Most methods for nucleic acid secondary structure prediction rely on a nearest neighbor thermodynamic model. A common method to determine the most probable structures given a sequence of nucleotides makes use of a dynamic programming algorithm that seeks to find structures with low free energy. Dynamic programming algorithms often forbid pseudoknots, or other cases in which base pairs are not fully nested, as considering these structures becomes computationally very expensive for even small nucleic acid molecules. Other methods, such as stochastic context-free grammars can also be used to predict nucleic acid secondary structure.
For many RNA molecules, the secondary structure is highly important to the correct function of the RNA &mdash; often more so than the actual sequence. This fact aids in the analysis of non-coding RNA sometimes termed "RNA genes". One application of bioinformatics uses predicted RNA secondary structures in searching a genome for noncoding but functional forms of RNA. For example, microRNAs have canonical long stem-loop structures interrupted by small internal loops.
RNA secondary structure applies in RNA splicing in certain species. In humans and other tetrapods, it has been shown that without the U2AF2 protein, the splicing process is inhibited. However, in zebrafish and other teleosts the RNA splicing process can still occur on certain genes in the absence of U2AF2. This may be because 10% of genes in zebrafish have alternating TG and AC base pairs at the 3 splice site (3ss) and 5 splice site (5ss) respectively on each intron, which alters the secondary structure of the RNA. This suggests that secondary structure of RNA can influence splicing, potentially without the use of proteins like U2AF2 that have been thought to be required for splicing to occur.