PSIV IRES-based eukaryotic kt-ON-riboswitches responsive to other ligands. (A,B) Predicted partial secondary structures of rationally designed eukaryotic kt-ON-riboswitches with the split tetracycline aptamer (tc-ISL) and the split FMN aptamer (FMN-ISL), respectively. The circled nucleotides probably hybridize to their complementary nucleotides to form the P3 duplex. The 5′ terminal A in the 5′-side tetracycline aptamer's core may hybridize to the 3′ terminal U in the 3′-side thereof in the ON structure. The A*–U* base pair is required for binding to FMN. It is impossible to design FMN-IS15 because the IS's loop would be only 2 nt. (C) Relative fluorescence intensities of YPet expressed from DNA templates encoding tc-ISL before the YPet gene (T7-tc-ISL-Y) in the absence or presence of 100 µM tetracycline (tc) in the WGE-cIVTT system. (D) Relative luminescence intensities of nLuc expressed from DNA templates encoding FMN-ISL before the nLuc gene (T7-FMN-ISL-nL) in the absence or presence of 300 µM FMN in the WGE-cIVTT system. The error bars represent the standard deviation (n = 4).
