siRNA Design to Silence the 3<mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M1"><mml:msup><mml:mrow /><mml:mrow><mml:mo>′</mml:mo></mml:mrow></mml:msup></mml:math>UTR Region of Zika Virus

Perez-Mendez, Maria Jose; Zárate-Segura, Paola; Salas-Benito, Juan Santiago; Bastida-González, Fernando

doi:10.1155/2020/6759346

Cited by 6 publications

(13 citation statements)

References 49 publications

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“…The long-term treatment of HCV replicon cells also did not lead to the emergence of escape mutant viruses which ensured the sustained effectiveness of the antiviral therapy over an extended time period [19]. We developed a novel in silico pipeline for predicting and validating siRNA molecules that combines multiple effective in silico methods used in the previous studies [38,39,23,19], which demonstrated successful inhibition of viral replication in vitro. This innovative pipeline con dently aims to identify and validate siRNAs with the potential to inhibit viral replication in in vitro experiments (Table 4).…”

Section: Discussionmentioning

confidence: 99%

“…The tertiary structures of guide strands of nal 17 siRNA molecules, which passed the validation criteria were modelled and viewed using UCSF Chimera 1.16 (Fig. [21][22][23][24][25][26][27]. The 3D models obtained were further validated using MolProbity server (Additional le 1: Table S8).…”

Section: Secondary Structure Prediction and Minimum Free Energy Deter...mentioning

confidence: 99%

“…In silico approaches utilizing computational biology tools and databases have facilitated the design of siRNAs for targeted gene silencing [16][17][18]. Earlier studies reported siRNAs and miRNAs as an effective defense against many viruses including Hepatitis C virus [19], Human immunode ciency virus [20], In uenza virus [21], Nipah virus [22] and Zika virus [23]. This in silico study includes conservation and phylogenetic analyses of NSP3, 4, and 6 sequences of SARS-CoV-2 strains across the globe along with the accessory genes including ORF3a, 6, 7a, 8, and 10.…”

Section: Introductionmentioning

confidence: 99%

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

Nawaz

Arif

Ahmad

et al. 2023

Preprint

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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.

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Section: Discussionmentioning

confidence: 99%

Section: Secondary Structure Prediction and Minimum Free Energy Deter...mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Nawaz

Arif

Ahmad

et al. 2023

Preprint

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“…These studies have proved that the efficiency of different siRNAs against the same target gene can be different; confirming that the random design of siRNAs is not a valid strategy when it comes to obtaining efficient inhibitors. One of the critical rules to take into account is related to the folding and accessibility of the target region [6][7][8][9][10]. Many of the highly conserved regions in viral genomes, which show a compact folding, can be refractory to siRNA targeting.…”

Section: Introductionmentioning

confidence: 99%

In Vitro Methods to Decipher the Structure of Viral RNA Genomes

2021

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RNA viruses encode essential information in their genomes as conserved structural elements that are involved in efficient viral protein synthesis, replication, and encapsidation. These elements can also establish complex networks of RNA-RNA interactions, the so-called RNA interactome, to shape the viral genome and control different events during intracellular infection. In recent years, targeting these conserved structural elements has become a promising strategy for the development of new antiviral tools due to their sequence and structural conservation. In this context, RNA-based specific therapeutic strategies, such as the use of siRNAs have been extensively pursued to target the genome of different viruses. Importantly, siRNA-mediated targeting is not a straightforward approach and its efficiency is highly dependent on the structure of the target region. Therefore, the knowledge of the viral structure is critical for the identification of potentially good target sites. Here, we describe detailed protocols used in our laboratory for the in vitro study of the structure of viral RNA genomes. These protocols include DMS (dimethylsulfate) probing, SHAPE (selective 2′-hydroxyl acylation analyzed by primer extension) analysis, and HMX (2′-hydroxyl molecular interference). These methodologies involve the use of high-throughput analysis techniques that provide extensive information about the 3D folding of the RNA under study and the structural tuning derived from the interactome activity. They are therefore a good tool for the development of new RNA-based antiviral compounds.

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“…In the recent past, there are reports available on the in silico design of siRNAs against different human viruses, e.g., Zika virus [14][15][16], MERS-CoV [17][18][19], Influenza virus [20][21][22], human adenovirus type-3 [23], hepatitis C virus [24], rabies virus [25], and respiratory syncytial virus [26]. siRNAs designed in silico from a majority of the studies mentioned above were tested in vitro and were found to be effective [14,[17][18][19][20][21][22]24]. A comprehensive review on the usage of siRNA for gene silencing of coronaviruses is given in Sajid et al [4] and Uludag et al [27].…”

mentioning

confidence: 99%

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

Ayyagari

2022

Journal of Genetic Engineering and Biotechnology

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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.

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siRNA Design to Silence the 3′UTR Region of Zika Virus

Cited by 6 publications

References 49 publications

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

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

In Vitro Methods to Decipher the Structure of Viral RNA Genomes

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

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