Riboswitches Based on Kissing Complexes for the Detection of Small Ligands

Biosensors derived from aptamers were designed for which folding into a hairpin shape is triggered by binding of the cognate ligand. These aptamers (termed aptaswitches) thus switch between folded and unfolded states in the presence and absence of the ligand, respectively. The apical loop of the folded aptaswitch is recognized by a second hairpin called the aptakiss through loop–loop or kissing interactions, whereas the aptakiss does not bind the unfolded aptaswitch. Therefore, the formation of a kissing complex signals the presence of the ligand. Aptaswitches were designed that enable the detection of GTP and adenosine in a specific and quantitative manner by surface plasmon resonance when using a grafted aptakiss or in solution by anisotropy measurement with a fluorescently labeled aptakiss. This approach is generic and can potentially be extended to the detection of any molecule for which hairpin aptamers have been identified, as long as the apical loop is not involved in ligand binding. Switch and kiss: Hairpin aptamers (gray) were engineered to switch between unfolded and folded conformations upon binding to their cognate ligand (orange). The folded conformation but not the unfolded one is recognized by a second hairpin termed the aptakiss (yellow) through loop–loop or kissing interactions (red). The detection of the aptakiss–aptamer–ligand ternary complex by SPR or fluorescence anisotropy signals the presence of the target ligand.