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Resolving Molecular Heterogeneity with Single-Molecule Centrifugation

[Image: see text] For many classes of biomolecules, population-level heterogeneity is an essential aspect of biological function—from antibodies produced by the immune system to post-translationally modified proteins that regulate cellular processes. However, heterogeneity is difficult to fully char...

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Autores principales: Luo, Yi, Chang, Jeffrey, Yang, Darren, Bryan, J. Shepard, MacIsaac, Molly, Pressé, Steve, Wong, Wesley P.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Chemical Society 2023
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9936575/
https://www.ncbi.nlm.nih.gov/pubmed/36716175
http://dx.doi.org/10.1021/jacs.2c11450
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author Luo, Yi
Chang, Jeffrey
Yang, Darren
Bryan, J. Shepard
MacIsaac, Molly
Pressé, Steve
Wong, Wesley P.
author_facet Luo, Yi
Chang, Jeffrey
Yang, Darren
Bryan, J. Shepard
MacIsaac, Molly
Pressé, Steve
Wong, Wesley P.
author_sort Luo, Yi
collection PubMed
description [Image: see text] For many classes of biomolecules, population-level heterogeneity is an essential aspect of biological function—from antibodies produced by the immune system to post-translationally modified proteins that regulate cellular processes. However, heterogeneity is difficult to fully characterize for multiple reasons: (i) single-molecule approaches are needed to avoid information lost by ensemble-level averaging, (ii) sufficient statistics must be gathered on both a per-molecule and per-population level, and (iii) a suitable analysis framework is required to make sense of a potentially limited number of intrinsically noisy measurements. Here, we introduce an approach that overcomes these difficulties by combining three techniques: a DNA nanoswitch construct to repeatedly interrogate the same molecule, a benchtop centrifuge force microscope (CFM) to obtain thousands of statistics in a highly parallel manner, and a Bayesian nonparametric (BNP) inference method to resolve separate subpopulations with distinct kinetics. We apply this approach to characterize commercially available antibodies and find that polyclonal antibody from rabbit serum is well-modeled by a mixture of three subpopulations. Our results show how combining a spatially and temporally multiplexed nanoswitch-CFM assay with BNP analysis can help resolve complex biomolecular interactions in heterogeneous samples.
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spelling pubmed-99365752023-02-18 Resolving Molecular Heterogeneity with Single-Molecule Centrifugation Luo, Yi Chang, Jeffrey Yang, Darren Bryan, J. Shepard MacIsaac, Molly Pressé, Steve Wong, Wesley P. J Am Chem Soc [Image: see text] For many classes of biomolecules, population-level heterogeneity is an essential aspect of biological function—from antibodies produced by the immune system to post-translationally modified proteins that regulate cellular processes. However, heterogeneity is difficult to fully characterize for multiple reasons: (i) single-molecule approaches are needed to avoid information lost by ensemble-level averaging, (ii) sufficient statistics must be gathered on both a per-molecule and per-population level, and (iii) a suitable analysis framework is required to make sense of a potentially limited number of intrinsically noisy measurements. Here, we introduce an approach that overcomes these difficulties by combining three techniques: a DNA nanoswitch construct to repeatedly interrogate the same molecule, a benchtop centrifuge force microscope (CFM) to obtain thousands of statistics in a highly parallel manner, and a Bayesian nonparametric (BNP) inference method to resolve separate subpopulations with distinct kinetics. We apply this approach to characterize commercially available antibodies and find that polyclonal antibody from rabbit serum is well-modeled by a mixture of three subpopulations. Our results show how combining a spatially and temporally multiplexed nanoswitch-CFM assay with BNP analysis can help resolve complex biomolecular interactions in heterogeneous samples. American Chemical Society 2023-01-30 /pmc/articles/PMC9936575/ /pubmed/36716175 http://dx.doi.org/10.1021/jacs.2c11450 Text en © 2023 The Authors. Published by American Chemical Society https://creativecommons.org/licenses/by-nc-nd/4.0/Permits non-commercial access and re-use, provided that author attribution and integrity are maintained; but does not permit creation of adaptations or other derivative works (https://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Luo, Yi
Chang, Jeffrey
Yang, Darren
Bryan, J. Shepard
MacIsaac, Molly
Pressé, Steve
Wong, Wesley P.
Resolving Molecular Heterogeneity with Single-Molecule Centrifugation
title Resolving Molecular Heterogeneity with Single-Molecule Centrifugation
title_full Resolving Molecular Heterogeneity with Single-Molecule Centrifugation
title_fullStr Resolving Molecular Heterogeneity with Single-Molecule Centrifugation
title_full_unstemmed Resolving Molecular Heterogeneity with Single-Molecule Centrifugation
title_short Resolving Molecular Heterogeneity with Single-Molecule Centrifugation
title_sort resolving molecular heterogeneity with single-molecule centrifugation
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9936575/
https://www.ncbi.nlm.nih.gov/pubmed/36716175
http://dx.doi.org/10.1021/jacs.2c11450
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