(228cd) Selection of Methylxanthine-Responsive Aptamers

RNA has been known to possess the abilities of catalysis and molecular recognition. In fact, the theophylline (1,3-methylxanthine) aptamer has found great utility in synthetic biology, with the generation of many theophylline-dependent riboswitches. The theophylline aptamer is selective against caffeine and other related methylxanthines, but a single nucleotide mutation can change the selectivity, resulting in an aptamer that recognizes 3-methylxanthine. However, there are currently no riboswitches or aptamers for any other methylxanthine. The objective of this study was to develop new RNA aptamers to recognize theobromine (3,7-dimethylxanthine) and paraxanthine (1,7-dimethylxanthine) through iterative rounds of in vitro selection. We first generated a pool of RNA molecules containing 40 random nucleotides affixed to the stem II loop of the hammerhead ribozyme. The initial C-G base pair on the stem II was left in place in order to retain ribozyme activity. After an initial negative selection step to remove ribozymes that self-cleaved in the absence of ligands, the remaining uncleaved molecules were incubated in a positive selection reaction with ligand molecules (i.e., theobromine or paraxanthine). The 5â?? cleavage products were purified and used as a template for subsequent rounds. After 8-10 rounds of allosteric selection, we found aptamers specific for theobromine and paraxanthine. These aptamers are expected to provide new tools to control gene expression and select for cells capable of producing high-value methylxanthines.