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Anal Chem. 2017 Jan 3;89(1):862-870. doi: 10.1021/acs.analchem.6b03926. Epub 2016 Dec 15.

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

Author information

1
Georgia State University , Department of Chemistry, 50 Decatur St. SE, Atlanta, Georgia 30303, United States.
2
Georgia State University , Department of Biology, P.O. 4010, Atlanta, Georgia 30303, United States.
3
University of Konstanz , Department of Chemistry, Physical Chemistry, Universitätsstraße 10, Box 714, D-78457 Konstanz, Germany.
4
The University of Texas Health Science Center at San Antonio , 7703 Floyd Curl Drive, MC 7760, San Antonio, Texas 78229-3901, United States.

Abstract

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.

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