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Nat Methods. 2016 May;13(5):453-8. doi: 10.1038/nmeth.3807. Epub 2016 Mar 21.

Reprogramming eukaryotic translation with ligand-responsive synthetic RNA switches.

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Department of Chemistry, Columbia University, New York, New York, USA.
Department of Systems Biology, Columbia University, New York, New York, USA.
Department of Biomedical Informatics, Columbia University College of Physicians and Surgeons, New York, New York, USA.


Protein synthesis in eukaryotes is regulated by diverse reprogramming mechanisms that expand the coding capacity of individual genes. Here, we exploit one such mechanism, termed -1 programmed ribosomal frameshifting (-1 PRF), to engineer ligand-responsive RNA switches that regulate protein expression. First, efficient -1 PRF stimulatory RNA elements were discovered by in vitro selection; then, ligand-responsive switches were constructed by coupling -1 PRF stimulatory elements to RNA aptamers using rational design and directed evolution in Saccharomyces cerevisiae. We demonstrate that -1 PRF switches tightly control the relative stoichiometry of two distinct protein outputs from a single mRNA, exhibiting consistent ligand response across whole populations of cells. Furthermore, -1 PRF switches were applied to build single-mRNA logic gates and an apoptosis module in yeast. Together, these results showcase the potential for harnessing translation-reprogramming mechanisms for synthetic biology, and they establish -1 PRF switches as powerful RNA tools for controlling protein synthesis in eukaryotes.

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