Single-Step Selection of Bivalent Aptamers Validated by Comparison with SELEX Using High-Throughput Sequencing

Abstract

The identification of nucleic acid aptamers would be advanced if they could be obtained after fewer rounds of selection and amplification. In this paper the identification of bivalent aptamers for thrombin by SELEX and single-step selection are compared using next generation sequencing and motif finding informatics. Results show that similar aptamers are identified by both methods. This is significant because it shows that next generation sequencing and motif finding informatics have the potential to simplify the selection of aptamers by avoiding multiple rounds of enzymatic transcription and amplification.

Trends in aptamer selection methods and applications

“…For instance, Wilson et al 27 recently reported bivalent thrombin aptamers selected at single cycle using 454 GS FLX NGS platform that enabled comprehensive data analysis obtained from the sequential dissociation rounds. Identification of candidate aptamers based on their consensus sequences and the secondary structures is considered as a fundamental change in the field.…”

The Clinical Application of Aptamers: Future Challenges and Prospects

“…NGS possesses unprecedented sequencing speed, thereby enabling impressive scientific achievements and novel biological applications [41,42]. Accordingly, this high-throughput NGS has also been introduced into aptamer selection [43][44][45][46][47][48]. In traditional aptamer selection, Sanger sequencing can only identify some hundred clones, requiring the successive shrinking of the enriched library to a small number of sequences through many rounds of selection.…”

Devices and approaches for generating specific high-affinity nucleic acid aptamers

“…55 A variation on this technology uses magnetic beads in a slightly different manner. In a method called Aptamer Selection Express (ASExp), target molecules and the library are mixed together in solution just like in filter binding or EMSA.…”

“…Combining the advantages of several technologies discussed may be an approach for further improving selection efficiencies, while minimizing each technology's unique sources of background binding, nucleic acid sequence bias, or other undesirable artefacts. In addition, other than using stringent or long washing conditions, 55 incorporating thermodynamic thresholds for binding and dissociation (although not yet commonplace) may allow for better discrimination between higher and lower affinity aptamers. For example, using salt or thermal gradients to elute bound sequences decreases their effective affinity (see Eq.…”