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Chaotropic Perturbation of Noncovalent Interactions of the Hemagglutinin Tag Monoclonal Antibody Fragment Enables Superresolution Molecular Census
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    Chaotropic Perturbation of Noncovalent Interactions of the Hemagglutinin Tag Monoclonal Antibody Fragment Enables Superresolution Molecular Census
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    ACS Nano

    Cite this: ACS Nano 2022, 16, 1, 129–139
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    https://doi.org/10.1021/acsnano.1c04237
    Published November 19, 2021
    Copyright © 2021 American Chemical Society

    Abstract

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    Antibody–antigen interactions represent one of the most exploited biomolecular interactions in experimental biology. While numerous techniques harnessed immobilized antibodies for nanoscale fluorescence imaging, few utilized their reversible binding kinetics. Here, we investigated noncovalent interactions of the monoclonal hemagglutinin (HA) epitope tag antibody, 12CA5, in the fixed cellular environment. We observed that the use of a chaotropic agent, potassium thiocyanate (KSCN), promoted the dissociation of the 12CA5 antibody fragment (Fab), which already displayed faster dissociation compared to its immunoglobulin G (IgG) counterpart. Molecular dynamic simulations revealed notable root-mean-square deviations and destabilizations in the presence of KSCN, while the hydrogen-bonding network remained primarily unaffected at the antigen-binding site. The reversible interactions enabled us to achieve a superresolution molecular census of local populations of 3xHA tagged microtubule fibers with improved molecular quantification consistency compared to single-molecule localization microscopy (SMLM) techniques utilizing standard immunofluorescence staining for sample labeling. Our technique, termed superresolution census of molecular epitope tags (SR-COMET), highlights the utilization of reversible antibody–antigen interactions for SMLM-based quantitative superresolution imaging.

    Copyright © 2021 American Chemical Society

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    • 12CA5-IgG dye labeling with Alexa Fluor 647 (AF647) and Fab production using papain digestion; representative time traces of single-molecule intensity profiles showing enhanced sampling rates with KSCN; A superresolution image acquired using 12CA5-Fab-AF647 in the presence of 200 mM KSCN; detected single-molecule localizations per 1000 frames over 12 000 frame image acquisition in the absence and presence of 200 mM KSCN; modeling revealed the potential formation of a secondary structure of 3xHA tag; MD simulations of the single HA tag; superresolution images from DNA-PAINT and SR-COMET using a 20 nm pixel size for the image reconstruction; schematic representation of SR-COMET detecting sterically hindered epitope tag sites unregistered by DNA-PAINT; SR-COMET analysis of microtubule fibers and nonspecific background bindings; SR-COMET enabled molecular census in “superresolution-limited” areas; SR-COMET on a wild-type U2OS cell and the corresponding intensity profiles across the marked lines; consistent specific interactions detected over an extended period; interactions between 3xHA and Fv-clasp complex; photobleaching and dissociation rate curve fitting statistics of the 12CA5-IgG and 12CA5-Fab; dissociation rate curve fitting statistics of the 12CA5-Fab as a function of the KSCN concentration; column statistics for the SR-COMET data in Figure 5c (PDF)

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    ACS Nano

    Cite this: ACS Nano 2022, 16, 1, 129–139
    Click to copy citationCitation copied!
    https://doi.org/10.1021/acsnano.1c04237
    Published November 19, 2021
    Copyright © 2021 American Chemical Society

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