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Int. J. Mol. Sci. 2018, 19(2), 642; https://doi.org/10.3390/ijms19020642

Refining the Results of a Classical SELEX Experiment by Expanding the Sequence Data Set of an Aptamer Pool Selected for Protein A

1
UFZ—Helmholtz Centre for Environmental Research, Department of Soil Ecology, 06120 Halle, Germany
2
UFZ—Helmholtz Centre for Environmental Research, Department of Environmental and Biotechnology Centre, 04318 Leipzig, Germany
*
Author to whom correspondence should be addressed.
Received: 9 January 2018 / Revised: 9 February 2018 / Accepted: 19 February 2018 / Published: 24 February 2018
(This article belongs to the Section Biochemistry, Molecular and Cellular Biology)
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Abstract

New, as yet undiscovered aptamers for Protein A were identified by applying next generation sequencing (NGS) to a previously selected aptamer pool. This pool was obtained in a classical SELEX (Systematic Evolution of Ligands by EXponential enrichment) experiment using the FluMag-SELEX procedure followed by cloning and Sanger sequencing. PA#2/8 was identified as the only Protein A-binding aptamer from the Sanger sequence pool, and was shown to be able to bind intact cells of Staphylococcus aureus. In this study, we show the extension of the SELEX results by re-sequencing of the same aptamer pool using a medium throughput NGS approach and data analysis. Both data pools were compared. They confirm the selection of a highly complex and heterogeneous oligonucleotide pool and show consistently a high content of orphans as well as a similar relative frequency of certain sequence groups. But in contrast to the Sanger data pool, the NGS pool was clearly dominated by one sequence group containing the known Protein A-binding aptamer PA#2/8 as the most frequent sequence in this group. In addition, we found two new sequence groups in the NGS pool represented by PA-C10 and PA-C8, respectively, which also have high specificity for Protein A. Comparative affinity studies reveal differences between the aptamers and confirm that PA#2/8 remains the most potent sequence within the selected aptamer pool reaching affinities in the low nanomolar range of KD = 20 ± 1 nM. View Full-Text
Keywords: aptamer; Protein A; Staphylococcus aureus; SELEX; NGS; sequence analysis; binding affinity; SPR aptamer; Protein A; Staphylococcus aureus; SELEX; NGS; sequence analysis; binding affinity; SPR
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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Stoltenburg, R.; Strehlitz, B. Refining the Results of a Classical SELEX Experiment by Expanding the Sequence Data Set of an Aptamer Pool Selected for Protein A. Int. J. Mol. Sci. 2018, 19, 642.

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