Fig. 4.

Using the Spinach riboswitch to identify agonists and antagonists of the TPP riboswitch. (A) Agonist activity of thiamine analogs on the Spinach riboswitch. Fluorescence emission was measured by incubating the Spinach riboswitch (100 nM) with 100 µM of each thiamine analog. Only PTPP, a known TPP riboswitch agonist, induced substantial fluorescence (14.8-fold increase). Shown are mean and SEM values of three independent replicates. The chemical structures of all compounds are indicated. (B) Characterization of thiamine analogs for TPP riboswitch antagonist activity. To identify antagonists, each candidate was incubated in the presence of the known riboswitch activator, TPP. Fluorescence emission was measured in the presence of 100 nM sensor, 2 µM TPP, and 100 µM of each thiamine analog drug candidate. An identical mixture of each drug candidate with TPP and 100 nM Spinach was used to normalize the fluorescence signal. Shown are mean and SEM values of three independent replicates. The chemical structures of all of the compounds are indicated.