Efficient Inhibition of SARS‐CoV‐2 Using Chimeric Antisense Oligonucleotides through RNase L Activation**
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Abstract
There is an urgent need to develop antiviral drugs and alleviate the current COVID‐19 pandemic. Herein we report the design and construction of chimeric oligonucleotides comprising a 2′‐OMe‐modified antisense oligonucleotide and a 5′‐phosphorylated 2′‐5′ poly(A) 4 (4A 2‐5 ) to degrade envelope and spike RNAs of SARS‐CoV‐2. The oligonucleotide was used for searching and recognizing target viral RNA sequence, and the conjugated 4A 2‐5 was used for guided RNase L activation to sequence‐specifically degrade viral RNAs. Since RNase L can potently cleave single‐stranded RNA during innate antiviral response, degradation efficiencies with these chimeras were twice as much as those with only antisense oligonucleotides for both SARS‐CoV‐2 RNA targets. In pseudovirus infection models, chimera‐S4 achieved potent and broad‐spectrum inhibition of SARS‐CoV‐2 and its N501Y and/or Δ H 69/Δ V 70 mutants, indicating a promising antiviral agent based on the nucleic acid‐hydrolysis targeting chimera (NATAC) strategy.
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SciScore for 10.1101/2021.03.04.433849: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Next, we screened three chimera candidates targeting S-RBD gene of SARS-CoV-2 for the most efficient one through evaluating their downregulation of S-RNA by RT-qPCR in Vero cells and the inhibition of SARS-CoV-2 pseudovirus packaging in HEK293T cells. HEK293Tsuggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)Cell culture and Transfection Procedure: Vero cells and A549 cells were grown at 37 C, 5% CO2 in DMEM (M&C) supplemented with 10% … SciScore for 10.1101/2021.03.04.433849: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Next, we screened three chimera candidates targeting S-RBD gene of SARS-CoV-2 for the most efficient one through evaluating their downregulation of S-RNA by RT-qPCR in Vero cells and the inhibition of SARS-CoV-2 pseudovirus packaging in HEK293T cells. HEK293Tsuggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)Cell culture and Transfection Procedure: Vero cells and A549 cells were grown at 37 C, 5% CO2 in DMEM (M&C) supplemented with 10% fetal bovine serum (PAN), 100 units/mL penicillin, and 100 μg/mL streptomycin. A549suggested: NoneSRB Assay: Oligonucleotides and plasmids were transfected into Vero cells (seeded in 96-well plates with the density of 2×104 cells per well). Verosuggested: RRID:CVCL_ZW93)In order to confirm the titration of pseudovirus, HEK293T-hACE2 cells were seeded into 6-well plates. HEK293T-hACE2suggested: NoneSoftware and Algorithms Sentences Resources Preparation of oligonucleotides: Chimeric oligonucleotides (Chimera-E or Chimera-S), ASO-S control oligonucleotides and 3’-Cy3 labeled E-RNA segment were purchased from Biosyntech. Biosyntechsuggested: NoneFull length RNase L gene was synthesized and subcloned into pGEX-4T-3 vector (pGEX-4T-RNaseL-GST) by GENEWIZ as previously described ( GENEWIZsuggested: (GENEWIZ, RRID:SCR_003177)Statistical Analysis: GraphPad Prism 7.04 was used for statistical analysis and graphing. GraphPadsuggested: (GraphPad Prism, RRID:SCR_002798)Results from OddPub: Thank you for sharing your data.
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
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