Modular RNA engineering of RNA molecular switches (riboswitches)

核糖开关不仅作为新型抗生素的药物靶点，而且作为控制基因表达的模块化工具具有吸引力。核糖开关在其适体模块中通常具有GNRA环/受体相互作用模块。在这项研究中，研究了GAAA环受体相互作用在c-diGMP核开关中的功能重要性。我们检测了一系列在GAAA环/受体相互作用中突变的c-diGMP变异核开关的开关能力。这些分析有力地表明，GAAA环/受体的相互作用并不是简单地建立RNA的3D结构，而是P2 GAAA环的对接使P3元件僵化。利用这一机制设计了一种含有茶碱适体模块的人工核糖体开关，其结构刚性可以被茶碱小分子调节。我们还通过体外定向进化产生了一类新的适体模块，可以特异性识别GAAC RNA四环。

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.