Multivalent Protein-Nucleic Acid Interactions Probed by Composition-Gradient Multiangle Light Scattering.

Abstract

Many RNA-binding proteins, such as TDP-43 or CELF1, interact multivalently with nucleic acid repetitive elements. The molecular stoichiometry of protein to nucleic acid is often difficult to assess, particularly by standard electrophoretic mobility shift assays (EMSAs). Here, we investigate the use of composition-gradient multiangle light scattering (CG-MALS) for quantifying binding affinity and stoichiometry for two RNA-binding proteins with their nucleic acid partners of varied sequence and length: TDP43's N-terminal RNA recognition motifs with both TG/GU-repeat ssDNA and ssRNA, respectively, and CELF1's two N-terminal RNA recognition motifs with (TG/UGUU/GU) repeats and an experimentally defined cognate GU-rich element (GRE). Our CG-MALS data derived from each of these interactions is consistent with expected ranges of binding affinity and stoichiometry for proteins binding to shorter nucleic acid repeats. Furthermore, we conclude that CG-MALS can be an excellent method for obtaining quantitative estimates even for high (>2) protein-nucleic acid stoichiometric ratios.