In silico design of small RNA switches

The discovery of natural RNA sensors that respond to a change in the environment by a conformational switch can be utilized for various biotechnological and nanobiotechnological advances. One class of RNA sensors is the riboswitch: an RNA genetic control element that is capable of sensing small molecules, responding to a deviation in ligand concentration with a structural change. Riboswitches are modularly built from smaller components. Computational methods can potentially be utilized in assembling these building block components and offering improvements in the biochemical design process. We describe a computational procedure to design RNA switches from building blocks with favorable properties. To achieve maximal throughput for genetic control purposes, future designer RNA switches can be assembled based on a computerized preprocessing buildup of the constituent domains, namely the aptamer and the expression platform in the case of a synthetic riboswitch. Conformational switching is enabled by the RNA versatility to possess two highly stable states that are energetically close to each other but topologically distinct, separated by an energy barrier between them. Initially, computer simulations can produce a list of short sequences that switch between two conformers when trigerred by point mutations or temperature. The short sequences should possess an additional desirable property; when these selected small RNA switch segments are attached to various aptamers, the ligand binding mechanism should replace the aforementioned event triggers, which will no longer be effective for crossing the energy barrier. In the assembled RNA sequence, energy minimization folding predictions should then show no difference between the folded structure of the entire sequence relative to the folded structure of each of its constituents. Moreover, energy minimization methods applied on the entire sequence could aid at this preprocessing stage by exhibiting high mutational robustness to capture the stability of the formed hairpin in the expression platform. The above computer-assisted assembly procedure together with application specific considerations may further be tailored for therapeutic gene regulation.