RNA-Affinity Chromatography

Abstract

RNA-affinity chromatography assays are used to identify proteins binding specific RNA sequences. These proteins represent potential factors contributing to the function of RNA molecules. In our lab, we have used this protocol to identify proteins binding sequence motifs involved in replication and transcription of positive strand RNA viruses. The assay described in this protocol consists on the immobilization of 5’-biotinylated RNA oligonucleotides (30-40 nt) on a streptavidin-conjugated, paramagnetic solid matrix. Then, cytoplasmic protein extracts pre-cleared on the solid matrix to decrease nonspecific binding, were incubated with the immobilized RNA molecules in the presence of a nonspecific competitor. RNA-protein complexes immobilized on the paramagnetic solid matrix were isolated using a magnet and the bound proteins were separated by polyacrylamide gel electrophoresis for proteomic analysis.