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

Lin, Ya-Hui; Chang, Kung-Yao

Given the structural information available for SARS-PK and theophylline aptamer, in-line probing was used to evaluate theophylline-binding activity as well as monitor the extent of ligand-dependent spontaneous RNA cleavage of Switch-1 RNA. The results were then compared to those of Switch-0 RNA (Figure 2 and Supplementary Figure S3 ). By tracking hydrolyzed RNA patterns with increased amounts of theophylline, dramatic changes in cleavage patterns were observed in regions corresponding to theophylline-binding pockets in both RNAs. This result was consistent with ligand-binding mediated conformational change or protection of cleavage with an apparent Kd value of 1.31 M for Switch-1 RNA (Supplementary Figure S2C) . Extra prominent RNA hydrolysis signals were observed in sequences involved in aptamer lower stem formation (corresponding to S3-1 to S3-11 in Figure 1D ) as well as in sequences cor- Figure 1 . SARS-PK as a scaffold for engineering a theophylline-dependent −1 PRF stimulator. (A) A schematic drawing shows the replacement of stem 3 (S3) of SARS-PK with a theophylline aptamer (boxed in blue) to form Switch-0. The drawing is based on characterized secondary structures of SARS-PK and ligand-bound theophylline aptamer. The secondary structures are designated by 'S' for a stem and 'L' for a loop with given numbers corresponding to appearance order from the 5 -end. (B) A scheme shows coupling of pseudoknot stem 2 formation with theophylline binding in Switch-1 by designing a switch hairpin. The 5 -and 3 -complementary sequences of the hairpin stem (in the free form) are designed to participate in the formations of ligandbinding pocket (colored in green) and stem 2 (colored in blue) upon the binding of theophylline, respectively. (C) Sequences and secondary structural models of SARS-PK and Switch-0 RNA (in theophylline-bound form). The numbering of sequence in SARS-PK follows the one described previously (22) . Numbering system in the SARS-PK part of Switch-0 follows that of SARS-PK while numbering in the aptamer domain starts at S3-1 and ends at S3-42 with S3 standing for stem 3. Characterized secondary structures of SARS-PK and theophylline aptamer are used as templates to build the models. (D) Sequences and secondary structural models of free and ligand-bound forms of Switch-1. Characterized secondary structures of stem1/2 of SARS-PK and theophylline aptamer are used as templates to build the models. Numbering logic is the same as that of Switch-0. The eight nucleotides different from Switch-0 are typed in lower case in both forms.