Split Aptamer-SENTR Integration: Conditional Gene Expression Through RNA Splicing

• Hamza Alam, Chemical Engineering, University of Delaware

• Mark Blenner, Chemical and Biomolecular Engineering, University of Delaware

Conditional gene expression is an important tool to regulate cellular behavior. Precise targeted control is critical for improving the specificity, safety, and functional robustness in synthetic biology applications such as biosensing or gene therapy. Traditional systems often struggle with unwanted background activity or rely on the burdensome expression of multiple regulator components, limiting their usefulness in real-world scenarios. 

To address this, we developed a split aptamer-SENTR (Split-Intron Enabled Trans-splicing Riboregulator) system that significantly enhances the specificity and dynamic range of conditional gene expression. The original SENTR system uses complementary RNA base pairing to drive reconstitution of a split RNA intron that splices together a split gene product. For our design, we replaced the RNA detection mechanism in the original SENTR with our split aptamers. By designing split RNA aptamers that only reassemble in the presence of user-defined input molecules, our platform ensures that gene activation occurs strictly when the intended input is present. This high level of control reduces off-target effects and false positives. To evaluate the efficiency of this approach, we are testing multiple insertion positions within the SENTR molecule, substituting the split aptamer at the middle, closest, and furthest parts of the complementary RNA parts in the SENTR. These designs test the stability of the split aptamer to see if binding favorability is affected. All tests are performed with sfGFP so that we can monitor fluorescence as an indicator of gene expression. This allows us to compare the performance of the split aptamer based on its location within the SENTR. We expect these experimental results in E. coli demonstrate that split aptamer-SENTR circuits deliver a lower background reading and strong activation only upon detection of correct signal combinations. These findings could be used to develop future biological devices where conditional gene control could be laying the groundwork for smarter, safer molecular biology applications.