Lawrence Berkeley National Laboratory, Physical Biosciences Division, Berkeley, CA 94702
Aptamers are sequences of RNA that bind to ligand molecules and consequently regulate that gene’s expression levels. They are frequently employed in nature to couple fluctuations in the concentration of a metabolite with changes in gene expression. Upon binding a small molecule, aptamers sequester the ribosome binding site [RBS] of a cis mRNA and repress translation. Thus, synthetic aptamers eliminate the need to rely upon pre-existing biological molecules as the source of binding structure. Generation of aptamers occurs through in vitro selection, iterative rounds of enrichment and amplification which eventually select for an RNA molecule with high binding affinity and specificity. Through a combination of directed evolution and rational design we generated functional, ligand-binding RNA structures that control the cis-expression of mRNA transcripts in response to tetramethylrhodamine [TMR], a small fluorescent dye that is cell permeable. Iterative rounds of reselections and binding assays in in vivo like conditions have generated 3 isolates of TMR-binding aptamers. Such molecules responsive to TMR will provide general tools for simultaneously varying the expression levels of toxic intermediates in synthetic and naturally-occurring pathways such as that of the antimalarial drug, artemisinin. When incorporated into constructs with self-cleaving hammerhead ribozymes, they will allow us to regulate and adjust multiple gene expression levels in a ligand-dependent fashion.