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Combining capillary electrophoresis and next-generation sequencing for aptamer selection

Guthold, Martin

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abstract
Next-generation sequencing (NGS) machines can sequence millions of DNA strands in a single run, such as oligonucleotide (oligo) libraries comprising millions to trillions of discrete oligo sequences. Capillary electrophoresis is an attractive technique to select tight binding oligos or “aptamers” because it requires minimal sample volumes (e.g., 100 nL) and offers a solution-phase selection environment through which enrichment of target-binding oligos can be determined quantitatively. We describe here experiments using capillary transient isotachophoresis (ctITP)-based nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) as a method for selecting aptamers from a randomized library containing a known (29mer) thrombin-binding aptamer. Our capillary electrophoresis (CE)-selected samples were sequenced by the Ion Torrent Personal Genome Machine (PGM) and analyzed for selection efficiency. We show that a single round of CE selection can enrich a randomer synthetic DNA oligo mixture for thrombin-binding activity from 0.4 % aptamer content before selection to >15 % aptamer content.
We are grateful to the North Carolina Biotechnology Center for their support of this work through two grants (NC Biotech Grant Award # 2011-MRG-1115) and the Kenan Fellowship Award (NC Biotech Grant Award # 2012-CFG-8008) to primary co-author Jason Gagliano. This work was also supported by the NIH (5R43GM102987) and by a collaborative pilot grant from the Wake Forest University Translational Science Institute and UNC Charlotte. (sponsorship)
subject
Separations
Instrumentation
Capillary electrophoresis
Electrophoresis
Next-generation sequencing
Bioanalytical methods
citation
6 (issue)
407 (volume)
contributor
Riley, Kathryn R. (author)
Gagliano, Jason (author)
Xiao, Jiajie (author)
Libby, Kara (author)
Saito, Shingo (author)
Yu, Guo (author)
Cubicciotti, Roger (author)
Macosko, Jed (author)
Colyer, Christa L. (author)
Guthold, Martin (author)
Bonin, Keith (author)
date
2015-10-09T18:54:23Z (accessioned)
2015-10-09T18:54:23Z (available)
2015 (issued)
http://dx.doi.org/10.1007/s00216-014-8427-y (doi)
identifier
http://hdl.handle.net/10339/57377 (uri)
publisher
Springer
rights
https://creativecommons.org/licenses/by/4.0/ (uri)
source
Analytical and Bioanalytical Chemistry
title
Combining capillary electrophoresis and next-generation sequencing for aptamer selection
type
Article

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