De Novo Design of RNA Switches for Conditional Transcription Repression

Controlling transcriptional synthesis of RNA provides a powerful means to design molecular circuitry and reprogram cellular behaviors. We report the computationally de novo design of a transcriptional RNA switch, termed RNA BRAKE, to precisely repress transcriptional RNA synthesis in response to a cellular RNA trigger in live cells. The design principle uses an introduced RNA trigger to regulate the co-transcriptional folding pathway of nascently transcribed RNA BRAKEs into an RNA terminator, thereby repressing downstream RNA transcription. Without the RNA trigger, the RNA polymerase can pass through the RNA BRAKE and continue transcribing downstream genes. We validated the designed RNA BRAKE's performance by encoding the downstream gene with GFP and found that the presence of trigger RNA can significantly repress the GFP expression and GFP mRNA transcription in Escherichia coli cells. We further investigated the mechanism of RNA BRAKE design and studied its compatibility with different RNA polymerases and ribozymes for gene regulation. To demonstrate the generality of RNA BRAKE in response to cellular mRNA, we successfully developed RNA BRAKEs that enable mCherry mRNA to repress the transcription of its encoded downstream gene. This developed RNA BRAKE offers a new strategy for manipulating cellular gene expression with broad biomedical applications.