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A Spinach molecular beacon triggered by strand displacement

We have re-engineered the fluorescent RNA aptamer Spinach to be activated in a sequence-dependent manner. The original Spinach aptamer was extended at its 5′- and 3′-ends to create Spinach.ST, which is predicted to fold into an inactive conformation and thus prevent association with the small molecu...

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Detalles Bibliográficos
Autores principales: Bhadra, Sanchita, Ellington, Andrew D.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Cold Spring Harbor Laboratory Press 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4105745/
https://www.ncbi.nlm.nih.gov/pubmed/24942625
http://dx.doi.org/10.1261/rna.045047.114
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author Bhadra, Sanchita
Ellington, Andrew D.
author_facet Bhadra, Sanchita
Ellington, Andrew D.
author_sort Bhadra, Sanchita
collection PubMed
description We have re-engineered the fluorescent RNA aptamer Spinach to be activated in a sequence-dependent manner. The original Spinach aptamer was extended at its 5′- and 3′-ends to create Spinach.ST, which is predicted to fold into an inactive conformation and thus prevent association with the small molecule fluorophore DFHBI. Hybridization of a specific trigger oligonucleotide to a designed toehold leads to toehold-initiated strand displacement and refolds Spinach into the active, fluorophore-binding conformation. Spinach.ST not only specifically detects its target oligonucleotide but can discriminate readily against single-nucleotide mismatches. RNA amplicons produced during nucleic acid sequence-based amplification (NASBA) of DNA or RNA targets could be specifically detected and reported in real-time by conformational activation of Spinach.ST generated by in vitro transcription. In order to adapt any target sequence to detection by a Spinach reporter we used a primer design technique that brings together otherwise distal toehold sequences via hairpin formation. The same techniques could potentially be used to adapt common Spinach reporters to non-nucleic acid analytes, rather than by making fusions between aptamers and Spinach.
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spelling pubmed-41057452015-08-01 A Spinach molecular beacon triggered by strand displacement Bhadra, Sanchita Ellington, Andrew D. RNA Report We have re-engineered the fluorescent RNA aptamer Spinach to be activated in a sequence-dependent manner. The original Spinach aptamer was extended at its 5′- and 3′-ends to create Spinach.ST, which is predicted to fold into an inactive conformation and thus prevent association with the small molecule fluorophore DFHBI. Hybridization of a specific trigger oligonucleotide to a designed toehold leads to toehold-initiated strand displacement and refolds Spinach into the active, fluorophore-binding conformation. Spinach.ST not only specifically detects its target oligonucleotide but can discriminate readily against single-nucleotide mismatches. RNA amplicons produced during nucleic acid sequence-based amplification (NASBA) of DNA or RNA targets could be specifically detected and reported in real-time by conformational activation of Spinach.ST generated by in vitro transcription. In order to adapt any target sequence to detection by a Spinach reporter we used a primer design technique that brings together otherwise distal toehold sequences via hairpin formation. The same techniques could potentially be used to adapt common Spinach reporters to non-nucleic acid analytes, rather than by making fusions between aptamers and Spinach. Cold Spring Harbor Laboratory Press 2014-08 /pmc/articles/PMC4105745/ /pubmed/24942625 http://dx.doi.org/10.1261/rna.045047.114 Text en © 2014 Bhadra and Ellington; 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 Report
Bhadra, Sanchita
Ellington, Andrew D.
A Spinach molecular beacon triggered by strand displacement
title A Spinach molecular beacon triggered by strand displacement
title_full A Spinach molecular beacon triggered by strand displacement
title_fullStr A Spinach molecular beacon triggered by strand displacement
title_full_unstemmed A Spinach molecular beacon triggered by strand displacement
title_short A Spinach molecular beacon triggered by strand displacement
title_sort spinach molecular beacon triggered by strand displacement
topic Report
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4105745/
https://www.ncbi.nlm.nih.gov/pubmed/24942625
http://dx.doi.org/10.1261/rna.045047.114
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