RNase footprinting to map sites of RNA-protein interactions

The binding of a protein to an RNA sequence protects that the region of the RNA from ribonuclease (RNase) digestion; this protected region is known as the protein's "footprint." In this protocol, end-labeled RNAs with and without bound protein are digested with RNase, and the products of digestion are analyzed by gel electrophoresis on denaturing polyacrylamide gels. If the experiment is performed properly, a comparison of the banding patterns from the two samples will reveal the binding site of the protein. The binding site-or footprint-will be detected as a region without bands in the protein-bound sample. In the sample without bound protein, the bands should cover the entirety of the RNA molecule. To establish appropriate digestion conditions for the procedure (i.e., ≤1 cleavage event per molecule), it is necessary to titrate the amount of RNase under a range of time and temperature conditions. RNase I cleaves after every nucleotide of RNA and works well under many assay conditions, but other enzymes with different cleavage specificities can also be used. RNase VI is preferable when analyzing structured RNA; RNase A is preferable when using pyrimidine-rich RNAs; and RNase T1 is useful for G-rich RNAs. Choosing enzymes with preference for double-stranded (such as RNase VI) versus single-stranded (such as RNase I) RNA may be helpful. Often, a combination of nucleases is advantageous.