RNA Structure Determination Using Nuclease Digestion

Abstract

Determining RNA structures (i.e., single- and double-strand regions) is often useful when assessing the potential for certain RNAs to interact with proteins or when determining whether RNAs that are dissimilar in sequence can form the same structure. A number of ribonucleases (RNases) have been used to map RNA structure, but many of these are no longer available. However, three commonly available RNA endonucleases (RNase T1, RNase I, and RNase V1) can provide a wealth of structural information. Cleavages of end-labeled RNA are initiated by one of the RNases (H₂O is used for mock-treated controls), terminated with aurintricarboxylic acid (a potent RNase inhibitor), and detected by electrophoresis on denaturing polyacrylamide gels. Because there are enzymes that can cleave only when the RNA is single stranded (e.g., RNase T1) or double stranded (e.g., RNase V1), it is possible to do parallel analyses.