Cargando…

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Kennedy, Andrew B., Vowles, James V., d'Espaux, Leo, Smolke, Christina D.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2014
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
_version_ 1782344479367233536
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
work_keys_str_mv AT kennedyandrewb proteinresponsiveribozymeswitchesineukaryoticcells
AT vowlesjamesv proteinresponsiveribozymeswitchesineukaryoticcells
AT despauxleo proteinresponsiveribozymeswitchesineukaryoticcells
AT smolkechristinad proteinresponsiveribozymeswitchesineukaryoticcells