Modular RNA engineering of RNA molecular switches (riboswitches)

Riboswitches are attractive not only as drug targets for novel antibiotics but also as modular tools for controlling gene expression. Riboswitches frequently have GNRA loop/receptor interaction modules in thier aptamer modules. In this study, the functional importance of the GAAA loop-receptor interaction in the c-diGMP riboswitch was examined. A series of variant c-diGMP riboswitches with mutations in the GAAA loop/receptor interaction were assayed for their switching abilities. These analyses strongly suggested that the GAAA loop/receptor interaction does not simply establish the RNA 3D structure but docking of P2 GAAA loop rigidifies the P3 element. This mechanism was applied to design an artificial riboswitch bearing a theophylline aptamer module in P3 the structural rigidity of which could be modulated by the small molecule theophylline. We also generated a new class of aptamer module that specifically recognizes a GAAC RNA tetraloop by employing in vitro directed evolution.