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

Lin, Ya-Hui; Chang, Kung-Yao

We then explored if the stability of a switch hairpin can be used as a guide to improving the design. We first stabilized the switch hairpin of Switch-1 by engineering two extra GC base pairs in the terminal end of the switch stem to form a Switch-lock construct (Supplementary Figure S5A) and found that it possessed low −1 PRF activity in theophylline (Supplementary Figure S5B-D) . This is consistent with the stabilized switch hairpin (predicted free-energy of −17.6 kcal/mole) being locked even in the presence of theophylline. Switch-2 was then designed by removing 3 base pairs from lower aptamer stem of the ligand-bound Switch-1. It resulted in three base pairs disruption in the upper stem of switch hairpin in the free form of Switch-2 (compare Figure 1D with Supplementary Figure S5A) . This was done with the assumption that the destabilized switch hairpin (predicted free-energy of −8.6 kcal/mole) could still compete with the formation of a pseudoknot stem 2 without theophylline, while binding of theophylline would facilitate stem 2 formation. Switch-2 possessed 4-fold increase in −1 PRF stimulation in response to 1 mM theophylline (Supplementary Figure S5B-D) . Furthermore, the dynamic range was increased to 6-fold in the presence of 2 mM theophylline, whereas the dynamic range of Switch-1 did not increase further (Supplementary Figure S5C) . In-line probing analysis of Switch-2 RNA indicated similar theophyllinedependent RNA hydrolysis patterns as those of Switch-1 RNA in ligand-binding-pockets and 5 -side sequences of stem 2 (Supplement Figure S6A and B) . A Kd value of 10folds higher than that of Switch-1 RNA (Supplement Figure S6C) helps explain higher theophylline concentration requirement to activate −1 PRF stimulation efficiency of Switch-2. However, experiments showed that Switch-2 behaves similarly to Switch-1 in 293T cells (data not shown), indicating a missing link between in vitro and cellular experiments.