The specificity and selectivity of aptamer depends on the binding epitope: insights from aptamer Hislxl-2a.

Aptamers, as a crucial class of recognition molecules, play a pivotal role in biomedical research. However, due to their structural diversity and the still-limited understanding of their recognition mechanisms, they continue to face challenges related to specificity and selectivity in practical applications. In this study, through an in-depth analysis of the structure of the aptamer Hislxl-2a and the features of its recognized epitope, we demonstrate for the first time that this aptamer adopts G-quadruplex structure and specifically binds to an epitope comprising at least four histidine residues. Although Hislxl-2a exhibits clear binding specificity for this four-histidine motif, this short sequence is widely present in numerous proteins. As a result, the aptamer fails to achieve high target discrimination on the cell membrane surface. This phenomenon may explain the underlying cause of the poor selectivity observed for certain aptamers in practical applications. Furthermore, these findings provide a new theoretical basis and valuable insights for future strategies aimed at screening and applying highly efficient and specific aptamers.