A number of analyses require estimates of the folding free energy changes of specific RNA secondary structures. These predictions are often based on a set of nearest neighbor parameters that models the folding stability of a RNA secondary structure as the sum of folding stabilities of the structural elements that comprise the secondary structure. In the software suite RNAstructure, the free energy change calculation is implemented in the program efn2. The efn2 program estimates the folding free energy change and the experimental uncertainty in the folding free energy change. It can be run through the graphical user interface for RNAstructure, from the command line, or a web server. This chapter provides detailed protocols for using efn2.

The purpose of this section is to detail methods for the computational prediction of RNA secondary structure. This protocol is intended to provide an easy entry into the field of RNA structure prediction for those wishing to utilize it in their research and to suggest \'best practices\' for going from sequence to secondary structure depending on the available data.