Reengineering orthogonally selective riboswitches.
The ability to independently control the expression of multiple genes by addition of distinct small-molecule modulators has many applications from synthetic biology, functional genomics, pharmaceutical target validation, through to gene therapy. Riboswitches are relatively simple, small-molecule-dependent, protein-free, mRNA genetic switches that are attractive targets for reengineering in this context. Using a combination of chemical genetics and genetic selection, we have developed riboswitches that are selective for synthetic "nonnatural" small molecules and no longer respond to the natural intracellular ligands. The orthogonal selectivity of the riboswitches is also demonstrated in vitro using isothermal titration calorimetry and x-ray crystallography. The riboswitches allow highly responsive, dose-dependent, orthogonally selective, and dynamic control of gene expression in vivo. It is possible that this approach may be further developed to reengineer other natural riboswitches for application as small-molecule responsive genetic switches in both prokaryotes and eukaryotes.
School of Chemistry and Manchester Interdisciplinary Biocentre, The University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom.
Neil
N
Dixon
John N
JN
Duncan
Torsten
T
Geerlings
Mark S
MS
Dunstan
John E G
JE
McCarthy
David
D
Leys
Jason
J
Micklefield
D052157:Aptamers, Nucleotide / Q000378:metabolism; D002151:Calorimetry; D018360:Crystallography, X-Ray; D005786:Gene Expression Regulation / Q000502:physiology*; D005818:Genetic Engineering / Q000379:methods*; D008958:Models, Molecular; D015394:Molecular Structure; D016337:RNA, Catalytic / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*
D052157:Aptamers, Nucleotide; D016337:RNA, Catalytic; D012333:RNA, Messenger