Inverse RNA folding workflow to design and test ribozymes that include pseudoknots

Ribozymes are RNAs that catalyze reactions. They occur in nature, and can also be evolved in vitro to catalyze novel reactions. This chapter provides detailed protocols for using inverse folding software to design a ribozyme sequence that will fold to a known ribozyme secondary structure and for testing the catalytic activity of the sequence experimentally. This protocol is able to design sequences that include pseudoknots, which is important as all naturally occurring full-length ribozymes have pseudoknots. The starting point is the known pseudoknot-containing secondary structure of the ribozyme and knowledge of any nucleotides whose identity is required for function. The output of the protocol is a set of sequences that have been tested for function. Using this protocol, we were previously successful at designing highly active double-pseudoknotted HDV ribozymes.