PT  - JOURNAL ARTICLE
AU  - Charles Limouse
AU  - Jason C. Bell
AU  - Colin J. Fuller
AU  - Aaron F. Straight
AU  - Hideo Mabuchi
TI  - Intramolecular dynamics of single molecules in free diffusion
AID  - 10.1101/120311
DP  - 2017 Jan 01
TA  - bioRxiv
PG  - 120311
4099  - http://biorxiv.org/content/early/2017/03/24/120311.short
4100  - http://biorxiv.org/content/early/2017/03/24/120311.full
AB  - 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.