Probing RNA–protein interactions with single-molecule pull-down assays

Abstract

Recent advances in single-molecule techniques allow for dynamic observations of the interactions between various protein assemblies and RNA molecules with high spatiotemporal resolution. However, it remains challenging to obtain functional eukaryotic protein complexes and cost-effective fluorescently labeled RNAs to study their interactions at the single-molecule level. Here, we describe protocols combining single-molecule fluorescence with various protein complex pull-down techniques to determine the function of RNA-interacting protein complexes of interest. We provide step-by-step guidance for using novel single-molecule techniques including RNA labeling, protein complexes purification, an..