Structure-based design of antisense oligonucleotides that inhibit SARS-CoV-2 replication

  1. Yan Li
  2. Gustavo Garcia
  3. Vaithilingaraja Arumugaswami
  4. Feng Guo

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Abstract

Antisense oligonucleotides (ASOs) are an emerging class of drugs that target RNAs. Current ASO designs strictly follow the rule of Watson-Crick base pairing along target sequences. However, RNAs often fold into structures that interfere with ASO hybridization. Here we developed a structure-based ASO design method and applied it to target severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Our method makes sure that ASO binding is compatible with target structures in three-dimensional (3D) space by employing structural design templates. These 3D-ASOs recognize the shapes and hydrogen bonding patterns of targets via tertiary interactions, achieving enhanced affinity and specificity. We designed 3D-ASOs that bind to the frameshift stimulation element and transcription regulatory sequence of SARS-CoV-2 and identified lead ASOs that strongly inhibit viral replication in human cells. We further optimized the lead sequences and characterized structure-activity relationship. The 3D-ASO technology helps fight coronavirus disease-2019 and is broadly applicable to ASO drug development.

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  1. ScreenIT

    SciScore for 10.1101/2021.08.23.457434: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    For immunostaining, cells were incubated overnight with the primary antibody (Monoclonal Anti-SARS-CoV S Protein (similar to 240C), NR-616 from the BEI Resources, NIAID, NIH) at 4°C.
    Anti-SARS-CoV S Protein
    suggested: (BioLegend Cat# 944301, RRID:AB_2890871)
    Experimental Models: Cell Lines
    SentencesResources
    SARS-CoV-2 was passaged once in Vero E6 cells and viral stocks were aliquoted and stored at −80°C.
    Vero E6
    suggested: RRID:CVCL_XD71)
    HEK293-hACE2 cells were cultured in 96-well plate at 37°C in 5% CO2.
    HEK293-hACE2
    suggested: None
    Programmed ribosomal frameshifting reporter assays: HEK293 cells were seeded at 5,000 cells per well in 96-well plates.
    HEK293
    suggested: None
    Recombinant DNA
    SentencesResources
    The PCR product was digested by HindIII and BlgII and ligated into pSGDLuc 3.0 44
    pSGDLuc 3.0
    suggested: None
    Sixteen hours after ASO transfection, when cells usually reached confluency of around 70%, the CoV2-FSE119 plasmid was transfected using lipofectamine 3000 (Thermo Fisher Scientific) at 100 ng per well.
    CoV2-FSE119
    suggested: None
    Software and Algorithms
    SentencesResources
    The data were analyzed using Origin 7.0 (Microcal)
    Origin
    suggested: (Origin, RRID:SCR_014212)
    The data were analyzed using the Thermo Fisher Connect Platform.
    Thermo Fisher Connect Platform
    suggested: None
    5c–e were fit to the equation using PRISM (version 7, GraphPad).
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    6e–i were fit to the equation using PRISM.
    PRISM
    suggested: (PRISM, RRID:SCR_005375)
    Statistical analyses: The Student t-tests, two-tailed and two-sample equal variance, were performed using Excel (Microsoft).
    Excel
    suggested: None

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    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 did not find any issues relating to the usage of bar graphs.

    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.
    • No funding statement was detected.
    • No protocol registration statement was detected.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  2. Version published to 10.1101/2021.08.23.457434v1 on bioRxiv