DNA aptamers discovered via an in vitro evolution process (called SELEX) typically do not form simple double helixes but rather complex tertiary structures that include many non-canonical base interactions. It is hypothesized that these interactions, including triplexes between A-T=A, G-quadruplexes, and non-Watson-Crick base pairs (A-C or G-T), provide aptamers with their high level of specificity, selectivity, and activity. Non-canonical base interactions are typically studied via structural determination through methods such as X-ray crystallography, NMR spectroscopy, and Cryo-EM. However, these methods require significant amounts of time and resources, and not all aptamers are amenable to each method of structure determination. A promising alternative is mutational analysis of the aptamer. However, it is extremely laborious to individually characterize each mutant sequence, which typically results in hand selecting a small subset of mutants. Using a novel imaging and screening platform we developed in the lab, we studied over 3,000 single and double mutants which allowed us to study far more mutants in parallel than previously done for the model system of the lettuce aptamer. We were able to sequence and estimate a dissociation constant (KD) for all mutants simultaneously using a flowcell. We chose the lettuce aptamer because its tertiary structure has been experimentally determined by Passalacqua, et. al. (Nature, 2023) and was shown to have many examples of non-canonical base interactions. Knowing the 3D structure of the aptamer allowed us to individually characterize each mutation by its interactions with other bases. With our novel platform, we compared changes in KD’s relative to the wild-type sequence which revealed that most mutations decreased affinity, although a small subset surprisingly enhanced it. As we explored these interactions, we were able to compare to the ground truth tertiary structure of the aptamer to understand which types of interactions are most important in this system. Mapping these effects onto the aptamer’s known tertiary structure highlighted the critical roles of specific stems, long-range contacts, G-quadruplexes, and triplexes. These findings not only shed light on the fundamental interactions governing aptamer function but also provide a valuable dataset for developing improved predictive models of the complex tertiary structures inherent to DNA aptamers.
