A Novel RNA Aptamer as Synthetic Inducer of DasR Controlled Transcription.

Progress in the synthetic biology field is driven by the development of new tools for synthetic circuit engineering. Traditionally, the focus has relied on protein-based designs. In recent years, the use of RNA-based tools has tremendously increased, due to their versatile functionality and applicability. A promising class of molecules is RNA aptamers, small, single-stranded RNA molecules that bind to a target molecule with high affinity and specificity. When targeting bacterial repressors, RNA aptamers allow one to add a new layer to an established protein-based regulation. In the present study, we selected an RNA aptamer binding the bacterial repressor DasR, preventing its binding to its operator sequence and activating DasR-controlled transcription in vivo. This was made possible only by the combination of an in vitro selection and subsequent in vivo screening. Next-generation sequencing of the selection process proved the importance of the in vivo screening for the discovery of aptamers functioning in the cell. Mutational and biochemical studies led to the identification of the minimal necessary binding motif. Taken together, the resulting combination of bacterial repressor and RNA aptamer enlarges the synthetic biology toolbox by adding a new level of regulation.

Structural Changes in Aptamers are Essential for Synthetic Riboswitch Engineering.

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.