RNA Structural Analysis by Enzymatic Digestion

Enzymatic probing is a rapid, straightforward method for determining which regions of a folded RNA are structurally constrained. It can be carried out using very small amounts of material, and is especially suitable for short RNAs. Here we report a protocol that we have found to be useful and readily adaptable to the evaluation of RNAs up to 150–200 nucleotides in length. Considerations for optimization are also included. In brief, the method includes folding end-labeled RNA into its native conformation, partial digestion with structure-sensitive nucleases, and identification of the cleavage sites by electrophoretic separation of the cleavage fragments.