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

Lin, Ya-Hui; Chang, Kung-Yao

In regards to the theophylline aptamer used in this study, the successes in the designs of Switch-1 and Switch-lock suggest that the stabilities of the switch hairpins affect the regulatory dynamic ranges of designed variants in vitro. Indeed, further destabilizing the switch hairpin in Switch-1 led to improvement in ligand responsiveness of Switch-2 in vitro. However, this improvement might be due to reasons other than the original design. In particular, design in Switch-2 could disrupt potential stacking between stem 1 and the lower aptamer stem in the absence of theophylline (see Figure 1D for a predicted secondary structure model), leading to reduced basal frameshifting activity and improved dynamic range. Additionally, this design could also reduce the base-pairing number of the lower aptamer stem in the presence of theophylline thereby affecting theophylline-binding affinity (35) , and raising the concentration of theophylline required to fully stabilize aptamer conformation. Consistently, increased spontaneous RNA hydrolysis bands appeared in the lower aptamer stem of Switch-2 RNA in higher theophylline concentration (Supplement Figure S6A and B), whereas no theophyllinedependent in-line cleavage in corresponding regions of Switch-1 RNA (Figure 2 and Supplement Figure S3 ). Finally, we observed a gap between in vitro and cellular results for Switch-2. As the theophylline-dependent activation ranges for Switch-1 were well-conserved between in vitro translation and 293T cells, this made it possible to predict that the discrepancy observed in Switch-2 could be caused by the six nucleotides removed from Switch-1. The predicted free energy for the switch hairpin of Switch-2 (−8.6 kcal/mole) suggests it could be unstable under cell culturing conditions (37 • C). By contrast, the switch hairpin (predicted free energy of −12.7 kcal/mole) in Switch-1 should populate significantly at both 30 • C and 37 • C. A more comprehensive analysis in energy contribution from the formation of stem 2 as well as from theophylline binding would also be required to address this problem. It will be interesting to see if Switch-2 retains its dynamic range in other eukaryotic systems requiring habitation temperatures lower than 37 • C.