![]() ![]() There are many variations especially with respect to the composition of the SELEX library, the adaption of process conditions for directional selection of aptamers with desired features, and the efficient separation of unbound from target-bound oligonucleotides and their recovery as the most crucial steps of a SELEX process. The SELEX technology has become a powerful standard method for the selection of high-affinity aptamers, and has been extensively modified and optimized over the years. This evolutionary approach leads to the enrichment of specific target-binding aptamers from a highly diverse oligonucleotide library with up to 10 15 different molecules during iterative selection cycles. Īptamers are generated by an in vitro method termed SELEX. Their high potential as therapeutic or diagnostic agents, delivery agents, molecular imaging tools, as capture or reporter molecules in analytical systems, and as recognition elements in biosensors has been demonstrated. Aptamers are currently in great demand in many fields of application from basic to medical and pharmaceutical research, or in the different areas of analytics. These features make aptamers functionally comparable to the widely used antibodies, but they also offer several notable advantages like high availability by chemical synthesis, none or low batch-to-batch variation, versatile chemical modification, high storage stability, regenerability, none or low immunogenicity, small size, flexible structure, and high affinity and specificity to their target, that can compete with that of antibodies. The ability to fold into a unique three-dimensional structure is necessary for their molecular interactions with the target and for forming a stable aptamer-target complex. To date, aptamers have been described for a wide variety of target molecules ranging from small molecules to proteins and composites of targets. Aptamers are artificial short single-stranded DNA or RNA molecules that are selected for recognition and binding of a specific target. During this time, they have received a growing interest in diverse scientific fields. It is now more than 25 years since the advent of a new kind of affinity molecules called aptamers. 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 K D = 20 ± 1 nM. 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. 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. 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. 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. 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. 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. New, as yet undiscovered aptamers for Protein A were identified by applying next generation sequencing (NGS) to a previously selected aptamer pool.
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