TY - JOUR
T1 - Resolving Molecular Heterogeneity with Single-Molecule Centrifugation
AU - Luo, Yi
AU - Chang, Jeffrey
AU - Yang, Darren
AU - Bryan, J. Shepard
AU - Macisaac, Molly
AU - Pressé, Steve
AU - Wong, Wesley P.
N1 - Publisher Copyright:
© 2023 American Chemical Society.
PY - 2023/2/15
Y1 - 2023/2/15
N2 - 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.
AB - 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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U2 - 10.1021/jacs.2c11450
DO - 10.1021/jacs.2c11450
M3 - Article
C2 - 36716175
AN - SCOPUS:85147224552
SN - 0002-7863
VL - 145
SP - 3276
EP - 3282
JO - Journal of the American Chemical Society
JF - Journal of the American Chemical Society
IS - 6
ER -