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Site-specific terminal and internal labeling of RNA by poly(A) polymerase tailing and copper-catalyzed or copper-free strain-promoted click chemistry
The modification of RNA with fluorophores, affinity tags and reactive moieties is of enormous utility for studying RNA localization, structure and dynamics as well as diverse biological phenomena involving RNA as an interacting partner. Here we report a labeling approach in which the RNA of interest...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2012
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3378897/ https://www.ncbi.nlm.nih.gov/pubmed/22344697 http://dx.doi.org/10.1093/nar/gks062 |
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author | Winz, Marie-Luise Samanta, Ayan Benzinger, Dirk Jäschke, Andres |
author_facet | Winz, Marie-Luise Samanta, Ayan Benzinger, Dirk Jäschke, Andres |
author_sort | Winz, Marie-Luise |
collection | PubMed |
description | The modification of RNA with fluorophores, affinity tags and reactive moieties is of enormous utility for studying RNA localization, structure and dynamics as well as diverse biological phenomena involving RNA as an interacting partner. Here we report a labeling approach in which the RNA of interest—of either synthetic or biological origin—is modified at its 3′-end by a poly(A) polymerase with an azido-derivatized nucleotide. The azide is later on conjugated via copper-catalyzed or strain-promoted azide–alkyne click reaction. Under optimized conditions, a single modified nucleotide of choice (A, C, G, U) containing an azide at the 2′-position can be incorporated site-specifically. We have identified ligases that tolerate the presence of a 2′-azido group at the ligation site. This azide is subsequently reacted with a fluorophore alkyne. With this stepwise approach, we are able to achieve site-specific, internal backbone-labeling of de novo synthesized RNA molecules. |
format | Online Article Text |
id | pubmed-3378897 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2012 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-33788972012-06-20 Site-specific terminal and internal labeling of RNA by poly(A) polymerase tailing and copper-catalyzed or copper-free strain-promoted click chemistry Winz, Marie-Luise Samanta, Ayan Benzinger, Dirk Jäschke, Andres Nucleic Acids Res Methods Online The modification of RNA with fluorophores, affinity tags and reactive moieties is of enormous utility for studying RNA localization, structure and dynamics as well as diverse biological phenomena involving RNA as an interacting partner. Here we report a labeling approach in which the RNA of interest—of either synthetic or biological origin—is modified at its 3′-end by a poly(A) polymerase with an azido-derivatized nucleotide. The azide is later on conjugated via copper-catalyzed or strain-promoted azide–alkyne click reaction. Under optimized conditions, a single modified nucleotide of choice (A, C, G, U) containing an azide at the 2′-position can be incorporated site-specifically. We have identified ligases that tolerate the presence of a 2′-azido group at the ligation site. This azide is subsequently reacted with a fluorophore alkyne. With this stepwise approach, we are able to achieve site-specific, internal backbone-labeling of de novo synthesized RNA molecules. Oxford University Press 2012-05 2012-02-16 /pmc/articles/PMC3378897/ /pubmed/22344697 http://dx.doi.org/10.1093/nar/gks062 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 Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Methods Online Winz, Marie-Luise Samanta, Ayan Benzinger, Dirk Jäschke, Andres Site-specific terminal and internal labeling of RNA by poly(A) polymerase tailing and copper-catalyzed or copper-free strain-promoted click chemistry |
title | Site-specific terminal and internal labeling of RNA by poly(A) polymerase tailing and copper-catalyzed or copper-free strain-promoted click chemistry |
title_full | Site-specific terminal and internal labeling of RNA by poly(A) polymerase tailing and copper-catalyzed or copper-free strain-promoted click chemistry |
title_fullStr | Site-specific terminal and internal labeling of RNA by poly(A) polymerase tailing and copper-catalyzed or copper-free strain-promoted click chemistry |
title_full_unstemmed | Site-specific terminal and internal labeling of RNA by poly(A) polymerase tailing and copper-catalyzed or copper-free strain-promoted click chemistry |
title_short | Site-specific terminal and internal labeling of RNA by poly(A) polymerase tailing and copper-catalyzed or copper-free strain-promoted click chemistry |
title_sort | site-specific terminal and internal labeling of rna by poly(a) polymerase tailing and copper-catalyzed or copper-free strain-promoted click chemistry |
topic | Methods Online |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3378897/ https://www.ncbi.nlm.nih.gov/pubmed/22344697 http://dx.doi.org/10.1093/nar/gks062 |
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