Structure-switching aptamers (SSAs) are short oligonucleotides that undergo a large conformational change on binding their specific target. This change can be used to read out target levels and SSA-based sensors have been developed for targets including drugs, metabolites, and toxins. Despite this potential as molecular sensors, we cannot predict how an SSA folds, how it binds its target, or the final SSA:target conformation and there are very few solved SSA structures. Here we use deep mutational scanning (DMS) to probe SSA structure and function, focusing on three published SSAs that detect cortisol. We show that DMS delivers rich data: it identifies which bases are required for activity but also maps base-pairing either in the presence or absence of target. We find all three SSAs have a similar structure when bound to their target despite having different predicted structures. To broaden our understanding of how SSAs can see cortisol, we selected many new cortisol-binding SSAs. Surprisingly, we identify diverse sequences that all have similar cores to the published cortisol SSAs - many diverse sequences thus converge on the same mode of target recognition. Finally, we use DMS to show we can change target specificity with a small number of mutations and map key specificity determinants. We conclude DMS can provide rich information on how SSAs work and that generating this for many SSAs could lead to greatly improved models for SSA mechanism.
Tan, J. H., Fraser, A. G.
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