Modular RNA engineering of RNA molecular switches (riboswitches)

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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.