Large-Scale Analysis of RNA–Protein Interactions for Functional RNA Motif Discovery Using FOREST

Abstract

RNA transcripts can form a variety of higher-order structures. We developed a large-scale affinity analysis system, FOREST (Folded RNA Element Profiling with Structure Library), to investigate the function of these RNA structures on transcriptome-wide scale. Here we describe a protocol to analyze RNA–protein interactions using FOREST. Users of the protocol prepare an RNA structure library comprised of diverse species of transcripts and perform high-throughput characterization of the RNA–protein interactions to obtain quantitative and comprehensive information on the binding affinities and specificities. Moreover, we demonstrate how FOREST can be used to analyze a non-canonical structure, the RNA G-quadruplex, without sequencing bias, because the quantification is performed directly on a microarray without sequence amplification. FOREST will contribute to the discovery of RNA structure motifs that determine RNA–protein interactions.