Mapping specificity landscapes of RNA-protein interactions by high throughput sequencing

•HiTS-Kin determines rate constants for interactions of RBPs with thousands of RNAs.•HiTS-EQ determines equilibrium binding constants for RBPs to thousands of RNAs.•A combination of HiTS-Kin and HiTS-EQ reveals RBP specificity landscapes.•Practical and conceptual considerations for HiTS-Kin and HiTS-EQ are discussed.

To function in a biological setting, RNA binding proteins (RBPs) have to discriminate between alternative binding sites in RNAs. This discrimination can occur in the ground state of an RNA-protein binding reaction, in its transition state, or in both. The extent by which RBPs discriminate at these reaction states defines RBP specificity landscapes. Here, we describe the HiTS-Kin and HiTS-EQ techniques, which combine kinetic and equilibrium binding experiments with high throughput sequencing to quantitatively assess substrate discrimination for large numbers of substrate variants at ground and transition states of RNA-protein binding reactions. We discuss experimental design, practical considerations and data analysis and outline how a combination of HiTS-Kin and HiTS-EQ allows the mapping of RBP specificity landscapes.