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Fluorophore ligand binding and complex stabilization of the RNA Mango and RNA Spinach aptamers

The effective tracking and purification of biological RNAs and RNA protein complexes is currently challenging. One promising strategy to simultaneously address both of these problems is to develop high-affinity RNA aptamers against taggable small molecule fluorophores. RNA Mango is a 39-nucleotide,...

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Autores principales: Jeng, Sunny C.Y., Chan, Hedy H.Y., Booy, Evan P., McKenna, Sean A., Unrau, Peter J.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Cold Spring Harbor Laboratory Press 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5113208/
https://www.ncbi.nlm.nih.gov/pubmed/27777365
http://dx.doi.org/10.1261/rna.056226.116
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author Jeng, Sunny C.Y.
Chan, Hedy H.Y.
Booy, Evan P.
McKenna, Sean A.
Unrau, Peter J.
author_facet Jeng, Sunny C.Y.
Chan, Hedy H.Y.
Booy, Evan P.
McKenna, Sean A.
Unrau, Peter J.
author_sort Jeng, Sunny C.Y.
collection PubMed
description The effective tracking and purification of biological RNAs and RNA protein complexes is currently challenging. One promising strategy to simultaneously address both of these problems is to develop high-affinity RNA aptamers against taggable small molecule fluorophores. RNA Mango is a 39-nucleotide, parallel-stranded G-quadruplex RNA aptamer motif that binds with nanomolar affinity to a set of thiazole orange (TO1) derivatives while simultaneously inducing a 10(3)-fold increase in fluorescence. We find that RNA Mango has a large increase in its thermal stability upon the addition of its TO1-Biotin ligand. Consistent with this thermal stabilization, RNA Mango can effectively discriminate TO1-Biotin from a broad range of small molecule fluorophores. In contrast, RNA Spinach, which is known to have a substantially more rigid G-quadruplex structure, was found to bind to this set of fluorophores, often with higher affinity than to its native ligand, 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI), and did not exhibit thermal stabilization in the presence of the TO1-Biotin fluorophore. Our data suggest that RNA Mango is likely to use a concerted ligand-binding mechanism that allows it to simultaneously bind and recognize its TO1-Biotin ligand, whereas RNA Spinach appears to lack such a mechanism. The high binding affinity and fluorescent efficiency of RNA Mango provides a compelling alternative to RNA Spinach as an RNA reporter system and paves the way for the future development of small fluorophore RNA reporter systems.
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spelling pubmed-51132082017-12-01 Fluorophore ligand binding and complex stabilization of the RNA Mango and RNA Spinach aptamers Jeng, Sunny C.Y. Chan, Hedy H.Y. Booy, Evan P. McKenna, Sean A. Unrau, Peter J. RNA Article The effective tracking and purification of biological RNAs and RNA protein complexes is currently challenging. One promising strategy to simultaneously address both of these problems is to develop high-affinity RNA aptamers against taggable small molecule fluorophores. RNA Mango is a 39-nucleotide, parallel-stranded G-quadruplex RNA aptamer motif that binds with nanomolar affinity to a set of thiazole orange (TO1) derivatives while simultaneously inducing a 10(3)-fold increase in fluorescence. We find that RNA Mango has a large increase in its thermal stability upon the addition of its TO1-Biotin ligand. Consistent with this thermal stabilization, RNA Mango can effectively discriminate TO1-Biotin from a broad range of small molecule fluorophores. In contrast, RNA Spinach, which is known to have a substantially more rigid G-quadruplex structure, was found to bind to this set of fluorophores, often with higher affinity than to its native ligand, 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI), and did not exhibit thermal stabilization in the presence of the TO1-Biotin fluorophore. Our data suggest that RNA Mango is likely to use a concerted ligand-binding mechanism that allows it to simultaneously bind and recognize its TO1-Biotin ligand, whereas RNA Spinach appears to lack such a mechanism. The high binding affinity and fluorescent efficiency of RNA Mango provides a compelling alternative to RNA Spinach as an RNA reporter system and paves the way for the future development of small fluorophore RNA reporter systems. Cold Spring Harbor Laboratory Press 2016-12 /pmc/articles/PMC5113208/ /pubmed/27777365 http://dx.doi.org/10.1261/rna.056226.116 Text en © 2016 Jeng 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 Article
Jeng, Sunny C.Y.
Chan, Hedy H.Y.
Booy, Evan P.
McKenna, Sean A.
Unrau, Peter J.
Fluorophore ligand binding and complex stabilization of the RNA Mango and RNA Spinach aptamers
title Fluorophore ligand binding and complex stabilization of the RNA Mango and RNA Spinach aptamers
title_full Fluorophore ligand binding and complex stabilization of the RNA Mango and RNA Spinach aptamers
title_fullStr Fluorophore ligand binding and complex stabilization of the RNA Mango and RNA Spinach aptamers
title_full_unstemmed Fluorophore ligand binding and complex stabilization of the RNA Mango and RNA Spinach aptamers
title_short Fluorophore ligand binding and complex stabilization of the RNA Mango and RNA Spinach aptamers
title_sort fluorophore ligand binding and complex stabilization of the rna mango and rna spinach aptamers
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5113208/
https://www.ncbi.nlm.nih.gov/pubmed/27777365
http://dx.doi.org/10.1261/rna.056226.116
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