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

Lin, Ya-Hui; Chang, Kung-Yao

The genes of designed pseudoknot constructs, with their corresponding slippery sequences and bridging spacer sequences, were generated by a fragment overlapping extension polymerase chain reaction (PCR) (20, 21) . Nucleotide sequences corresponding to SARS-CoV pseudoknot or core missing 3 -half sequences were PCR-amplified by designed primers using previous SARS-CoV plasmids (22) as the templates. Different fragments were assembled by PCR via overlapping sequences located in the 3 -and 5ends of each fragment. A theophylline-responsive element combining theoOFF2 with Switch-1 was generated using the same strategy. The final assembled fragment flanked by SalI and BamHI restriction sites was restriction-enzymes digested, purified and cloned into compatible sites of PUC18, p2luc dual luciferase reporter (23) or pNinsertC-Venus fluorescence reporter (24) (for pNtheoOFF2-Switch1C-Venus). A theoOFF2-Switch1 containing Venus was further amplified from pNtheoOFF2-Switch1C-Venus and constructed downstream of the tetracycline responsive element (TRE) promoter of PB-T-PAF vector (25) to form plasmid PBTPAF-theoOFF2-Switch1. The PB-RN plasmid that carries reverse tetracycline transactivator gene (rtTA), the helper plasmid PBCy43 that expresses PB transposase and PB-T-PAF were gifts from Prof. J. M. Rini at the University of Toronto, Canada (25) . Mutants with theophylline binding pocket disruption or read-through control used for calibrating frameshifting efficiency were generated using the quick-change mutagenesis kit from Stratagene according to manufacturer's instructions. All the primers were chemically synthesized and purchased from Genomics BioSci & Tech, Taiwan. Identities of all cloned and mutated genes were confirmed by DNA sequencing.