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A versatile cis-blocking and trans-activation strategy for ribozyme characterization
Synthetic RNA control devices that use ribozymes as gene-regulatory components have been applied to controlling cellular behaviors in response to environmental signals. Quantitative measurement of the in vitro cleavage rate constants associated with ribozyme-based devices is essential for advancing...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2013
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3553993/ https://www.ncbi.nlm.nih.gov/pubmed/23155065 http://dx.doi.org/10.1093/nar/gks1036 |
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author | Kennedy, Andrew B. Liang, Joe C. Smolke, Christina D. |
author_facet | Kennedy, Andrew B. Liang, Joe C. Smolke, Christina D. |
author_sort | Kennedy, Andrew B. |
collection | PubMed |
description | Synthetic RNA control devices that use ribozymes as gene-regulatory components have been applied to controlling cellular behaviors in response to environmental signals. Quantitative measurement of the in vitro cleavage rate constants associated with ribozyme-based devices is essential for advancing the molecular design and optimization of this class of gene-regulatory devices. One of the key challenges encountered in ribozyme characterization is the efficient generation of full-length RNA from in vitro transcription reactions, where conditions generally lead to significant ribozyme cleavage. Current methods for generating full-length ribozyme-encoding RNA rely on a trans-blocking strategy, which requires a laborious gel separation and extraction step. Here, we develop a simple two-step gel-free process including cis-blocking and trans-activation steps to support scalable generation of functional full-length ribozyme-encoding RNA. We demonstrate our strategy on various types of natural ribozymes and synthetic ribozyme devices, and the cleavage rate constants obtained for the RNA generated from our strategy are comparable with those generated through traditional methods. We further develop a rapid, label-free ribozyme cleavage assay based on surface plasmon resonance, which allows continuous, real-time monitoring of ribozyme cleavage. The surface plasmon resonance-based characterization assay will complement the versatile cis-blocking and trans-activation strategy to broadly advance our ability to characterize and engineer ribozyme-based devices. |
format | Online Article Text |
id | pubmed-3553993 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2013 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-35539932013-01-24 A versatile cis-blocking and trans-activation strategy for ribozyme characterization Kennedy, Andrew B. Liang, Joe C. Smolke, Christina D. Nucleic Acids Res Methods Online Synthetic RNA control devices that use ribozymes as gene-regulatory components have been applied to controlling cellular behaviors in response to environmental signals. Quantitative measurement of the in vitro cleavage rate constants associated with ribozyme-based devices is essential for advancing the molecular design and optimization of this class of gene-regulatory devices. One of the key challenges encountered in ribozyme characterization is the efficient generation of full-length RNA from in vitro transcription reactions, where conditions generally lead to significant ribozyme cleavage. Current methods for generating full-length ribozyme-encoding RNA rely on a trans-blocking strategy, which requires a laborious gel separation and extraction step. Here, we develop a simple two-step gel-free process including cis-blocking and trans-activation steps to support scalable generation of functional full-length ribozyme-encoding RNA. We demonstrate our strategy on various types of natural ribozymes and synthetic ribozyme devices, and the cleavage rate constants obtained for the RNA generated from our strategy are comparable with those generated through traditional methods. We further develop a rapid, label-free ribozyme cleavage assay based on surface plasmon resonance, which allows continuous, real-time monitoring of ribozyme cleavage. The surface plasmon resonance-based characterization assay will complement the versatile cis-blocking and trans-activation strategy to broadly advance our ability to characterize and engineer ribozyme-based devices. Oxford University Press 2013-01 2012-11-15 /pmc/articles/PMC3553993/ /pubmed/23155065 http://dx.doi.org/10.1093/nar/gks1036 Text en © The Author(s) 2012. Published by Oxford University Press. http://creativecommons.org/licenses/by-nc/3.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial reuse, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com. |
spellingShingle | Methods Online Kennedy, Andrew B. Liang, Joe C. Smolke, Christina D. A versatile cis-blocking and trans-activation strategy for ribozyme characterization |
title | A versatile cis-blocking and trans-activation strategy for ribozyme characterization |
title_full | A versatile cis-blocking and trans-activation strategy for ribozyme characterization |
title_fullStr | A versatile cis-blocking and trans-activation strategy for ribozyme characterization |
title_full_unstemmed | A versatile cis-blocking and trans-activation strategy for ribozyme characterization |
title_short | A versatile cis-blocking and trans-activation strategy for ribozyme characterization |
title_sort | versatile cis-blocking and trans-activation strategy for ribozyme characterization |
topic | Methods Online |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3553993/ https://www.ncbi.nlm.nih.gov/pubmed/23155065 http://dx.doi.org/10.1093/nar/gks1036 |
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