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Resolving Mixtures in Solution by Single-Molecule Rotational Diffusivity
journal contribution
posted on 2018-07-12, 00:00 authored by Hsiang-Yu Yang, W. E. MoernerSensing
the size of individual molecules in an ensemble has proven
to be a powerful tool to investigate biomolecular interactions and
association–dissociation processes. In biologically relevant
solution environments, molecular size is often sensed by translational
or rotational diffusivity. The rotational diffusivity is more sensitive
to the size and conformation of the molecules as it is inversely proportional
to the cube of the hydrodynamic radius, as opposed to the inverse
linear dependence of the translational diffusion coefficient. Single-molecule
rotational diffusivity has been measured with time-resolved fluorescence
anisotropy decay, but the ability to sense different sizes has been
restricted by the limited number of photons available or has required
surface attachment to observe each molecule longer, and the attachment
may be perturbative. To address these limitations, we show how to
measure and monitor single-molecule rotational diffusivity by combining
the solution-phase Anti-Brownian ELectrokinetic (ABEL) trap and maximum
likelihood analysis of time-resolved fluorescence anisotropy based
on the information inherent in each detected photon. We demonstrate
this approach by resolving a mixture of single- and double-stranded
fluorescently labeled DNA molecules at equilibrium, freely rotating
in a native solution environment. The rotational diffusivity, fluorescence
brightness and lifetime, and initial and steady-state anisotropy are
simultaneously determined for each trapped single DNA molecule. The
time resolution and precision of this method are analyzed using statistical
signal analysis and simulations. We present key parameters that define
the usefulness of a particular fluorescent label for extracting molecular
size information from single-molecule rotational diffusivity measurements.
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solution environmenthydrodynamic radiuslikelihood analysistime-resolved fluorescence anisotropy decaysurface attachmentABELtranslational diffusion coefficientfluorescence brightnessDNA moleculesdiffusivity measurementsbiomolecular interactionsSingle-Molecule Rotational Diffusivitydouble-stranded fluorescentlytime resolutionResolving Mixturessize informationsolution environmentssignal analysistime-resolved fluorescence anisotropyDNA moleculesolution-phase Anti-Brownian ELectrokinetic
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