A Bioluminescent Biosensor for Quantifying the Interaction of SARS-CoV-2 and Its Receptor ACE2 in Cells and In Vitro

  1. Xiaolong Yang
  2. Lidong Liu
  3. Yawei Hao
  4. Eva So
  5. Sahar Sarmasti Emami
  6. Derek Zhang
  7. Yanping Gong
  8. Prameet M. Sheth
  9. Yutian Wang

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Abstract

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is currently spreading and mutating with increasing speed worldwide. Therefore, there is an urgent need for a simple, sensitive, and high-throughput (HTP) assay to quantify virus–host interactions in order to quickly evaluate the infectious ability of mutant viruses and to develop or validate virus-inhibiting drugs. Here, we developed an ultrasensitive bioluminescent biosensor to evaluate virus–cell interactions by quantifying the interaction between the SARS-CoV-2 receptor binding domain (RBD) and its cellular receptor angiotensin-converting enzyme 2 (ACE2) both in living cells and in vitro. We have successfully used this novel biosensor to analyze SARS-CoV-2 RBD mutants and evaluated candidate small molecules (SMs), antibodies, and peptides that may block RBD:ACE2 interaction. This simple, rapid, and HTP biosensor tool will significantly expedite the detection of viral mutants and the anti-COVID-19 drug discovery process.

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  1. Version published to 10.3390/v13061055
  2. ScreenIT

    SciScore for 10.1101/2020.12.29.424698: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    The blot was first probed with anti-6×His mouse monoclonal antibody (Abcam ab18184), followed by incubation with goat anti-mouse IgG secondary antibody (Jackson ImmunoResearch).
    anti-6×His
    suggested: None
    anti-mouse IgG
    suggested: None
    Validation of SRAE2-BS using SMs, antibodies, and peptides: About 6 μg of Sm-ACE2 or RBD-Lg plasmids were transfected into a 100 mm plate using PolyJet.
    SMs,
    suggested: None
    For examination of the effect of candidate drugs on biosensor activity, triplicate of increasing amounts (0-1000 μM) of SMs [Baicalin, Hesperidin, Sculellarin, Theaflavin, Heparin from TargetMol] or freshly prepared synthesized peptides [ACE2-P1~2] or 1-50 μg/ml of IgG or VHH72 anti-SARS-CoV-2 Spike RBD LIamabody monoclonal antibody (R&D#LMAB10541) were preincubated with 1 μg of Sm-ACE2 protein lysate in 10 μl in 96-well plates on a rocker at RT for 30 min.
    μM) of SMs [Baicalin, Hesperidin, Sculellarin, Theaflavin, Heparin from TargetMol]
    suggested: None
    anti-SARS-CoV-2
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    NanoLuc luciferase (NanoBiT) assay: For analysis of biosensor activity in living cell in vivo, 2×104 HEK293T cells were seeded in triplicate in 96-well plate 24 hours before transfection.
    HEK293T
    suggested: None
    Software and Algorithms
    SentencesResources
    Materials: Chemicals were purchased from Sigma-Aldrich or Bioshop unless otherwise stated.
    Bioshop
    suggested: None
    Validation of SRAE2-BS using SMs, antibodies, and peptides: About 6 μg of Sm-ACE2 or RBD-Lg plasmids were transfected into a 100 mm plate using PolyJet.
    PolyJet
    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.
    • 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.

    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.

  3. Version published to 10.1101/2020.12.29.424698v1 on bioRxiv