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Intramolecular dynamics of single molecules in free diffusion

View ORCID ProfileCharles Limouse, View ORCID ProfileJason C. Bell, View ORCID ProfileColin J. Fuller, View ORCID ProfileAaron F. Straight, Hideo Mabuchi
doi: https://doi.org/10.1101/120311
Charles Limouse
1Department of Applied Physics, Stanford University, Stanford, CA 94305
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Jason C. Bell
2Department of Biochemistry, Stanford University, Stanford, CA 94305
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Colin J. Fuller
2Department of Biochemistry, Stanford University, Stanford, CA 94305
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Aaron F. Straight
2Department of Biochemistry, Stanford University, Stanford, CA 94305
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  • For correspondence: hmabuchi@stanford.edu astraigh@stanford.edu
Hideo Mabuchi
1Department of Applied Physics, Stanford University, Stanford, CA 94305
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  • For correspondence: hmabuchi@stanford.edu astraigh@stanford.edu
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Abstract

Biomolecular systems such as multiprotein complexes or biopolymers can span several tens to several hundreds of nanometers, but the dynamics of such “mesocale” molecules remain challenging to probe. We have developed a single-molecule technique that uses Tracking Fluorescence Correlation Spectroscopy (tFCS) to measure the conformation and dynamics of molecular assemblies specifically at the mesoscale level (~100-1000 nm). tFCS is non-perturbative, as molecules, which are tracked in real-time, are untethered and freely diffusing. To achieve sub-diffraction spatial resolution, we use a feedback scheme which allows us to maintain the molecule at an optimal position within the laser intensity gradient. We find that tFCS is sufficiently sensitive to measure the distance fluctuations between two sites within a DNA molecule separated by distances as short as 1000 bp. We demonstrate that tFCS detects changes in the compaction of reconstituted chromatin, and can assay transient protein mediated interactions between distant sites in an individual DNA molecule. Our measurements highlight the impact that tFCS can have in the study of a wide variety of biochemical processes involving mesoscale conformational dynamics.

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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-NC-ND 4.0 International license.
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Posted March 24, 2017.
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Intramolecular dynamics of single molecules in free diffusion
Charles Limouse, Jason C. Bell, Colin J. Fuller, Aaron F. Straight, Hideo Mabuchi
bioRxiv 120311; doi: https://doi.org/10.1101/120311
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Intramolecular dynamics of single molecules in free diffusion
Charles Limouse, Jason C. Bell, Colin J. Fuller, Aaron F. Straight, Hideo Mabuchi
bioRxiv 120311; doi: https://doi.org/10.1101/120311

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