Secondary structural features of alternative translation initiation sites in 5'-UTR regions of mRNA. Folding of a representative alternative start site in a stem-loop model using UNAFold centered on the start site is illustrated in this figure. The alternative start site, CUG, is circled in red. Features of this secondary structure served as inputs to the ANN. For each 50 base pair window surrounding the putative alternative initiation site (as shown in this figure), the local stability of the start codon itself and the free energy of the structure were recorded. The window size (50 bp) was experimentally determined as the minimum window size which produced consistent foldings through shifts in the folding window. Based on the scale of 0 to 3 scale, the stability would be measured as a 3 since the codon (all three bases) are present entirely in the loop structure.