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

Lin, Ya-Hui; Chang, Kung-Yao

In order to engineer a ligand-responsive stimulator with efficiency to rival that of a viral −1 PRF stimulator in mammalian cells, we looked for a potent −1 PRF stimulator to be our designing template since the integration of an RNA aptamer could compromise stimulation activity. Previously, a three-stem pseudoknot, SARS-PK was characterized as the −1 PRF stimulator of SARS coronavirus (22, 30, 31) ( Figure 1A and C). Mutagenesis analysis of stems indicated that stem 3 of SARS-PK could tolerate modification without severe reduction in −1 PRF stimulation activity (22) , while an intermolecular kissing-loop interaction involving the loop of stem 3 was shown to affect frameshifting activity (32) . Given that solution NMR and limited nuclease digestion analyses have supported three-stem formation in SARS-PK (22, (30) (31) (32) , using it as a scaffold could also provide advantages in detection of ligand-dependent conformational switch during the designing process.