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Ribonucleoprotein purification and characterization using RNA Mango

The characterization of RNA–protein complexes (RNPs) is a difficult but increasingly important problem in modern biology. By combining the compact RNA Mango aptamer with a fluorogenic thiazole orange desthiobiotin (TO1-Dtb or TO3-Dtb) ligand, we have created an RNA tagging system that simplifies the...

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Detalles Bibliográficos
Autores principales: Panchapakesan, Shanker Shyam S., Ferguson, Matthew L., Hayden, Eric J., Chen, Xin, Hoskins, Aaron A., Unrau, Peter J.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Cold Spring Harbor Laboratory Press 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5602116/
https://www.ncbi.nlm.nih.gov/pubmed/28747322
http://dx.doi.org/10.1261/rna.062166.117
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author Panchapakesan, Shanker Shyam S.
Ferguson, Matthew L.
Hayden, Eric J.
Chen, Xin
Hoskins, Aaron A.
Unrau, Peter J.
author_facet Panchapakesan, Shanker Shyam S.
Ferguson, Matthew L.
Hayden, Eric J.
Chen, Xin
Hoskins, Aaron A.
Unrau, Peter J.
author_sort Panchapakesan, Shanker Shyam S.
collection PubMed
description The characterization of RNA–protein complexes (RNPs) is a difficult but increasingly important problem in modern biology. By combining the compact RNA Mango aptamer with a fluorogenic thiazole orange desthiobiotin (TO1-Dtb or TO3-Dtb) ligand, we have created an RNA tagging system that simplifies the purification and subsequent characterization of endogenous RNPs. Mango-tagged RNP complexes can be immobilized on a streptavidin solid support and recovered in their native state by the addition of free biotin. Furthermore, Mango-based RNP purification can be adapted to different scales of RNP isolation ranging from pull-down assays to the isolation of large amounts of biochemically defined cellular RNPs. We have incorporated the Mango aptamer into the S. cerevisiae U1 small nuclear RNA (snRNA), shown that the Mango-snRNA is functional in cells, and used the aptamer to pull down a U1 snRNA-associated protein. To demonstrate large-scale isolation of RNPs, we purified and characterized bacterial RNA polymerase holoenzyme (HE) in complex with a Mango-containing 6S RNA. We were able to use the combination of a red-shifted TO3-Dtb ligand and eGFP-tagged HE to follow the binding and release of the 6S RNA by two-color native gel analysis as well as by single-molecule fluorescence cross-correlation spectroscopy. Together these experiments demonstrate how the Mango aptamer in conjunction with simple derivatives of its flurophore ligands enables the purification and characterization of endogenous cellular RNPs in vitro.
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spelling pubmed-56021162018-10-01 Ribonucleoprotein purification and characterization using RNA Mango Panchapakesan, Shanker Shyam S. Ferguson, Matthew L. Hayden, Eric J. Chen, Xin Hoskins, Aaron A. Unrau, Peter J. RNA Method The characterization of RNA–protein complexes (RNPs) is a difficult but increasingly important problem in modern biology. By combining the compact RNA Mango aptamer with a fluorogenic thiazole orange desthiobiotin (TO1-Dtb or TO3-Dtb) ligand, we have created an RNA tagging system that simplifies the purification and subsequent characterization of endogenous RNPs. Mango-tagged RNP complexes can be immobilized on a streptavidin solid support and recovered in their native state by the addition of free biotin. Furthermore, Mango-based RNP purification can be adapted to different scales of RNP isolation ranging from pull-down assays to the isolation of large amounts of biochemically defined cellular RNPs. We have incorporated the Mango aptamer into the S. cerevisiae U1 small nuclear RNA (snRNA), shown that the Mango-snRNA is functional in cells, and used the aptamer to pull down a U1 snRNA-associated protein. To demonstrate large-scale isolation of RNPs, we purified and characterized bacterial RNA polymerase holoenzyme (HE) in complex with a Mango-containing 6S RNA. We were able to use the combination of a red-shifted TO3-Dtb ligand and eGFP-tagged HE to follow the binding and release of the 6S RNA by two-color native gel analysis as well as by single-molecule fluorescence cross-correlation spectroscopy. Together these experiments demonstrate how the Mango aptamer in conjunction with simple derivatives of its flurophore ligands enables the purification and characterization of endogenous cellular RNPs in vitro. Cold Spring Harbor Laboratory Press 2017-10 /pmc/articles/PMC5602116/ /pubmed/28747322 http://dx.doi.org/10.1261/rna.062166.117 Text en © 2017 Panchapakesan et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society http://creativecommons.org/licenses/by-nc/4.0/ This article is distributed exclusively by the RNA Society for the first 12 months after the full-issue publication date (see http://rnajournal.cshlp.org/site/misc/terms.xhtml). After 12 months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.
spellingShingle Method
Panchapakesan, Shanker Shyam S.
Ferguson, Matthew L.
Hayden, Eric J.
Chen, Xin
Hoskins, Aaron A.
Unrau, Peter J.
Ribonucleoprotein purification and characterization using RNA Mango
title Ribonucleoprotein purification and characterization using RNA Mango
title_full Ribonucleoprotein purification and characterization using RNA Mango
title_fullStr Ribonucleoprotein purification and characterization using RNA Mango
title_full_unstemmed Ribonucleoprotein purification and characterization using RNA Mango
title_short Ribonucleoprotein purification and characterization using RNA Mango
title_sort ribonucleoprotein purification and characterization using rna mango
topic Method
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5602116/
https://www.ncbi.nlm.nih.gov/pubmed/28747322
http://dx.doi.org/10.1261/rna.062166.117
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