Programming anti-ribozymes to sense trigger RNAs for modulating gene expression in mammalian cells

Abstract

Synthetic RNA-based switches provide distinctive merits in modulating gene expression. Simple and flexible RNA-based switches are crucial for advancing the field of gene regulation, paving the way for innovative tools that can sense and manipulate cellular processes. In this research, we have developed programmable ribozymes that are capable of suppressing gene expression in response to specific, endogenously expressed trigger RNAs. We engineer ribozymes by introducing upstream antisense sequences (anti-ribozymes) to inhibit the self-cleaving activity of the hammerhead ribozyme and open the expression of the target gene. The trigger RNA is designed to recognize and bind to complementary sequences within the anti-ribozymes, thereby inhibiting their ability to direct protein synthesis. The anti-ribozyme performance is optimized by regulating the essential sequence modules that play a crucial role in determining the specificity and efficiency of the anti-ribozyme's interaction with its trigger RNA. By applying this switch mechanism to various ribozyme designs, we have shown that it is possible to achieve control over gene expression across a wide range of trigger RNAs. By exploiting these programmable anti-ribozymes, we aim to create a powerful tool for controlling gene expression in mammalian cells, which could have important implications for basic research, disease diagnosis, and therapeutic interventions.