The discovery of ribozymes has inspired exploration of RNA’s potential to serve as primordial catalysts in a hypothesized RNA world. Modern oxidoreductase enzymes employ differential binding between reduced and oxidized forms of redox cofactors to alter cofactor reduction potential and enhance the enzyme’s catalytic capabilities. The utility of differential affinity has been underexplored as a chemical strategy for RNA. Here we show an RNA aptamer that preferentially binds oxidized forms of flavin over reduced forms and markedly shifts flavin reduction potential by −40 mV, similar to shifts for oxidoreductases. Nuclear magnetic resonance structural analysis revealed π–π and donor atom–π interactions between the aptamer and flavin that cause unfavorable contacts with the electron-rich reduced form, suggesting a mechanism by which the local environment of the RNA-binding pocket drives the observed shift in cofactor reduction potential. It seems likely that primordial RNAs could have used similar strategies in RNA world metabolisms.

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Benzodiazepines are a class of psychoactive compounds that are commonly prescribed for conditions such as anxiety, depression, and chronic pain. The use and abuse of benzodiazepines is slowly rising and requires reliable detection methods to monitor patient use and compliance. Most current screening methods are antibody-based and have inherent issues, including limited stability and shelf life. The use of non-antibodybased detection methods that overcome these challenges would be useful for testing purposes. Using in vitro selection, we have identified three DNA aptamers for benzodiazepines with K d values between 0.2-0.6 μM for diazepam and alprazolam and their metabolites but little to no binding for lorazepam or clonazepam. One aptamer, 20DF42, was used to develop a strand-displacement fluorescence polarization biosensor for detection of benzodiazepines in synthetic urine. The resulting sensor had a limit of detection of 231 ng/mL with temazepam as the calibrant.