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An on-bead tailing/ligation approach for sequencing resin-bound RNA libraries

Nucleic acids possess the unique property of being enzymatically amplifiable, and have therefore been a popular choice for the combinatorial selection of functional sequences, such as aptamers or ribozymes. However, amplification typically requires known sequence segments that serve as primer bindin...

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Detalles Bibliográficos
Autores principales: Wiesmayr, Anna, Fournier, Pierre, Jäschke, Andres
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3351178/
https://www.ncbi.nlm.nih.gov/pubmed/22298510
http://dx.doi.org/10.1093/nar/gks004
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author Wiesmayr, Anna
Fournier, Pierre
Jäschke, Andres
author_facet Wiesmayr, Anna
Fournier, Pierre
Jäschke, Andres
author_sort Wiesmayr, Anna
collection PubMed
description Nucleic acids possess the unique property of being enzymatically amplifiable, and have therefore been a popular choice for the combinatorial selection of functional sequences, such as aptamers or ribozymes. However, amplification typically requires known sequence segments that serve as primer binding sites, which can be limiting for certain applications, like the screening of on-bead libraries. Here, we report a method to amplify and sequence on-bead RNA libraries that requires not more than five known nucleotides. A key element is the attachment of the starting nucleoside to the synthesis resin via the nucleobase, which leaves the 3′-OH group accessible to subsequent enzymatic manipulations. After split-and-mix synthesis of the oligonucleotide library and deprotection, a poly(A)-tail can be efficiently added to this free 3′-hydroxyl terminus by Escherichia coli poly(A) polymerase that serves as an anchored primer binding site for reverse transcription. The cDNA is joined to a DNA adapter by T4 DNA ligase. PCR amplification yielded single-band products that could be cloned and sequenced starting from individual polystyrene beads. The method described here makes the selection of functional RNAs from on-bead RNA libraries more attractive due to increased flexibility in library design, higher yields of full-length sequence on bead and robust sequence determination.
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spelling pubmed-33511782012-05-14 An on-bead tailing/ligation approach for sequencing resin-bound RNA libraries Wiesmayr, Anna Fournier, Pierre Jäschke, Andres Nucleic Acids Res Methods Online Nucleic acids possess the unique property of being enzymatically amplifiable, and have therefore been a popular choice for the combinatorial selection of functional sequences, such as aptamers or ribozymes. However, amplification typically requires known sequence segments that serve as primer binding sites, which can be limiting for certain applications, like the screening of on-bead libraries. Here, we report a method to amplify and sequence on-bead RNA libraries that requires not more than five known nucleotides. A key element is the attachment of the starting nucleoside to the synthesis resin via the nucleobase, which leaves the 3′-OH group accessible to subsequent enzymatic manipulations. After split-and-mix synthesis of the oligonucleotide library and deprotection, a poly(A)-tail can be efficiently added to this free 3′-hydroxyl terminus by Escherichia coli poly(A) polymerase that serves as an anchored primer binding site for reverse transcription. The cDNA is joined to a DNA adapter by T4 DNA ligase. PCR amplification yielded single-band products that could be cloned and sequenced starting from individual polystyrene beads. The method described here makes the selection of functional RNAs from on-bead RNA libraries more attractive due to increased flexibility in library design, higher yields of full-length sequence on bead and robust sequence determination. Oxford University Press 2012-05 2012-01-31 /pmc/articles/PMC3351178/ /pubmed/22298510 http://dx.doi.org/10.1093/nar/gks004 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
Wiesmayr, Anna
Fournier, Pierre
Jäschke, Andres
An on-bead tailing/ligation approach for sequencing resin-bound RNA libraries
title An on-bead tailing/ligation approach for sequencing resin-bound RNA libraries
title_full An on-bead tailing/ligation approach for sequencing resin-bound RNA libraries
title_fullStr An on-bead tailing/ligation approach for sequencing resin-bound RNA libraries
title_full_unstemmed An on-bead tailing/ligation approach for sequencing resin-bound RNA libraries
title_short An on-bead tailing/ligation approach for sequencing resin-bound RNA libraries
title_sort on-bead tailing/ligation approach for sequencing resin-bound rna libraries
topic Methods Online
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3351178/
https://www.ncbi.nlm.nih.gov/pubmed/22298510
http://dx.doi.org/10.1093/nar/gks004
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