Fig. 1
From: Massively parallel RNA device engineering in mammalian cells with RNA-Seq
A high-throughput, quantitative pipeline for engineering ribozyme switches. a A schematic illustrating the mechanism by which ribozyme switches achieve conditional gene expression regulation. In the absence of ligand, the switch undergoes self-cleavage, destabilizing the transcript and resulting in low protein expression. Ligand binding to the switch interferes with the loop–loop tertiary interactions of the ribozyme to inhibit self-cleavage, in turn stabilizing the transcript and leading to increased protein expression. The differential mRNA levels resulting from ribozyme switch activity can be assayed with RNA-Seq. b Schematic of the architecture of a ribozyme switch. The switch comprises a ribozyme actuator domain (black) and a ligand-sensing aptamer domain (blue). In the illustrated library design, an aptamer is grafted onto stem I of the hammerhead ribozyme and 4–6 bases of stem loop II are randomized. The library is screened for loop sequences that interact with the integrated aptamer to enable ribozyme self-cleavage, while allowing ligand binding at the aptamer to disrupt the tertiary interactions and inhibit cleavage. c RNA-Seq workflow for measuring differential mRNA expression levels associated with a ribozyme switch library
