https://hdl.handle.net/10012/22131

DNA aptamers for small molecules hold transformative promise in biosensing, diagnostics, and therapeutics, yet their in vitro evolution has been hampered by incomplete knowledge of the parameters that drive efficient enrichment. In recent years, the development of library-immobilization based method, so called capture-SELEX, has generated over 100 high-quality DNA aptamers for various types of small molecules. Importantly, capture-SELEX allows systematic investigation of fundamental problems in the selection of aptamers. This thesis studies the capture-SELEX platform by dissecting thermodynamic, kinetic, and methodological variables to accelerate the discovery of high-affinity DNA aptamers. Using adenosine/ATP as targets for selection has repeatedly produced the same guanine-rich aptamer motif that was first reported by the Szostak group in 1995. This aptamer has been considered as the adenosine/ATP aptamer by the field. First, by gradually increasing the selection stringency on classical targets (adenosine and ATP), we selected two new aptamers with Kd ≈ 230 nM, 35-fold tighter than that of the classical aptamer sequence. This was achieved through gradual reduction of target concentration from 5 mM to the low-micromolar range. The evolution of the sequence abundance cross different rounds was traced by deep sequencing, and the reason for the previous repeated report of the classical sequence was attributed to its short 12-nucleotide conserved binding regions, whereas the two new aptamers have approximately 16 conserved nucleotides. This study highlights the importance of using low target concentration in order to enrich high affinity aptamers. During aptamer selection, using a lower target concentration tends to favor the enrichment of higher affinity binders, raising the question of whether a practical lower limit exists. Next, we performed three capture-SELEX campaigns using 5 µM, 500 nM, and 50 nM guanine as the target, respectively, to investigate it. Both the 5 µM and 500 nM selections successfully enriched the same guanine aptamer-requiring eight rounds at 5 µM guanine versus 17 rounds at 500 nM guanine. However, the 50 nM selection failed to yield any aptamers. The highest affinity and most enriched aptamer from these selections displayed a Kd of 200 nM, indicating that if the target concentration is much lower than Kd can lead to failed selections. Mutation analysis further revealed a critical cytosine in the guanine binding pocket: substituting this cytosine with a thymine switched selectivity from guanine to adenine. A similar specificity switching was previously seen in the natural guanine riboswitches. These findings define a lower limit for target concentration in capture-SELEX and offer a practical guidance for selecting target levels to isolate high-affinity aptamers. Selection of high-affinity aptamers underpins all downstream applications, yet most protocols emphasize thermodynamic factors-such as target concentration-while overlooking binding kinetics. Third, we performed a library-immobilization selection against ampicillin to dissect these influences. Under typical gravity-flow conditions (1-2 min interaction), a low-affinity aptamer (Kd = 12.7 µM) dominated the enriched pool. In contrast, extending the incubation time to 10 min enriched a higher affinity sequence (Kd = 1.8 µM), differing by only three nucleotides from the weaker Kd aptamer. Systematic comparison of library immobilization efficiency, release fraction, and release kinetics confirmed that dissociation rate from the capture duplex was the primary determinant of the selection outcome. We observed the same kinetic bias in parallel adenosine selections, demonstrating the generality of this effect. Based on these findings, we recommend combining low target concentrations with extended incubation time to favor the enrichment of high-affinity aptamers. This study not only yields a robust, high affinity and selective ampicillin aptamer but also highlights a critical interplay between thermodynamics and kinetics during in vitro aptamer selection. Since 1990, numerous aptamer-selection techniques have been developed, yet quantitative comparisons of their enrichment efficiencies remain scarce. Finally, we evaluated three library‐immobilization SELEX methods, capture‐SELEX, GO‐SELEX, and gold‐SELEX, using a spiked library containing DNA aptamers with varying affinities for adenosine. Using 100 µM adenosine as target, all three methods showed that <1 % of the library was released by adenosine as revealed by qPCR, with gold‐SELEX showing virtually no DNA elution. Deep sequencing of three model aptamers (Ade1301, Ade1304, and the classical adenosine aptamer) revealed 30-50‐fold enrichment in capture-SELEX, whereas GO‐SELEX and gold‐SELEX both yielded enrichment factors below 1, indicating a lack of aptamer enrichment. Blocking the primer‐binding regions improved GO‐SELEX enrichment to ~14 % but still fell far short of capture‐SELEX’s performance. Finally, we compared nonspecific versus target‐induced release and elucidated why capture‐SELEX’s structural-switching mechanism offers superior aptamer enrichment. Overall, capture‐SELEX is a markedly more efficient strategy for isolating high‐affinity aptamers. Collectively, this work establishes a quantitative framework for capture-SELEX-balancing target concentration, kinetic control, and partitioning strategy-to reliably isolate nanomolar-class DNA aptamers for small molecules.

https://hdl.handle.net/10012/22131