A Single-Component Luminescent Biosensor for the SARS-CoV-2 Spike Protein

  1. Matthew Ravalin
  2. Heegwang Roh
  3. Rahul Suryawanshi
  4. G. Renuka Kumar
  5. John E. Pak
  6. Melanie Ott
  7. Alice Y. Ting

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Abstract

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  1. ASAPbio crowd review

    Review coordinated via ASAPbio’s crowd preprint review

    This review reflects comments and contributions by Ruchika Bajaj, Rasmus Norrild and Akihito Inoue. Review synthesized by Bianca Trovò.

    Antibody-based technologies for the detection and quantification of analytes in complex biological samples present challenges regarding the infrastructure and chemical modifications involved. There is therefore interest in developing alternative biosensor platforms that leverage split luciferase enzymes. Single-component luminescent biosensors can be more easily produced and work in both homogenous and immobilized assay formats. The manuscript reports the design of BAT, a single-component, NanoLuc-based, Binding Activated Tandem split enzyme biosensor for the detection of the SARS-CoV-2 spike protein in multiple assay formats.

    The reviewers praised the efficiency and practical value of the reporter system described, as the reporter protein works even in crude bacterial cell lysate, as well as the novelty of the mechanism of action for the reporter system. A few comments and suggestions raised are outlined below.

    Major comments

    1. In the introduction, the manuscript mentions the single-component, NanoLuc-based, Binding Activated Tandem split enzyme (BAT) biosensor, which is said to “not rely on a large conformational change in the binding module or competition with a tethered decoy as with other single component platforms”. The manuscript argues for the uniqueness and generality of the BAT approach based on a mechanism that does not rely on conformational change. Further references and explanation for the mode of action would be helpful to support the argument. Given the lack of conformational change in the binding module, can an explanation be included for what causes the split components to come closer and reconstitute again?
    2. Results, Design and optimization of SARS-CoV-2 biosensor “S-BAT”: the explanation of Figure 1a and Figure S1f provided in the context of the model for the mechanism of BATs could be strengthened with crystal diffraction data to validate the hypothesis, especially for clarifying how steric hindrance occurs when binding with the antigen (although this will not elucidate any conformational change happening in LCB1 upon binding).
    3. Results, Design and optimization of SARS-CoV-2 biosensor “S-BAT”: the manuscript reports a full mutational analysis, or deep mutational scanning (DMS) leading to the generation of “a point mutant in the S-BAT binding module at Asp30 (“S-BAT”), designed to ablate salt bridges formed with Lys417 and Arg403 in the Spike receptor binding domain (RBD)*”. Would it be possible to have this mutation motivated in the manuscript, and why was it chosen over other possible mutations in that context?
    4. Results, Design and optimization of SARS-CoV-2 biosensor “S-BAT”: the statement that “the absence of a hook effect at super-stoichiometric concentrations of Spike binding sites to sensor copies supports a predominantly cis activation mechanism” is a strong point but further clarification on this point is recommended, for example, further context on the Hook effect, and what would have been expected if trans activation was the major mode of action.
    5. Discussion: the manuscript has shown different versions of the same assay, so a discussion on advantages of one version over the other would be important.

    Minor comments

    1. Results, Design and optimization of SARS-CoV-2 biosensor “S-BAT”: “In addition to having high thermal stability, rigidity, and no disulfide bonds to complicate purification” - Please clarify which protein these qualities refer to.
    2. Results, Design and optimization of SARS-CoV-2 biosensor “S-BAT”: “This suggests that cis activation is likely the predominant source of signal in the assay, but we cannot rule out the contribution of a trans activating mechanism. In the trans mechanism, simultaneous binding to multiple protomers in a single Spike might increase the effective concentration, driving activation.” - Please provide further comments on the cis and trans mechanisms.
    3. Results, S-BAT is functional in multiple assay formats: “Adsorption-based immobilization is advantageous in that it requires no chemical modification to the protein reagent”, recommend reporting the efficiency of this step and how much signal is left after the washing steps.
    4. Methods, Cloning: the manuscript reports that all BAT constructs were subcloned using NcoI and BamHI restriction sites via Gibson assembly. Restriction site cloning and Gibson assembly seem to be two orthogonal methods, suggest providing further information on the cloning procedure.
    5. Methods, Recombinant Protein Production: "The un-cleaved BAT sensors were concentrated to ∼1.0 mL, and the concentration was calculated from the A280 value”. It is unclear if this was done using highly pure imidazole or if the signal was subtracted from Imidazole? A280 quantification is known to be difficult with Imidazole present.
    6. In Figure 1c: “Performance (Signal to Noise (S/N) multiplied by the magnitude of signal change (S-N))” please provide the mathematical expression for this analysis.
  2. Version published to 10.1021/jacs.2c04192
  3. Version published to 10.1101/2022.06.15.496006v1 on bioRxiv