Development of a novel tobramycin dependent riboswitch.

We herein report the selection and characterization of a new riboswitch dependent on the aminoglycoside tobramycin. Its dynamic range rivals even the tetracycline dependent riboswitch to be the current best performing, synthetic riboswitch that controls translation initiation. The riboswitch was selected with RNA Capture-SELEX, a method that not only selects for binding but also for structural changes in aptamers on binding. This study demonstrates how this method can fundamentally reduce the labour required for the de novo identification of synthetic riboswitches. The initially selected riboswitch candidate harbours two distinct tobramycin binding sites with KDs of 1.1 nM and 2.4 µM, respectively, and can distinguish between tobramycin and the closely related compounds kanamycin A and B. Using detailed genetic and biochemical analyses and 1H NMR spectroscopy, the proposed secondary structure of the riboswitch was verified and the tobramycin binding sites were characterized. The two binding sites were found to be essentially non-overlapping, allowing for a separate investigation of their contribution to the activity of the riboswitch. We thereby found that only the high-affinity binding site was responsible for regulatory activity, which allowed us to engineer a riboswitch from only this site with a minimal sequence size of 33 nt and outstanding performance.

Next-level riboswitch development-implementation of Capture-SELEX facilitates identification of a new synthetic riboswitch.

The development of synthetic riboswitches has always been a challenge. Although a number of interesting proof-of-concept studies have been published, almost all of these were performed with the theophylline aptamer. There is no shortage of small molecule-binding aptamers; however, only a small fraction of them are suitable for RNA engineering since a classical SELEX protocol selects only for high-affinity binding but not for conformational switching. We now implemented RNA Capture-SELEX in our riboswitch developmental pipeline to integrate the required selection for high-affinity binding with the equally necessary RNA conformational switching. Thus, we successfully developed a new paromomycin-binding synthetic riboswitch. It binds paromomycin with a KD of 20 nM and can discriminate between closely related molecules both in vitro and in vivo. A detailed structure-function analysis confirmed the predicted secondary structure and identified nucleotides involved in ligand binding. The riboswitch was further engineered in combination with the neomycin riboswitch for the assembly of an orthogonal Boolean NOR logic gate. In sum, our work not only broadens the spectrum of existing RNA regulators, but also signifies a breakthrough in riboswitch development, as the effort required for the design of sensor domains for RNA-based devices will in many cases be much reduced.

Riboswitching with ciprofloxacin-development and characterization of a novel RNA regulator.

RNA molecules play important and diverse regulatory roles in the cell. Inspired by this natural versatility, RNA devices are increasingly important for many synthetic biology applications, e.g. optimizing engineered metabolic pathways, gene therapeutics or building up complex logical units. A major advantage of RNA is the possibility of de novo design of RNA-based sensing domains via an in vitro selection process (SELEX). Here, we describe development of a novel ciprofloxacin-responsive riboswitch by in vitro selection and next-generation sequencing-guided cellular screening. The riboswitch recognizes the small molecule drug ciprofloxacin with a KD in the low nanomolar range and adopts a pseudoknot fold stabilized by ligand binding. It efficiently interferes with gene expression both in lower and higher eukaryotes. By controlling an auxotrophy marker and a resistance gene, respectively, we demonstrate efficient, scalable and programmable control of cellular survival in yeast. The applied strategy for the development of the ciprofloxacin riboswitch is easily transferrable to any small molecule target of choice and will thus broaden the spectrum of RNA regulators considerably.