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. 2023 Dec 12;23(1):872.
doi: 10.1186/s12879-023-08870-0.

An ncRNA transcriptomics-based approach to design siRNA molecules against SARS-CoV-2 double membrane vesicle formation and accessory genes

Rabia Nawaz #  1   2 , Muhammad Ali Arif #  3 , Zainab Ahmad #  3 , Ammara Ahad  3 , Muhammad Shahid  4 , Zohal Hassan  3 , Ali Husnain  3 , Ali Aslam  3 , Muhammad Saad Raza  3 , Uqba Mehmood  3 , Muhammad Idrees  4   5
Affiliations

An ncRNA transcriptomics-based approach to design siRNA molecules against SARS-CoV-2 double membrane vesicle formation and accessory genes

Rabia Nawaz et al. BMC Infect Dis. .

Abstract

Background: The corona virus SARS-CoV-2 is the causative agent of recent most global pandemic. Its genome encodes various proteins categorized as non-structural, accessory, and structural proteins. The non-structural proteins, NSP1-16, are located within the ORF1ab. The NSP3, 4, and 6 together are involved in formation of double membrane vesicle (DMV) in host Golgi apparatus. These vesicles provide anchorage to viral replicative complexes, thus assist replication inside the host cell. While the accessory genes coded by ORFs 3a, 3b, 6, 7a, 7b, 8a, 8b, 9b, 9c, and 10 contribute in cell entry, immunoevasion, and pathological progression.

Methods: This in silico study is focused on designing sequence specific siRNA molecules as a tool for silencing the non-structural and accessory genes of the virus. The gene sequences of NSP3, 4, and 6 along with ORF3a, 6, 7a, 8, and 10 were retrieved for conservation, phylogenetic, and sequence logo analyses. siRNA candidates were predicted using siDirect 2.0 targeting these genes. The GC content, melting temperatures, and various validation scores were calculated. Secondary structures of the guide strands and siRNA-target duplexes were predicted. Finally, tertiary structures were predicted and subjected to structural validations.

Results: This study revealed that NSP3, 4, and 6 and accessory genes ORF3a, 6, 7a, 8, and 10 have high levels of conservation across globally circulating SARS-CoV-2 strains. A total of 71 siRNA molecules were predicted against the selected genes. Following rigorous screening including binary validations and minimum free energies, final siRNAs with high therapeutic potential were identified, including 7, 2, and 1 against NSP3, NSP4, and NSP6, as well as 3, 1, 2, and 1 targeting ORF3a, ORF7a, ORF8, and ORF10, respectively.

Conclusion: Our novel in silico pipeline integrates effective methods from previous studies to predict and validate siRNA molecules, having the potential to inhibit viral replication pathway in vitro. In total, this study identified 17 highly specific siRNA molecules targeting NSP3, 4, and 6 and accessory genes ORF3a, 7a, 8, and 10 of SARS-CoV-2, which might be used as an additional antiviral treatment option especially in the cases of life-threatening urgencies.

Keywords: Accessory genes; Gene silencing; NSPs; Non-structural genes; Open Reading frame ORF; SARS-CoV-2; ncRNA; siRNA.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Phylogenetic analysis of NSP3 gene sequences of SARS-CoV-2
Fig. 2
Fig. 2
Phylogenetic analysis of NSP4 gene sequences of SARS-CoV-2
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Fig. 3
Phylogenetic analysis of NSP6 gene sequences of SARS-CoV-2
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Fig. 4
Phylogenetic analysis of accessory genes sequences of SARS-CoV-2
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Fig. 5
Secondary structures and MFE of siRNA molecules targeting NSP3
Fig. 6
Fig. 6
Secondary structures and MFE of siRNA molecules targeting NSP4
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Fig. 7
Secondary structure and MFE of siRNA molecule targeting NSP6
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Fig. 8
Secondary structures and MFE of siRNA molecules targeting ORF3a
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Fig. 9
Secondary structure and MFE of siRNA molecule targeting ORF6
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Fig. 10
Secondary structure and MFE of siRNA molecule targeting ORF7a
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Fig. 11
Secondary structures and MFE of siRNA molecules targeting ORF8
Fig. 12
Fig. 12
Secondary structure and MFE of siRNA molecule targeting ORF10
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Fig. 13
Secondary structures and MFE of siRNA-target duplexes for NSP3 regions
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Fig. 14
Secondary structures and MFE of siRNA-target duplexes for NSP4 regions
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Fig. 15
Secondary structure and MFE of siRNA-target duplex for NSP6 regions
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Fig. 16
Secondary structures and MFE of siRNA-target duplexes for ORF3a regions
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Fig. 17
Secondary structures and MFE of siRNA-target duplex for ORF6 regions
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Fig. 18
Secondary structure and MFE of siRNA-target duplex for ORF7a regions
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Fig. 19
Secondary structures and MFE of siRNA-target duplexes for ORF8 regions
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Secondary structure and MFE of siRNA-target duplex for ORF10 regions
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Tertiary structures of siRNA molecules targeting NSP3
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Fig. 22
Tertiary structures of siRNA molecules targeting NSP4
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Tertiary structure of siRNA molecule targeting NSP6
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Tertiary structures of siRNA molecules targeting ORF3a
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Tertiary structure of siRNA molecule targeting ORF7a
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Fig. 26
Tertiary structure of siRNA molecules targeting ORF8
Fig. 27
Fig. 27
Tertiary structure of siRNA molecule targeting ORF10
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