Improvement of Aptamers by High-Throughput Sequencing of Doped-SELEX

Abstract

Although SELEX can identify high-affinity aptamers, Doped-SELEX is often performed post-selection for the identification of better variants. Starting from a partially randomized (doped) library derived from an already identified aptamer, this method can screen rapidly several thousand substitutions in order to identify those that can improve the binding of the aptamers. It can also highlight the positions that do not tolerate substitutions, which suggest they are crucial for the interaction of the aptamer with its target. High-throughput sequencing (HTS), also named next-generation sequencing (NGS), can dramatically improve this method by studying millions of sequences. This high number of sequences ensures a statistically robust analysis of variants even for those with a low frequency in the library. It can reduce the number of selection rounds and provide a more in-depth analysis of the positions that are crucial for the aptamer affinity. In this chapter, we provide a protocol to simultaneously study and improve an aptamer using Doped-SELEX and HTS analysis, including the design of the doped library, the selection, HTS, and analysis. This protocol could be useful to improve the affinity of an aptamer and to reduce its size as well as to improve ribozyme.

Keywords: Aptamer; Doped-SELEX; High-throughput sequencing; Next-generation sequencing.