Synthetic Mammalian RNA-Based Auxiliary Module for High Gene Expression

Synthetic and native switchable genetic expression systems are extensively used in basic research and a wide range of applications in biomedicine and biotechnology. Leakiness, which represents promoter activity prior to the presence of an appropriate instigator, could have negative consequences ranging from ineffective biological sensor systems to toxic side effects in medical applications. Here, we construct an auxiliary RNA-based augmentation system rooted in mutual inhibition that can be added to existing systems without modifying the involved transcription factors, promoters, or output genes. This system utilizes a constitutively expressed small hairpin RNA to reduce the expression level of a target gene, which is governed by a responsive promoter. Additionally, a second promoter, identical to the output promoter, drives the expression of a binding-site sponge specifically designed to sequester the shRNA, thereby mitigating its inhibitory effect. Following mathematical modeling, we examine the experimental effectiveness of the system in reducing the leakiness and enhancing the fold change of the doxycycline-inducible Tet-On system tested in a two- and three-dimensional spheroid setting, a GAL4-inducible system, and a native cancer-specific promoter-pH2A1.