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

Lin, Ya-Hui; Chang, Kung-Yao

We also used a split fluorescent reporter with the coding region of its C terminal domain shifted to the −1 frame (24) to monitor theophylline-dependent −1 PRF activity in 293T cells by using theoOFF2-Switch1 to link the split N and C domains of fluorescent protein. Consistently, elevated fulllength fluorescent protein expression was induced by theophylline treatment, whereas the construct of read-through control (theoOFF2-RFC1) expressed constitutively ( Figure 5E and F). Importantly, these transiently expressed results indicate that a combination of theoOFF2 and Switch-1 provides tighter theophylline-dependent regulation of −1 PRF compared with theoOFF2 alone (Figure 5E and F) . Finally, a stable cell-line (293T-theo1) harboring a split fluorescent reporter gene embedded with theoOFF2-Switch1 was established via a PiggBac-based approach (25) with the transcription of reporter mRNA controlled by tetracycline. We found that prominent Venus activity could be observed in the presence of both theophylline and tetracycline ( Figure 6A and B), whereas low Venus activity existed in the absence of theophylline. Together, these results clearly demonstrate that the engineered theophylline-responsive −1 PRF stimulator is robust and compatible with existing tools to build a regulatory circuit in the 293T human cell-line.