Probing the SELEX Process with Next-Generation Sequencing

Abstract

BackgroundSELEX is an iterative process in which highly diverse synthetic nucleic acid libraries are selected over many rounds to finally identify aptamers with desired properties. However, little is understood as how binders are enriched during the selection course. Next-generation sequencing offers the opportunity to open the black box and observe a large part of the population dynamics during the selection process.MethodologyWe have performed a semi-automated SELEX procedure on the model target streptavidin starting with a synthetic DNA oligonucleotide library and compared results obtained by the conventional analysis via cloning and Sanger sequencing with next-generation sequencing. In order to follow the population dynamics during the selection, pools from all selection rounds were barcoded and sequenced in parallel.ConclusionsHigh affinity aptamers can be readily identified simply by copy number enrichment in the first selection rounds. Based on our results, we suggest a new selection scheme that avoids a high number of iterative selection rounds while reducing time, PCR bias, and artifacts.

Quantitative selection and parallel characterization of aptamers

“…Many different approaches have been described in the literature to discriminate high-affinity aptamers from undesired background sequences arising from selection biases. These methods include enrichment fold (18), repeating motif (8), and copy number (19,20) analysis. We chose copy number analysis to test our capacity to directly distinguish high-affinity aptamers from background sequences via parallel binding measurements.…”

“…NGS allows us to analyze vastly larger number of molecules (∼10 7 sequences) compared with conventional cloning-based methods (∼10 2 sequences), such that putative high-affinity aptamers that become enriched over the course of selection can be identified earlier and without fully converging the enriched pool. This is important, because increasing the number of selection rounds introduces and amplifies undesirable biases that may confound the discovery of targetspecific sequences (20,30). Most importantly, the aptamer arrays make it possible to perform quantitative measurement of both affinity and specificity for thousands of aptamer candidates simultaneously, even in complex samples such as undiluted serum.…”

“…Traditional selection techniques depend on the convergence of the aptamer pool toward a relatively small number of sequences, such that consensus motifs can be obtained from a few hundred sequences. NGS can analyze millions of sequences, thus identifying enriched sequences much earlier in the selection process; this reduces the number of selection rounds required, and enables the identification and elimination of artifacts that typically arise over repeated rounds of PCR or during the cloning process (18)(19)(20). Despite these advances, the generation of high-quality aptamers remains a time-consuming and low-throughput process.…”

New SPR-based methods for analysis of allergenic agents used in wine treatment

“…To increase the selectivity of the aptamers, it is necessary to improve the SELEXprocedure. Recent developments in this field of research indicate that a continuous monitoring of the SELEXprocess by techniques like next-generation sequencing is advisable [17]. Moreover, the use of bioinformatics to identify binding motives and the subsequent enhancement of affinity by the introduction of additional features (artificial nucleic bases or directed extension of the motives) are promising developments [22].…”

“…An identification of potential candidates is conducted through a SELEX (systematic evolution of ligands by exponential enrichment), which must be performed one time. A broad variety of different procedures have been described in the literature since 1990, but the basic principles remain the same [17,18]. The starting point is always an aptamer library with a high number of nucleic acids which are composed of a randomized region flanked by constant primer regions.…”