Yurdusev, E.; Nicole, Y.; Pan Du, F.; Suslu, N. E.; Perreault, J.

Nucleic acid-based regulatory elements capable of modulating gene expression in response to specific molecular cues have gained increasing attention in synthetic biology. These systems, which include riboswitches, allosteric DNAzymes, and aptazymes, function as Gene Expression Nucleic Allosteric actuators (GENAs) by coupling molecular recognition with genetic regulation. Their versatility can enable applications in diagnostics, therapeutics, and metabolic engineering. This study presents a novel \"semi-trans\" aptazyme-based system for gene regulation in bacteria, expanding the range of GENAs. The system employs theophylline-responsive hammerhead ribozyme aptazymes positioned in the 5\' UnTranslated Region (UTR), designed to cleave within the coding sequence of the target gene, thereby modulating gene expression in a ligand-dependent manner. Using the tetA gene in Escherichia coli (E. coli) as a proof of concept, we demonstrate ligand-controlled regulation of tetracycline resistance and nickel sensitivity. The system\'s effectiveness is validated through in vitro cleavage assays and in vivo phenotypic studies in two E. coli strains, highlighting its portability across genetic backgrounds. Furthermore, the ability to design multiple aptazymes targeting different coding regions enables complex and fine-tuned regulation. This work broadens the landscape of synthetic gene regulation tools, facilitating the development of new aptazymes based on this approach.