A Step-by-Step Guide to Study Protein–RNA Interactions

Abstract

Protein–RNA complex formation is at the center of RNA metabolism and leads to the modulation of protein and RNA functions. We propose here a step-by-step guide to investigate these interactions including the identification of the protein and RNA parts involved in complex formation, the determination of the affinity of the complex and the characterization of the protein–RNA interface at amino acid and nucleotide level. Moreover, we briefly review the methods that are the most often used to obtain this information using primarily examples from our lab and finally mention what we perceive as the next challenges in the field.

How to Find the Right RNA-Sensing CRISPR-Cas System for an In Vitro Application

“…Quantitative data on binding affinity are scarce but much preferred over qualitative results. These are often derived from densitometry quantification of EMSA bands (i.e., for Sau Cas9 [ 26 ]) or, more accurately, by biophysical methods, such as isothermal titration calorimetry, microscale thermophoresis, or surface plasmon resonance [ 60 ].…”

The Perturbed Free‐Energy Landscape: Linking Ligand Binding to Biomolecular Folding

“…Technologies for characterizing protein-RNA binding affinities, as well as mapping binding surfaces and detecting conformation changes, have been reviewed recently. [119] Though it might require more prior knowledge (as through crystallization trials) there could be some proteins for which single combinations of secondary/tertiary structure could be experimentally isolated and binding affinity could be measured. Perhaps more promising in the long run, is the prospect of blind prediction of protein binding affinities from structural data on a single complex.…”