Reverse Footprinting to Map Sites of RNA–Protein Interactions

Abstract

Nuclease protection of a site-specifically labeled RNA by an RNA-binding protein is an extremely powerful method for determining the site of an RNA–protein interaction. If a protein binds to the region that contains the site-specific label, it will protect the label and adjoining sequences from nuclease digestion. The protected region, or “footprint,” can then be characterized by extensive fragmentation and gel electrophoresis. As the name implies, reverse footprinting produces a mirror image of a conventional footprint; the footprint is revealed by the presence, not the absence, of bands. This method has a distinct advantage over conventional footprinting in that only a small fraction of the labeled RNA must be bound.