Reaction Conditions Promoting the Specific Detection of SARS-CoV-2 NendoU Enzymatic Activity

  1. Nodar Makharashvili
  2. James O. McNamara

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Abstract

Methods that enable rapid detection of SARS-CoV-2 provide valuable tools for detecting and controlling Covid-19 outbreaks and also facilitate more effective treatment of infected individuals. The predominant approaches developed use PCR to detect viral nucleic acids or immunoassays to detect viral proteins. Each approach has distinct advantages and disadvantages, but alternatives that do not share the same limitations could enable substantial improvements in outbreak detection and management. For instance, methods that have comparable sensitivity to PCR, but that are not prone to the false-positive results that stem from the tendency of PCR to detect molecular degradation products could improve accurate identification of infected individuals. An alternative approach with potential to achieve this entails harnessing the unique enzymatic properties of SARS-CoV-2 enzymes to generate SARS-Cov-2-specific signals that indicate the presence of the virus. This route benefits from the high sensitivity provided by enzymatic signal amplification and also the fact that signal is generated only by intact viral enzymes, not degradation products. Here, we demonstrate enzymatic reaction conditions that enable the preferential detection of NendoU of SARS-CoV-2, versus several of its orthologues, with a fluorogenic oligonucleotide substrate. These compositions provide a possible technical foundation for a novel approach for detecting SARS-CoV-2 that has distinct advantages from current approaches.

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  1. ScreenIT

    SciScore for 10.1101/2022.05.06.490915: (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.

    Table 2: Resources

    Experimental Models: Organisms/Strains
    SentencesResources
    Amino acid sequences of the NendoU (Nsp15) orthologues of the following human coronaviruses were obtained from the Uniprot.org database: SARS-CoV2; HCoV-229E; HCoV-NL63; HCoV-OC43; HCoV-HKU1.
    SARS-CoV2; HCoV-229E; HCoV-NL63; HCoV-OC43; HCoV-HKU1
    suggested: None
    Recombinant DNA
    SentencesResources
    DNA sequences encoding each protein were codon-optimized for bacterial expression, synthesized, and cloned into the pET28a expression plasmid as custom orders by GenScript Biotech.
    pET28a
    suggested: RRID:Addgene_139598)
    Software and Algorithms
    SentencesResources
    The following reagents were acquired from Fisher Scientific: 1 M Tris-HCl pH 7.0 stock solution, TCEP-HCl, MnCl2, beta-mercaptoethanol, NaCl.
    Fisher Scientific
    suggested: (Thermo Fisher Scientific, RRID:SCR_008452)

    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 10.1101/2022.05.06.490915v1 on bioRxiv