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Massively Parallel Interrogation of Aptamer Sequence, Structure and Function
BACKGROUND: Optimization of high affinity reagents is a significant bottleneck in medicine and the life sciences. The ability to synthetically create thousands of permutations of a lead high-affinity reagent and survey the properties of individual permutations in parallel could potentially relieve t...
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Formato: | Texto |
Lenguaje: | English |
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Public Library of Science
2008
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2444025/ https://www.ncbi.nlm.nih.gov/pubmed/18628955 http://dx.doi.org/10.1371/journal.pone.0002720 |
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author | Fischer, Nicholas O. Tok, Jeffrey B.-H. Tarasow, Theodore M. |
author_facet | Fischer, Nicholas O. Tok, Jeffrey B.-H. Tarasow, Theodore M. |
author_sort | Fischer, Nicholas O. |
collection | PubMed |
description | BACKGROUND: Optimization of high affinity reagents is a significant bottleneck in medicine and the life sciences. The ability to synthetically create thousands of permutations of a lead high-affinity reagent and survey the properties of individual permutations in parallel could potentially relieve this bottleneck. Aptamers are single stranded oligonucleotides affinity reagents isolated by in vitro selection processes and as a class have been shown to bind a wide variety of target molecules. METHODOLOGY/PRINCIPAL FINDINGS: High density DNA microarray technology was used to synthesize, in situ, arrays of approximately 3,900 aptamer sequence permutations in triplicate. These sequences were interrogated on-chip for their ability to bind the fluorescently-labeled cognate target, immunoglobulin E, resulting in the parallel execution of thousands of experiments. Fluorescence intensity at each array feature was well resolved and shown to be a function of the sequence present. The data demonstrated high intra- and inter-chip correlation between the same features as well as among the sequence triplicates within a single array. Consistent with aptamer mediated IgE binding, fluorescence intensity correlated strongly with specific aptamer sequences and the concentration of IgE applied to the array. CONCLUSION AND SIGNIFICANCE: The massively parallel sequence-function analyses provided by this approach confirmed the importance of a consensus sequence found in all 21 of the original IgE aptamer sequences and support a common stem:loop structure as being the secondary structure underlying IgE binding. The microarray application, data and results presented illustrate an efficient, high information content approach to optimizing aptamer function. It also provides a foundation from which to better understand and manipulate this important class of high affinity biomolecules. |
format | Text |
id | pubmed-2444025 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2008 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-24440252008-07-16 Massively Parallel Interrogation of Aptamer Sequence, Structure and Function Fischer, Nicholas O. Tok, Jeffrey B.-H. Tarasow, Theodore M. PLoS One Research Article BACKGROUND: Optimization of high affinity reagents is a significant bottleneck in medicine and the life sciences. The ability to synthetically create thousands of permutations of a lead high-affinity reagent and survey the properties of individual permutations in parallel could potentially relieve this bottleneck. Aptamers are single stranded oligonucleotides affinity reagents isolated by in vitro selection processes and as a class have been shown to bind a wide variety of target molecules. METHODOLOGY/PRINCIPAL FINDINGS: High density DNA microarray technology was used to synthesize, in situ, arrays of approximately 3,900 aptamer sequence permutations in triplicate. These sequences were interrogated on-chip for their ability to bind the fluorescently-labeled cognate target, immunoglobulin E, resulting in the parallel execution of thousands of experiments. Fluorescence intensity at each array feature was well resolved and shown to be a function of the sequence present. The data demonstrated high intra- and inter-chip correlation between the same features as well as among the sequence triplicates within a single array. Consistent with aptamer mediated IgE binding, fluorescence intensity correlated strongly with specific aptamer sequences and the concentration of IgE applied to the array. CONCLUSION AND SIGNIFICANCE: The massively parallel sequence-function analyses provided by this approach confirmed the importance of a consensus sequence found in all 21 of the original IgE aptamer sequences and support a common stem:loop structure as being the secondary structure underlying IgE binding. The microarray application, data and results presented illustrate an efficient, high information content approach to optimizing aptamer function. It also provides a foundation from which to better understand and manipulate this important class of high affinity biomolecules. Public Library of Science 2008-07-16 /pmc/articles/PMC2444025/ /pubmed/18628955 http://dx.doi.org/10.1371/journal.pone.0002720 Text en Fischer et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Fischer, Nicholas O. Tok, Jeffrey B.-H. Tarasow, Theodore M. Massively Parallel Interrogation of Aptamer Sequence, Structure and Function |
title | Massively Parallel Interrogation of Aptamer Sequence, Structure and Function |
title_full | Massively Parallel Interrogation of Aptamer Sequence, Structure and Function |
title_fullStr | Massively Parallel Interrogation of Aptamer Sequence, Structure and Function |
title_full_unstemmed | Massively Parallel Interrogation of Aptamer Sequence, Structure and Function |
title_short | Massively Parallel Interrogation of Aptamer Sequence, Structure and Function |
title_sort | massively parallel interrogation of aptamer sequence, structure and function |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2444025/ https://www.ncbi.nlm.nih.gov/pubmed/18628955 http://dx.doi.org/10.1371/journal.pone.0002720 |
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