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Published October 29, 2014 | Supplemental Material + Published
Journal Article Open

Protein-responsive ribozyme switches in eukaryotic cells


Genetic devices that directly detect and respond to intracellular concentrations of proteins are important synthetic biology tools, supporting the design of biological systems that target, respond to or alter specific cellular states. Here, we develop ribozyme-based devices that respond to protein ligands in two eukaryotic hosts, yeast and mammalian cells, to regulate the expression of a gene of interest. Our devices allow for both gene-ON and gene-OFF response upon sensing the protein ligand. As part of our design process, we describe an in vitro characterization pipeline for prescreening device designs to identify promising candidates for in vivo testing. The in vivo gene-regulatory activities in the two types of eukaryotic cells correlate with in vitro cleavage activities determined at different physiologically relevant magnesium concentrations. Finally, localization studies with the ligand demonstrate that ribozyme switches respond to ligands present in the nucleus and/or cytoplasm, providing new insight into their mechanism of action. By extending the sensing capabilities of this important class of gene-regulatory device, our work supports the implementation of ribozyme-based devices in applications requiring the detection of protein biomarkers.

Additional Information

© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Received July 22, 2014; Revised September 5, 2014; Accepted September 11, 2014. First published online: October 1, 2014. We thank K. Lee and the Stanford Cell Sciences Imaging Facility for providing fluorescence microscopy access (NIH grant SIG number 1S10OD01058001A1) and training, R. Green for providing His-MS2-MBP; M. McKeague for assistance with SPR-based assays; S. Culler, M. Mathur for providing plasmids. Funding: National Institutes of Health [RC1GM091298 to C.D.S.]; Defense Advanced Research Projects Agency [HR0011-11-2-0002 to C.D.S.]. The open access publication charge for this paper has been waived by Oxford University Press - NAR Editorial Board members are entitled to one free paper per year in recognition of their work on behalf of the journal. Conflict of interest statement. None declared.

Attached Files

Published - Nucl._Acids_Res.-2014-Kennedy-12306-21.pdf

Supplemental Material - nar-01977-r-2014-File009.pdf


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