Bioinformatics approach for the design of siRNAs against SARS-CoV-2

By Dr. Sanchari Sinha Dutta, Ph.D.Dec 14 2022Reviewed by Danielle Ellis, B.Sc.

In a recent study posted to the bioRxiv* preprint server, scientists have developed a set of siRNAs against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using computational approaches. The siRNAs demonstrate high antiviral activity against SARS-CoV-2 and are able to recognize all available viral sequences.

*Important notice: bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Background

RNA interference (RNAi) is a highly conserved mechanism among eukaryotes that uses small interfering RNAs (siRNAs) to suppress the expression of target RNAs exhibiting appropriate sequence complementarity.

The RNAi mechanism is widely used in laboratory setups to suppress the expression of target RNAs. Its clinical application is also slowly gaining attention. A total of five therapeutic siRNAs are currently available in the market. However, none of them targets a virus.

In the current study, scientists have used bioinformatics approaches to design siRNAs against SARS-CoV-2. They have validated the antiviral activities of these siRNAs using ex vivo models of SARS-CoV-2-infected cultured cells.

Design of antiviral siRNA

The scientists generated a set of positive strand-targeting siRNAs against SARS-CoV-2. Initially, they predicted accessible viral regions and selected them as possible targets. They specifically selected highly conserved viral regions to avoid the emergence of mutated resistant variants in the long run.

For the optimal base-paring between siRNAs and viral targets, the scientists selected 13-mer target regions that are highly conserved among different viral variants. They optimized siRNA duplex asymmetry by allowing mismatches at tolerable positions and forcing them at beneficial positions. To increase the binding affinity, they performed many other modifications, such as adding a 5’ uridine in each siRNA candidate.

A total of eight siRNA candidates with minimal off-targets were generated in the study. The off-targets refer to endogenous mRNA sequences other than the targeted viral sequences. The siRNAs were predicted to target viral sequences that are conserved in more than 99.8% of viral variants.

Validation of siRNAs

Scientists assessed the antiviral activity of candidate siRNAs by transfecting them in cells and subsequently infecting the cells with SARS-CoV-2. They measured viral RNA titer and cell morphological changes after 48 hours and 72 hours of infection, respectively.

The findings revealed that six of eight siRNAs have high antiviral activity against SARS-CoV-2 at nanomolar concentrations. The same siRNA concentrations were used to assess cytotoxic effects. The findings revealed that all siRNAs are safe for use.

Furthermore, scientists performed optimization procedures using viral sequences from publicly available databases to determine the variant coverage of candidate siRNAs. They analyzed more than 13,000,000 sequences and identified 12 distinct triplets of siRNAs that could recognize each of the sequenced variants.

They observed that the candidate siRNAs could recognize SARS-CoV-2 variants of concern (VOCs), which exhibit improved transmissibility, infectivity, immune fitness, and pathogenicity.

Study significance

The study describes the development and validation of a set of candidate siRNAs exhibiting high antiviral activity against SARS-CoV-2. The siRNAs can recognize all available viral sequences, including VOCs. In particular, multiple sets of 3-siRNA combinations developed in the study are sufficient to recognize all correctly sequenced SARS-CoV-2 sequences available to date.  

Mutations emerging in the SARS-CoV-2 genome are not expected to affect the antiviral activity of these siRNAs as they target highly conserved viral regions. As mentioned by the scientists, these siRNAs could serve as a potential therapeutic intervention against SARS-CoV-2 infection.

The only obstacle against the clinical application of antiviral siRNAs is the difficulty of delivery to the target organs. The in vivo delivery of therapeutic siRNA through inhalation is under investigation and showing promising outcomes. With the optimization of a robust strategy of on-target delivery, siRNAs are expected to serve as a simple, efficient, and easily adaptable therapeutic intervention to manage the ongoing pandemic as well as future viral outbreaks.

*Important notice: bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.