Deep Mutational Scanning of structure-switching aptamers identifies both base requirement and base-pairing and uncovers regions required for either sensitivity or specificity

Deep mutational scanning (DMS) has been employed to investigate the structure and function of structure-switching aptamers (SSAs), specifically three SSAs designed to detect cortisol. This advanced technique has yielded valuable insights into the bases required for SSA activity and how base-pairing occurs in both the presence and absence of the target.

Despite having differing predicted structures, all three SSAs exhibit a similar conformation upon binding the cortisol target. This finding demonstrates the potential of DMS to uncover structural similarities among seemingly disparate SSAs.

To further explore the mechanisms behind cortisol binding, the researchers generated numerous new SSAs capable of binding cortisol. Surprisingly, their findings revealed that diverse sequences converge on a similar core structure for recognizing the target. This suggests that there may be multiple ways to achieve the same mode of target recognition within SSAs.

Furthermore, DMS was utilized to modify target specificity through a limited number of mutations, thereby mapping out key determinants of specificity. This research highlights the power of DMS in providing detailed information on the inner workings of SSAs, potentially leading to the development of more accurate models for understanding the mechanisms behind SSA function.