Mechanism-guided library design and dual genetic selection of synthetic OFF riboswitches.
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.
Muranaka
Norihito
N
Department of Biomedical Engineering, University of California, Davis, California 95616, USA.
Abe
Koichi
K
Yokobayashi
Yohei
Y
D020413:3' Untranslated Regions / Q000235:genetics; D000595:Amino Acid Sequence; D052157:Aptamers, Nucleotide / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D015723:Gene Library; D008969:Molecular Sequence Data; D012270:Ribosomes / Q000235:genetics / Q000378:metabolism*; D013835:Thiamine Pyrophosphate / Q000378:metabolism
D020413:3' Untranslated Regions; D052157:Aptamers, Nucleotide; D013835:Thiamine Pyrophosphate