Canonical translation-modulating OFF-riboswitches with a single aptamer binding to a small molecule that function in a higher eukaryotic cell-free expression system.

We have found that OFF-riboswitches that ligand-dependently downregulate the canonical translation in a higher eukaryotic expression system (wheat germ extract) can be easily created by inserting a single aptamer into the 5' untranslated region (UTR) of mRNA, even if its ligand is as small as theophylline. The key is the position of the inserted aptamer: the 5' end (+0 position) is much better than other positions for inhibiting canonical translation with the aptamer-ligand complex. The data showed that ribosome loading is suppressed by a rigid structure in the 5' end, and this suppression is dependent on the structure's stability but not on its size. Although this preference of aptamer insertion point contradicts the results in a lower eukaryote, it accords with the fact that the 5'-end structural hindrance is more effective for blocking the ribosome in higher eukaryotes. Therefore, the present type of OFF-riboswitch would function in various higher eukaryotic expression systems.