To draw general conclusions regarding riboswitch function, we considered three representative regulatory mechanisms: transcriptional termination, translational repression, and mRNA destabilization. Modeling results suggest that riboswitch performance is highly dependent on the competition between irreversible rates such as mRNA degradation or transcriptional termination and reversible rates such as conformational switching or ligand binding. When reversible rates dominate (thermodynamically-driven), riboswitch performance is independent of the selected regulatory mechanism. When the rates are balanced (kinetically-driven), riboswitch performance generally decreases, transcriptional folding becomes important, and performance depends on the selected regulatory mechanism. When irreversible rates dominate (non-functional), riboswitch performance is insensitive to the input signal. In addition, imposing an upper limit on the input signal reduces the maximal dynamic range and establishes a bounded parameter space for optimal riboswitch performance. Model predictions are supported by published experimental data and physical modification of a synthetic riboswitch.
From our modeling efforts, we arrived at multiple design principles to guide the construction and modification of synthetic riboswitches. These principles address selection and modification of device components as well as formulation of composition frameworks. Current work is focused on applying these principles to improving the performance of a synthetic riboswitch actively used in our group for biomedical and biotechnological applications.