A novel hybrid approach to overcome defects of CE-SELEX and cell-SELEX in developing aptamers against aspartate β-hydroxylase.

Background and purpose: Aptamers, a new category of molecular probes, are overthrowing antibodies in molecular diagnostics. However, there are serious problems with using aptamers for this application including poor or non-specific binding in vivo conditions. Systematic evolution of aptamers is achieved through various approaches including CE-SELEX and Cell-SELEX, each suffering its inevitable weaknesses. The shortcomings of negative selection and the lengthy procedure are Cell-SELEX's main problems, while CE-SELEX is deprived of native targets. Here, we introduced a kind of hybrid CE-Cell-SELEX, named CEC hybrid-SELEX, for addressing these limitations in creating aptamer probes detecting human aspartate β-hydroxylase (ASPH), which is a well-established tumor biomarker, in cancer diagnostic investigations.

Experimental approach: In our approach, the selected oligomer pool from the last cycle of CE-SELEX was sequenced and then subjected to 3 additional rounds of Cell-SELEX which provides native ASPH (CEC hybrid-SELEX). High-throughput sequencing was applied to achieve a comprehensive sight of the enriched pools. Further confirmatory investigations on oligomers with higher copy numbers were performed using flow cytometry.

Findings/results: Three selected oligomers, AP-CEC 1, AP-CEC 2, and AP-CEC 3, showing Kd values of 43.09 nM, 34.85 nM, and 35.92 nM, respectively, were achieved based on the affinity assessment of the ASPH-expressing cells.

Conclusion and implications: Our research suggested that CEC hybrid-SELEX could help recognize which oligomers from CE-SELEX are more capable of binding native ASPH in vivo.