert the best action based on GC content, free energy of folding, free energy of binding, melting temperature, heat capacity and molecular docking analysis with Human AGO2 protein. Our engineered siRNA candidates could be used as a genome-level therapeutic treatment against various sequenced SARS-CoV-2 strains in India. However, future applications will necessitate additional validations in vitro and in vivo animal models.

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Computational Design and Experimental Evaluation of MERS-CoV siRNAs in Selected Cell Lines.

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Computational Design and Experimental Evaluation of MERS-CoV siRNAs in Selected Cell Lines.

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