A synchronous counter-positive strategy via online CE-SELEX for high-efficiency aptamer selection

Capillary electrophoresis-based systematic evolution of ligands by exponential enrichment (CE-SELEX) has been widely recognized as an effective method for aptamer selection. In this study, we developed a CE-SELEX strategy to synchronize the counter and positive selection in a single online CE operation, involving the successive injections of the ssDNA library and two proteins as well as their online-modular reaction based on their differentiated electrophoretic mobilities, herein called online synchronous counter-positive CE-SELEX (oscpCE-SELEX). Two model proteins, human lactoferrin (HLF) and ferritin (Fer), were selected. Fer, as the counter protein, first passed through the ssDNA library zone and bound a portion of oligonucleotide sequences, after which HLF was introduced to perform positive selection. Through three rounds of oscpCE-SELEX and sequence analysis, Seq.H-2 was identified with a stem-loop structure and three motifs, exhibiting the highest affinity of 0.39 ± 0.02 μmol/L. Besides, the binding analysis showed that Seq.H-2 exhibited excellent specificity against Fer and other interfering proteins. Furthermore, the versatility of oscpCE-SELEX was confirmed by performing several groups of proteins, and its feasibility for the synchronous selection of two protein aptamers was also evaluated. With the advantages of being fast, efficient, and controllable, oscpCE-SELEX helps address the challenge of separating positive and counter selection in traditional offline SELEX and shows potential for efficient aptamer selection.