Introducing three GU base pairs into the mRNA/miRNA duplex did, with only minor changes to the binding energy, almost completely destroy the functionality of the binding site. While Doench and Sharp concluded that miRNA binding sites are not functional because of the GU base pairs, testing the dimer with RNAcofold shows that there is also a significant difference in the cofolding structure that might account for the activity difference without invoking sequence specificities: Because of the secondary structure of the target, the binding at the 5' end of the miRNA is much weaker than in the wild type, Fig. 4.