NGS-Guided Aptamer Re-Selection for Improved Sensor Applications: Biotin as a Modification Tag in the Amplification of Enriched Pools

Aptasensors are biosensors that rely on an aptamer’s ability to selectively bind targets. To produce a signal indicative of successful binding, aptamers are frequently modified with reporter agents. However, modification of aptamers with specific reporter agents affects subsequent aptamer-target binding, resulting in time-consuming screening assays to identify suitable aptamers capable of binding, once modified. To address this, this study proposes a SELEX approach that amplifies an enriched aptamer pool using a 5′-biotin-C6-phosphoramidite modification and using that for subsequent selection of suitable sequences capable of binding when modified. For this study, fractions from an existing enriched aptamer pool from a previous SELEX for hCG aptamers were separately amplified utilising biotinylated and non-biotinylated (unmodified) primers and sequenced via nanopore next-generation sequencing. While several enriched sequences were represented within the pools, bioinformatic analysis of the pools indicated subtle clustering of sequences between pools. However, the disparity in the number of sequences between both pools may indicate a possible amplification or sequencing-based bias caused by the biotinylation. This approach has merit to support aptamer SELEX strategies but may require further validation.