Sequence Design Using RNAstructure.

RNA is present in all domains of life. It was once thought to be solely involved in protein expression, but recent advances have revealed its crucial role in catalysis and gene regulation through noncoding RNA. With a growing interest in exploring RNAs with specific structures, there is an increasing focus on designing RNA structures for in vivo and in vitro experimentation and for therapeutics. The development of RNA secondary structure prediction methods has also spurred the growth of RNA design software. However, there are challenges to designing RNA sequences that meet secondary structure requirements. One major challenge is that the secondary structure design problem is likely NP-hard, making it computationally intensive. Another issue is that objective functions need to consider the folding ensemble of RNA molecules to avoid off target structures. In this chapter, we provide protocols for two software tools from the RNAstructure package: "Design" for structured RNA sequence design and "orega" for unstructured RNA sequence design.