Spectral and Hydrodynamic Analysis of West Nile Virus RNA–Protein Interactions by Multiwavelength Sedimentation Velocity in the Analytical Ultracentrifuge

Interactions between
nucleic acids and proteins are critical for
many cellular processes, and their study is of utmost importance to
many areas of biochemistry, cellular biology, and virology. Here,
we introduce a new analytical method based on sedimentation velocity
(SV) analytical ultracentrifugation, in combination with a novel multiwavelength
detector to characterize such interactions. We identified the stoichiometry
and molar mass of a complex formed during the interaction of a West
Nile virus RNA stem loop structure with the human T cell-restricted
intracellular antigen-1 related protein. SV has long been proven as
a powerful technique for studying dynamic assembly processes under
physiological conditions in solution. Here, we demonstrate, for the
first time, how the new multiwavelength technology can be exploited
to study protein–RNA interactions, and show how the spectral
information derived from the new detector complements the traditional
hydrodynamic information from analytical ultracentrifugation. Our
method allows the protein and nucleic acid signals to be separated
by spectral decomposition such that sedimentation information from
each individual species, including any complexes, can be clearly identified
based on their spectral signatures. The method presented here extends
to any interacting system where the interaction partners are spectrally
separable.