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

Lin, Ya-Hui; Chang, Kung-Yao

In a first step to constructing a ligand-responsive −1 PRF stimulator, we designed Switch-0 RNA with a theophylline aptamer replacing the stem 3 of SARS-PK ( Figure 1A and C). A theophylline aptamer was used due to theophylline's cell permeability (33) and the well-characterized structural features of the aptamer (34) . The ligand-binding pocket of theophylline aptamer is composed of an internal-loop and an adjacent bulge with conserved key theophyllinecontact sequences distributed within the two motifs. Each motif is connected to duplex regions that serve as the carrier of the binding-pocket ( Figure 1A and C). Importantly, the conservation of primary sequences in the terminal duplex (the 'lower stem' in Figure 1A ) that closes the internal-loop is not absolutely required as long as basepairing complementarity of the duplex is maintained (35) . This feature thus provides flexibility in designing a liganddependent conformational switch. The −1 PRF activity of Switch-0 placed downstream of a slippery sequence is one third that of SARS-PK based on in vitro frameshifting assays performed in reticulocyte lysate. Furthermore, the −1 PRF efficiencies of both SARS-PK and Switch-0 remained virtually unchanged with or without 1 mM theophylline treatment (Supplementary Figure S1A-D) . However, results from in-line probing analysis of Switch-0 RNA (Figure 2A) indicated that the embedded theophylline aptamer remained theophylline binding competent (Supplementary Figure S1E) . Therefore, Switch-0 represents an ideal starting framework to build a theophylline-dependent −1 PRF stimulator.