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High-throughput smFRET analysis of freely diffusing nucleic acid molecules and associated proteins

View ORCID ProfileMaya Segal, View ORCID ProfileAntonino Ingargiola, View ORCID ProfileEitan Lerner, View ORCID ProfileSang Yoon Chung, View ORCID ProfileJonathan A. White, View ORCID ProfileAaron Streets, View ORCID ProfileS. Weiss, View ORCID ProfileX. Michalet
doi: https://doi.org/10.1101/651869
Maya Segal
aDepartment of Chemistry & Biochemistry, UCLA, Los Angeles, CA 90095, USA
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Antonino Ingargiola
aDepartment of Chemistry & Biochemistry, UCLA, Los Angeles, CA 90095, USA
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Eitan Lerner
aDepartment of Chemistry & Biochemistry, UCLA, Los Angeles, CA 90095, USA
bDepartment of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, Faculty of Mathematics & Science, The Edmond J. Safra Campus, The Hebrew University of Jerusalem, Jerusalem 9190401, Israel
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Sang Yoon Chung
aDepartment of Chemistry & Biochemistry, UCLA, Los Angeles, CA 90095, USA
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Jonathan A. White
cDepartment of Bioengineering, UC Berkeley, Berkeley, CA 94720, USA
dBioengineering, California Institute of Technology, Pasadena, CA 91125, USA
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Aaron Streets
cDepartment of Bioengineering, UC Berkeley, Berkeley, CA 94720, USA
eChan-Zuckerberg Biohub, San Francisco, CA 94158, USA
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S. Weiss
aDepartment of Chemistry & Biochemistry, UCLA, Los Angeles, CA 90095, USA
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X. Michalet
aDepartment of Chemistry & Biochemistry, UCLA, Los Angeles, CA 90095, USA
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  • For correspondence: michalet@chem.ucla.edu
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Abstract

Single-molecule Förster resonance energy transfer (smFRET) is a powerful technique for nanometer-scale studies of single molecules. Solution-based smFRET, in particular, can be used to study equilibrium intra- and intermolecular conformations, binding/unbinding events and conformational changes under biologically relevant conditions without ensemble averaging. However, single-spot smFRET measurements in solution are slow. Here, we detail a high-throughput smFRET approach that extends the traditional single-spot confocal geometry to a multispot one. The excitation spots are optically conjugated to two custom silicon single photon avalanche diode (SPAD) arrays. Two-color excitation is implemented using a periodic acceptor excitation (PAX), allowing distinguishing between singly- and doubly-labeled molecules. We demonstrate the ability of this setup to rapidly and accurately determine FRET efficiencies and population stoichiometries by pooling the data collected independently from the multiple spots. We also show how the high throughput of this approach can be used to increase the temporal resolution of single-molecule FRET population characterization from minutes to seconds. Combined with microfluidics, this high-throughput approach will enable simple real-time kinetic studies as well as powerful molecular screening applications.

Footnotes

  • Manuscript submitted to Methods and revised to address comments from reviewers.

  • https://doi.org/10.6084/m9.figshare.7838156

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
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Posted June 25, 2019.
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High-throughput smFRET analysis of freely diffusing nucleic acid molecules and associated proteins
Maya Segal, Antonino Ingargiola, Eitan Lerner, Sang Yoon Chung, Jonathan A. White, Aaron Streets, S. Weiss, X. Michalet
bioRxiv 651869; doi: https://doi.org/10.1101/651869
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High-throughput smFRET analysis of freely diffusing nucleic acid molecules and associated proteins
Maya Segal, Antonino Ingargiola, Eitan Lerner, Sang Yoon Chung, Jonathan A. White, Aaron Streets, S. Weiss, X. Michalet
bioRxiv 651869; doi: https://doi.org/10.1101/651869

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