RNA Aptamer-Mediated Gene Activation Systems for Inducible Transgene Expression in Animal Cells.

Abstract

RNA expression analyses can be used to obtain various information from inside cells, such as physical conditions, the chemical environment, and endogenous signals. For detecting RNA, the system regulating intracellular gene expression has the potential for monitoring RNA expression levels in real time within living cells. Synthetic biology provides powerful tools for detecting and analyzing RNA inside cells. Here, we devised an RNA aptamer-mediated gene activation system, RAMGA, to induce RNA-triggered gene expression activation by employing an inducible complex formation strategy grounded in synthetic biology. This methodology connects DNA-binding domains and transactivators through target RNA using RNA-binding domains, including phage coat proteins. MS2 bacteriophage coat protein fused with a transcriptional activator and PP7 bacteriophage coat protein fused with the tetracycline repressor (tetR) can be bridged by target RNA encoding MS2 and PP7 stem-loops, resulting in transcriptional activation. We generated recombinant CHO cells containing an inducible GFP expression module governed by a minimal promoter with a tetR-responsive element. Cells carrying the trigger RNA exhibited robust reporter gene expression, whereas cells lacking it exhibited no expression. GFP expression was upregulated over 200-fold compared with that in cells without a target RNA expression vector. Moreover, this system can detect the expression of mRNA tagged with aptamer tags and modulate reporter gene expression based on the target mRNA level without affecting the expression of the original mRNA-encoding gene. The RNA-triggered gene expression systems developed in this study have potential as a new platform for establishing gene circuits, evaluating endogenous gene expression, and developing novel RNA detectors.