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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 l...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2014
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4231745/ https://www.ncbi.nlm.nih.gov/pubmed/25274734 http://dx.doi.org/10.1093/nar/gku875 |
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author | Kennedy, Andrew B. Vowles, James V. d'Espaux, Leo Smolke, Christina D. |
author_facet | Kennedy, Andrew B. Vowles, James V. d'Espaux, Leo Smolke, Christina D. |
author_sort | Kennedy, Andrew B. |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-4231745 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-42317452014-11-21 Protein-responsive ribozyme switches in eukaryotic cells Kennedy, Andrew B. Vowles, James V. d'Espaux, Leo Smolke, Christina D. Nucleic Acids Res Synthetic Biology and Bioengineering 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. Oxford University Press 2014-10-29 2014-10-01 /pmc/articles/PMC4231745/ /pubmed/25274734 http://dx.doi.org/10.1093/nar/gku875 Text en © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by/4.0/ 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. |
spellingShingle | Synthetic Biology and Bioengineering Kennedy, Andrew B. Vowles, James V. d'Espaux, Leo Smolke, Christina D. Protein-responsive ribozyme switches in eukaryotic cells |
title | Protein-responsive ribozyme switches in eukaryotic cells |
title_full | Protein-responsive ribozyme switches in eukaryotic cells |
title_fullStr | Protein-responsive ribozyme switches in eukaryotic cells |
title_full_unstemmed | Protein-responsive ribozyme switches in eukaryotic cells |
title_short | Protein-responsive ribozyme switches in eukaryotic cells |
title_sort | protein-responsive ribozyme switches in eukaryotic cells |
topic | Synthetic Biology and Bioengineering |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4231745/ https://www.ncbi.nlm.nih.gov/pubmed/25274734 http://dx.doi.org/10.1093/nar/gku875 |
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