Riboswitches have gained attention as tools for syn- thetic biology, since they enable researchers of re- programming cells to sense and respond to exoge- nous molecules. In vitro evolutionary approaches produced numerous RNA aptamers that bind such small ligands, but their conversion into functional riboswitches remains difficult. We previously devel- oped a computational approach for the design of syn- thetic theophylline riboswitches based on secondary structure prediction. These riboswitches have been constructed to regulate ligand-dependent transcrip- tion termination in Escherichia coli. Here, we test the applicability of this design strategy by applying the approach to tetracycline and streptomycin aptamers. The resulting tetracycline riboswitches exhibit ro- bust regulatory properties in vivo. Tandem fusions of these riboswitches with theophylline riboswitches represent logic gates responding to two different in- put signals. In contrast, the conversion of the strepto- mycin aptamer into functional riboswitches proves to be difficult. Investigations of the underlying aptamer secondary structure revealed differences between in silico prediction and structure probing. We con- clude that only aptamers adopting the minimal free energy (MFE) structure are suitable targets for con- struction of synthetic riboswitches with design ap- proaches based on equilibrium thermodynamics of RNA structures. Further improvements in the design strategy are required to implement aptamer struc- tures not corresponding to the calculated MFE.

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