Multivalent Protein–Nucleic\nAcid Interactions\nProbed by Composition-Gradient Multiangle Light Scattering

Many RNA-binding proteins, such as TDP-43 or CELF1, interact\nmultivalently\nwith nucleic acid repetitive elements. The molecular stoichiometry\nof protein to nucleic acid is often difficult to assess, particularly\nby standard electrophoretic mobility shift assays (EMSAs). Here, we\ninvestigate the use of composition-gradient multiangle light scattering\n(CG-MALS) for quantifying binding affinity and stoichiometry for two\nRNA-binding proteins with their nucleic acid partners of varied sequence\nand length: TDP43’s N-terminal RNA recognition motifs with\nboth TG/GU-repeat ssDNA and ssRNA, respectively, and CELF1’s\ntwo N-terminal RNA recognition motifs with (TG/UGUU/GU) repeats and\nan experimentally defined cognate GU-rich element (GRE). Our CG-MALS\ndata derived from each of these interactions is consistent with expected\nranges of binding affinity and stoichiometry for proteins binding\nto shorter nucleic acid repeats. Furthermore, we conclude that CG-MALS\ncan be an excellent method for obtaining quantitative estimates even\nfor high (>2) protein–nucleic acid stoichiometric ratios.