Exampleunfolded RNA, such as the one shown at right, is zero. Formation of the four base pairs indicated would release 1.9 kcal/mol of energy into the surroundings. The flow of energy into the surroundings is customarily written as a negative value, so the free energy of the RNA is listed in game as -1.9 kcal/mol. Note that the energy listed is actually the change in free energy that accompanies folding of the RNA. The other way to look at it is as a measure of the potential energy in the system. A shape with a negative free energy would require additional energy from the environment to break the bonds and unfold it, hence it is at a lower potential energy than the unfolded string. Loops often have positive free energy. They would release that energy to the environment if broken open. It is the negative free energy (from the bonds) in the attached stacks that allow them to stay closed. Some boosted loops (e.g. a 1-1 loop with double G boost) will have a negative free energy. This free energy is actually coming from a non-Watson-Crick bond involving the boost nucleotides.
Calculating Free Energy
EteRNA uses the energy function in the Vienna package to calculate free energy. Energy parameters can be found at the Nearest Neighbor Database. There may be minor differences between energy functions listed at NNDB and those used in EteRNA.
Teaching about Free Energy
Understanding Free Energy (Using Legos) by Kevin Steppe