Using Aptazymes to Detect for Theophylline as a Test Pathway for Our Device

Aptazymes are a rapidly-developing technology in synthetic biology and biosensing. Aptazymes are made up of an aptamer (a single strand of RNA whose 3D folded structure can bind specifically to a target molecule) and a ribozymes (self-cleaving pieces of RNA which can be utilized to destabilize RNA transcripts). Both active and specific, it is possible to design aptazymes to detect specifically for a wide range of small molecules and proteins. In order to demonstrate a working prototype of our device (which uses yeast housed in a paper device to detect for a target molecule), we chose to use an aptazyme that binds specifically to theophylline, an asthma drug. This system was integrated into yeast (S. cerevisiae).

In our system, the theophylline aptazyme is transcribed with yellow fluorescent protein to visibly display when theophylline is present. When no theophylline is present, the aptazyme takes on a special conformation that activates the ribozyme subunit of the aptazyme to cleave its polyA tails, which allow for translation to occur. Thus, with no theophylline, no YFP is produced. However, in the presence of theophylline, the aptamer assumes a new conformation which stabilizes the mRNA so translation can proceed.

We used a flow cytometer (which measures fluorescence of each cell in a sample individually) to test fluorescence in our samples with and without theophylline. As seen below, we were able to show that there was a systematic difference between fluorescence in our samples with and without theophylline; in the presence of theophylline, fluorescence was always higher for each yeast transformant.

 

Also seen below, we were also able to show that our aptazyme was dosage-sensing (as theophylline concentration increased, so did fluorescence).

 

Our aptazyme was also highly specific; while caffeine is only one methyl group different from theophylline, our construct showed much higher fluorescence with theophylline than with caffeine.

 

Still, background fluorescence (when theophylline is not present) is still a problem in easy naked-eye detection of theophylline. We believe that this is in large part due to the accumulation of YFP, which is highly stable and does not degrade quickly. In order to optimize the difference between the "on" and "off" states of our system, we are now utilizing a PEST domain after our YFP domain in order to degrade it and reduce accumulation. But that's a story for another day...