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

Lin, Ya-Hui; Chang, Kung-Yao

Comparison of in vitro activities of Switch-1 and Switch-2 indicated that residual frameshifting activity in the absence of theophylline is the main cause of reduction in the dynamic range of ligand-responsiveness of designed stimulators (Supplementary Figure S7) . Recently, we have identified RNA hairpins upstream of a frameshifting site as a negative regulator of −1 PRF (40) and demonstrated that −1 PRF activity can be regulated by ligand-induced conformational rearrangements of this upstream attenuator (24) . To improve the dynamic range of ligand response and to see if theophylline aptamers can be functional while existing in both positive and negative regulators of −1 PRF, we fused previously designed theophylline-dependent upstream attenuator, theoOFF2 (24) with Switch-1 ( Figure 5A ) and examined theophylline-dependent −1 PRF activity in vitro. For comparison, a construct with an upstream theoOFF2 and a downstream SARS-PK was also generated. We found that the upstream theoOFF2 regulated −1 PRF stimulated by downstream SARS-PK in a theophylline-dependent way with a dynamic range better than that of Switch-1 (Figure 5B and C) . Furthermore, a 6-to 8-folds increase in in vitro −1 PRF stimulation was observed in the theoOFF2-Switch1 construct when theophylline was increased to 1 mM ( Figure 5C ), suggesting the existence of a synergetic ef-fect for theophylline-dependent −1 PRF stimulation. However, this dynamic range was reduced to 5-fold in 293T cells transfected with the theoOFF2-Switch1 construct ( Figure 5D ). Further analysis suggests that this was due to the reduced dynamic range of theoOFF2 in 293T cells because the dynamic range of Switch-1 remained virtually the same under both conditions ( Figure 5C and D) . Thus, the use of the same ligand-binding aptamer in both upstream attenuator and downstream stimulator result in further enhancement of ligand-responsiveness for −1 PRF activity regulation.