From Single Binders to Sequence Populations: SELEX-Based Molecular Recognition for Environmental Detection and Cancer Cell Profiling

Abstract

Aptamers are single-stranded DNA or RNA oligonucleotides selected through Systematic Evolution of Ligands by Exponential Enrichment (SELEX) that fold into defined three-dimensional structures capable of high-affinity, high-specificity target recognition. This thesis explores two complementary applications of SELEX-based aptamer technology: the isolation of a highly selective small-molecule aptamers for environmental biosensing, and the use of early-round, high-throughput sequencing data for computational cancer cell profiling. In the first study, capture-SELEX incorporating extensive negative selection against 17β-estradiol (E2) was used to isolate a DNA aptamer selective for 17α-ethinylestradiol (EE2), a synthetic estrogen and priority environmental contaminant that is structurally very similar to its natural counterparts, estrone (E1) and E2. After eighteen rounds of selection, the aptamer EE2-1 was identified, exhibiting nanomolar affinity for EE2 (Kd = 200 nM by thioflavin T fluorescence; 138 nM by isothermal titration calorimetry) with minimal cross-reactivity toward E1 and E2. Mutational analysis confirmed that binding is mediated by a conserved loop region, and binding was shown to be independent of Mg2+. EE2-1 was incorporated into a label-free fluorescent biosensor capable of detecting EE2 in both buffer and wastewater, representing the first experimentally validated aptamer selected directly against EE2. In the second study, single-round cell-SELEX against HeLa and MCF-7 cancer cell lines, combined with deep sequencing and automated 6-mer frequency analysis, was used to test whether minimally enriched sequence populations encode cell-type-discriminatory information. Differentially enriched motifs distinguishing the two cell lines were identified as early as Round 1, including several novel candidate MCF-7-associated sequence families. Longitudinal analysis across Rounds 1, 4, and 6 revealed that this discriminatory landscape evolves substantially during selection, with a broadly enriched “pan-cancer” motif emerging by Round6 in both cell lines. Comparison of 18- and 30-nucleotide randomized libraries further showed that library architecture influences the information recovered during early selection. Together, these studies demonstrate that SELEX can be exploited in two complementary ways: iterative enrichment with negative selection to isolate individual highly selective aptamers, and computational analysis of minimally enriched populations to extract biologically meaningful molecular fingerprints. This work broadens the scope of aptamer technology for both environmental monitoring and cancer cell profiling and highlights the growing role of bioinformatics in aptamer research.