Department of Biomedical Engineering, University of California, Davis, California 95616, USA.
Chembiochem. 2009 Sep 21;10(14):2375-81. doi: 10.1002/cbic.200900313.
After the recent discovery of bacterial riboswitches, synthetic riboswitches have been engineered by using natural and artificial RNA aptamers. In contrast to natural riboswitches, the majority of synthetic riboswitches in bacteria reported to date are ON switches that activate gene expression in response to the aptamer ligand. In this study, we adopted a mechanism-guided approach to design libraries predisposed to contain OFF riboswitches that respond to thiamine pyrophosphate (TPP). The first library design exploited a pseudo-Shine-Dalgarno (SD) sequence located near the 3'-end of the TPP aptamer, which would be less accessible to the ribosome when the aptamer is bound to TPP. In the second library, an SD sequence was strategically placed in the aptamer's P1 stem, which is stabilized upon ligand binding. OFF riboswitches were obtained by dual genetic selection of these libraries. The results underscore the importance of effective library design to achieve desired riboswitch functions.
最近发现了细菌核糖开关后，人们利用天然和人工 RNA 适体来设计合成的核糖开关。与天然核糖开关不同，迄今为止在细菌中报道的大多数合成核糖开关都是 ON 开关，它们会响应适体配体激活基因表达。在这项研究中，我们采用了一种基于机制的方法来设计文库，使文库倾向于包含响应焦磷酸硫胺素 (TPP) 的 OFF 核糖开关。第一个文库设计利用了位于 TPP 适体 3' 末端附近的伪 Shine-Dalgarno (SD) 序列，当适体与 TPP 结合时，该序列对核糖体的可及性较低。在第二个文库中，SD 序列被策略性地放置在适体的 P1 茎中，该茎在配体结合时稳定。通过对这两个文库进行双重遗传选择获得了 OFF 核糖开关。这些结果强调了有效文库设计对于实现所需核糖开关功能的重要性。
