Calculated folding free energies of mRNA sequences from 33 human genes were each compared to ten step-wise partially shuffled versions of the same sequences, keeping the dinucleotide, or "neighbor", composition preserved. The first ten base-swap steps of the shuffling process were dissected by examining the folding free energies after each base-swap. This whole procedure was repeated a hundred times for each gene. Most of the genes showed a tendency for the average free energy to decline dramatically, suggesting that these natural mRNAs have been subject to selection to maximize the folding free energy. For most of the genes, the direction of average change for these first ten steps was accurately predicted by the mRNA folding Z-scores calculated for thoroughly shuffled sequences. Estimates ranged from 12 to 69.2 as to how many swap steps would be required to match the average free energy for fully shuffled sequences.
