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Rationally Manipulating Aptamer Binding Affinities in a Stem-Loop Molecular Beacon

[Image: see text] Single-stranded DNA sequences that are highly specific for a target ligand are called aptamers. While the incorporation of aptamer sequences into stem-loop molecular beacons has become an essential tool in optical biosensors, the design principles that determine the magnitude of bi...

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Autores principales: Armstrong, Rachel E., Strouse, Geoffrey F.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Chemical Society 2014
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4198099/
https://www.ncbi.nlm.nih.gov/pubmed/25170558
http://dx.doi.org/10.1021/bc500286r
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author Armstrong, Rachel E.
Strouse, Geoffrey F.
author_facet Armstrong, Rachel E.
Strouse, Geoffrey F.
author_sort Armstrong, Rachel E.
collection PubMed
description [Image: see text] Single-stranded DNA sequences that are highly specific for a target ligand are called aptamers. While the incorporation of aptamer sequences into stem-loop molecular beacons has become an essential tool in optical biosensors, the design principles that determine the magnitude of binding affinity and its relationship to placement of the aptamer sequence in the stem-loop architecture are not well defined. By controlled placement of the aptamer along the loop region of the molecular beacon, it is observed that the binding affinity can be tuned over 4 orders of magnitude (1.3 nM – 203 μM) for the Huizenga and Szostak ATP DNA aptamer sequence. It is observed that the K(d) is enhanced for the fully exposed sequence, with reduced binding affinity when the aptamer is part of the stem region of the beacon. Analysis of the ΔG values indicate a clear correlation between the aptamer hybridized length in the stem and its observed K(d). The use of a nanometal surface energy transfer probe method for monitoring ATP binding to the aptamer sequence allows the observation of negative cooperativity between the two ATP binding events. Maintenance of the high binding affinity of this ATP aptamer and the observation of two separate K(d)’s for ATP binding indicate NSET as an effective, nonmanipulative, optical method for tracking biomolecular changes.
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spelling pubmed-41980992015-08-29 Rationally Manipulating Aptamer Binding Affinities in a Stem-Loop Molecular Beacon Armstrong, Rachel E. Strouse, Geoffrey F. Bioconjug Chem [Image: see text] Single-stranded DNA sequences that are highly specific for a target ligand are called aptamers. While the incorporation of aptamer sequences into stem-loop molecular beacons has become an essential tool in optical biosensors, the design principles that determine the magnitude of binding affinity and its relationship to placement of the aptamer sequence in the stem-loop architecture are not well defined. By controlled placement of the aptamer along the loop region of the molecular beacon, it is observed that the binding affinity can be tuned over 4 orders of magnitude (1.3 nM – 203 μM) for the Huizenga and Szostak ATP DNA aptamer sequence. It is observed that the K(d) is enhanced for the fully exposed sequence, with reduced binding affinity when the aptamer is part of the stem region of the beacon. Analysis of the ΔG values indicate a clear correlation between the aptamer hybridized length in the stem and its observed K(d). The use of a nanometal surface energy transfer probe method for monitoring ATP binding to the aptamer sequence allows the observation of negative cooperativity between the two ATP binding events. Maintenance of the high binding affinity of this ATP aptamer and the observation of two separate K(d)’s for ATP binding indicate NSET as an effective, nonmanipulative, optical method for tracking biomolecular changes. American Chemical Society 2014-08-29 2014-10-15 /pmc/articles/PMC4198099/ /pubmed/25170558 http://dx.doi.org/10.1021/bc500286r Text en Copyright © 2014 American Chemical Society
spellingShingle Armstrong, Rachel E.
Strouse, Geoffrey F.
Rationally Manipulating Aptamer Binding Affinities in a Stem-Loop Molecular Beacon
title Rationally Manipulating Aptamer Binding Affinities in a Stem-Loop Molecular Beacon
title_full Rationally Manipulating Aptamer Binding Affinities in a Stem-Loop Molecular Beacon
title_fullStr Rationally Manipulating Aptamer Binding Affinities in a Stem-Loop Molecular Beacon
title_full_unstemmed Rationally Manipulating Aptamer Binding Affinities in a Stem-Loop Molecular Beacon
title_short Rationally Manipulating Aptamer Binding Affinities in a Stem-Loop Molecular Beacon
title_sort rationally manipulating aptamer binding affinities in a stem-loop molecular beacon
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4198099/
https://www.ncbi.nlm.nih.gov/pubmed/25170558
http://dx.doi.org/10.1021/bc500286r
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