Boundary Analysis to Determine the Minimal RNA Sequence Required for Protein Binding

Abstract

Boundary analysis is a powerful and often overlooked method for determining the sequences within an RNA molecule that are required for a specific RNA–protein interaction. In this approach, 5′- and 3′-end-labeled RNAs are fragmented randomly (usually by limited alkaline hydrolysis, but nuclease fragmentation can also be used) such that a ladder of fragments covering the whole molecule of interest is produced. This mixture of fragments is then allowed to bind to the protein (or other molecule) of interest. Bound fragments are selected by affinity or antibody binding and then displayed on a gel. The point at which banding is lost for 5′-end-labeled RNAs defines the 3′ boundary of the binding site, and the point at which banding is lost for 3′-end-labeled RNAs defines the 5′ boundary of the binding site.