Synthetic riboswitches are powerful tools in synthetic biology in which sensing and execution are consolidated in a single RNA molecule. By using SELEX to select aptamers in vitro, synthetic riboswitches can in theory be engineered against any ligand of choice. Surprisingly, very few in vitro selected aptamers have been used for the engineering of synthetic riboswitches. In-depth studies of these aptamers suggest that the key characteristics of such regulatory active RNAs are their structural switching abilities and their binding dynamics. Conventional SELEX approaches seem to be inadequate to select for these characteristics, which may explain the lack of in vitro selected aptamers suited for engineering of synthetic riboswitches. In this review, we explore the functional principles of synthetic riboswitches, identify key characteristics of regulatory active in vitro selected aptamers and integrate these findings in context with available in vitro selection methods. Based on these insights, we propose to use a combination of capture-SELEX and subsequent functional screening for a more successful in vitro selection of aptamers that can be applied for the engineering of synthetic riboswitches.
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