Abstract 3761: A modified SELEX enables the efficient identification of DNA aptamers with potential for specific cancer targeting
Authors: Lin Yang , Chen Cho-Yi , Lin Zhi-Qian , Chen Bing-Hong , Sun Yi-Chen , Hung Kai-Feng 
Published: March 2024

Abstract
Background: Aptamers have sparked significant interest in cell recognition due to 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. However, the success rate of aptamer identification is low. This is possibly due to the presence of complementary sequences or sequences rich in quinine and cytosine that are less accessible for primers. Here, we use ovalbumin model antigen to explore whether aptamers with specificity for binding to a defined peptide-MHC complex can be identified. We developed DNA aptamers with modified SELEX by employing systemic consecutive selections with minimal PCR amplification. Furthermore, the other tumour antigen, claudin-6, was also used as a target to determine whether our modification for SELEX can be used with any selection technology.
Methods: To determine whether our modified SELEX has potential for selecting aptamer capable of cancer-specific targeting, we used ovalbumin presented by H-2Kb (to represent the specific antigen presented by MHC) and claudin-6 (a cancer-associated adhesion molecule) as a target for the selection of aptamers. This modified SELEX consisted of consecutive positive and negative selections with minimal PCR amplification. Meanwhile, the selection of candidate aptamers is based on their appearance in multiple rounds rather than enrichment, and is facilitated by several computational tools. The specificity and affinity of the candidate aptamer are validated by flow cytometry, immunohistochemistry, and immunofluorescence analysis with cells with or without these molecules.
Results: After high-throughput sequencing, a total of 211 candidate aptamers were selected for further analysis. The quadruplex forming G-rich sequences (QGRS) mapper, mFold modeling, and NUPACK web servers were used to predict the potential of G-quadruplex formation, resulting in only four potential aptamers. Using MTCQ1-OVA cells with FITC-conjugated aptamers, two aptamers showed selective binding to MTCQ1-OVA cells and had no binding specificity to MTCQ1-WT cells. The binding specificity was further validated by immunohistochemistry in tumour derived from mice inoculated with MTCQ1-WT or MTCQ1-OVA cells. In the pull-down assay, the binding affinity of these two candidate aptamers was 136.2 nM and 155.1 nM affinity.
Conclusions: This preliminary study demonstrated that our modification for SELEX is effective in the selection of aptamers with promising potential for specific cancer targeting. The identification of aptamers may facilitate further application in diverse biomedical fields.
Citation Format: Yang Lin, Cho-Yi Chen, Zhi-Qian Lin, Bing-Hong Chen, Yi-Chen Sun, Kai-Feng Hung. A modified SELEX enables the efficient identification of DNA aptamers with potential for specific cancer targeting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3761.