Assessing the in vitro Binding Affinity of Protein-RNA Interactions Using an RNA Pull-down Technique

RNA is a vital component of the cell and is involved in a diverse range of cellular processes through a variety of functions. However, many of these functions cannot be performed without interactions with proteins. There are currently several techniques used to study protein-RNA interactions, such as electrophoretic mobility shift assay, fluorescence anisotropy, and filter binding. RNA-pulldown is a technique that uses biotinylated RNA probes to capture protein-RNA complexes of interest. First, the RNA probe and a recombinant protein are incubated to allow the in vitro interaction to occur. The fraction of bound protein is then captured by a biotin pull-down using streptavidin-agarose beads, followed by elution and immunoblotting for the recombinant protein with a His-tag-reactive probe. Overall, this method does not require specialized equipment outside what is typically found in a modern molecular laboratory and easily facilitates the maintenance of an RNase-free environment. This protocol was validated in: Nucleic Acids Res (2020), DOI: 10.1093/nar/gkaa029 Graphical abstract.