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.
Highlights
It is more than 25 years since the advent of a new kind of affinity molecules called aptamers [1,2]
We have shown that the results of our classical SELEX experiment including cloning and Sanger sequencing of the enriched aptamer pool selected for binding to Protein A could be expanded by applying next generation sequencing (NGS) technology
Known characteristics of the aptamer pool like high complexity, high portion of orphans, relative frequency distribution of certain sequences, or co-enrichment of background binders were confirmed by the NGS pool
Summary
It is more than 25 years since the advent of a new kind of affinity molecules called aptamers [1,2]. Valenzano et al applied high throughput sequencing and bioinformatics analysis at specific stages of a multiple round SELEX process where changes of the selection conditions occurred [45] They were interested in deeply understanding the effects of increasing stringency on the enrichment of target-specific aptamers and their dynamics over the course of 21 SELEX rounds. Soldevilla et al combined high throughput sequencing with a strategy of Conserved Motif Accumulation (CMA) [46] They applied NGS after the last two rounds (6–7) in their SELEX experiment and identified the five most abundant aptamers. The authors postulate that aptamer species with a higher accumulation of potential binding motifs are likely to have a higher probability of being better binders These different approaches exemplify the multitude of possibilities to integrate generation sequencing into SELEX experiments with the aim to improve the process of aptamer development. It underlines that the aptamer development remains a complex process and a simple universal method or strategy does not exist
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