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-dep...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2010-02, Vol.107 (7), p.2830-2835
Main Authors: Dixon, Neil, Duncan, John N, Geerlings, Torsten, Dunstan, Mark S, McCarthy, John E.G, Leys, David, Micklefield, Jason
Format: Article
Language:English
Subjects:
Aptamers, Nucleotide - metabolism
Binding sites
Biological Sciences
Calorimetry
Crystallography, X-Ray
Eukaryotes
Gene expression
Gene expression regulation
Gene Expression Regulation - physiology
Gene therapy
Genes
Genetic Engineering - methods
Genetic mutation
Genomics
Inductive reasoning
Ligands
Messenger RNA
Models, Molecular
Molecular Structure
Nucleotides
Physical Sciences
Prokaryotes
Purines
Ribonucleic acid
RNA
RNA, Catalytic - metabolism
RNA, Messenger - metabolism
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Summary: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.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.0911209107