A modified SELEX approach to identify DNA aptamers with binding specificity to the major histocompatibility complex presenting ovalbumin model antigen

Aptamers have sparked significant interest in cell recognition because of their superior binding specificity and biocompatibility. Cell recognition can be mediated by targeting the major histocompatibility complex (MHC) that presents short peptides derived from intracellular antigens. Although numerous antibodies have demonstrated a specific affinity for the peptide-MHC complex, the number of aptamers that exhibit comparable characteristics is limited. Aptamers are usually selected from large libraries via the Systemic Evolution of Ligands by Exponential Enrichment (SELEX), an iterative process of selection and PCR amplification to enrich a pool of aptamers with high affinity. However, the success rate of aptamer identification is low, possibly due to the presence of complementary sequences or sequences rich in guanine and cytosine that are less accessible for primers. Here, we modified SELEX by employing systemic consecutive selections with minimal PCR amplification. We also modified the analysis by selecting aptamers that were identified in multiple selection rounds rather than those that are highly enriched. Using this approach, we were able to identify two aptamers with binding specificity to cells expressing the ovalbumin alloantigen as a proof of concept. These two aptamers were also discovered among the top 150 abundant candidates, despite not being highly enriched, by performing conventional SELEX. Additionally, we found that highly enriched aptamers tend to contain fractions of the primer sequence and have minimal target affinity. Candidate aptamers are easily missed in the conventional SELEX process. Therefore, our modification for SELEX may facilitate the identification of aptamers for more application in diverse biomedical fields. Significance : we modify the conventional method to improve the efficiency in the identification of the aptamer, a single strand of nucleic acid with binding specificity to the target molecule, showing as a proof of concept that this approach is particularly useful to select aptamers that can selectively bind to cells presenting a particular peptide by the major histocompatibility complex (MHC) on the cell surface. Given that cancer cells may express mutant peptide-MHC complexes that are distinct from those expressed by normal cells, this study sheds light on the potential application of aptamers to cancer cell targeting. A modified SELEX that employs consecutive selections with minimal PCR and selects aptamers that w