Understanding how aptamer structure and function are related is crucial in the design and development of aptamer-based biosensors. We have analyzed a series of cocaine-binding aptamers with different lengths of their stem 1 in order to understand the role that this stem plays in the ligand-induced structure-switching binding mechanism utilized in many of the sensor applications of this aptamer. In the cocaine-binding aptamer, the length of stem 1 controls whether the structure-switching binding mechanism for this aptamer occurs or not. We varied the length of stem 1 from being one to seven base pairs long and found that the structural transition from unfolded to folded in the unbound aptamer is when the aptamer elongates from 3 to 4 base pairs in stem 1. We then used this knowledge to achieve new binding selectivity of this aptamer for quinine over cocaine by using an aptamer with a stem 1 two base pairs long. This selectivity is achieved by means of the greater affinity quinine has for the aptamer compared with cocaine. Quinine provides enough free energy to both fold and bind the 2-base pair-long aptamer while cocaine does not. This tuning of binding selectivity of an aptamer by reducing its stability is likely a general mechanism that could be used to tune aptamer specificity for tighter binding ligands.

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