Rational design of a synthetic mammalian riboswitch as a ligand-responsive -1 ribosomal frame-shifting stimulator

Lin, Ya-Hui; Chang, Kung-Yao

Recent simulation studies have indicated that the stabilities of constituent secondary structures determined the folding of RNA pseudoknots (36) . This means, interference of the folding of stem 1 or stem 2 of a pseudoknot to affect pseudoknot formation could be controlled by a designed secondary structural element within the pseudoknot. As the 3 -side of pseudoknot stem 2 as well as that of the embedded theophylline aptamer in Switch-0 is bridged by UCU tri-nucleotides, we reasoned that a theophylline-responsive −1 PRF stimulator (Switch-1) could be constructed by coupling stem 2 formation with theophylline-binding pocket formation ( Figure 1B and D) . This was achieved by designing sequences flanking UCU to form a stable hairpin, while maintaining base pairing of the lower stem in the theophylline-bound aptamer ( Figure 1D ). We rationalized that such an engineered switch hairpin of reasonable stability (predicted free energy of −12.7 kcal/mole (37)) would be the dominant conformation that could interfere with the formation of pseudoknot stem 2 in the absence of theophylline (Supplementary Figure S2A) . As it is difficult to measure the free energy contribution of stem 2 formation, we mimicked it by a hairpin of UCU loop closed by the stem 2 (36) with a predicted free energy of −7.0 kcal/mole (Supplementary Figure S2B) . By contrast, the addition of theophylline could interfere with switch hairpin formation via theophylline aptamer stabilization and help release the trapped 3 -side of stem 2 to facilitate stem 2 pairing for generation of a pseudoknot. In the design of Switch-1, only the eight nucleotides constituting its lower aptamer stem are different from those of Switch-0 ( Figure 1C and D) .