Dynamic signal processing by ribozyme-mediated RNA circuits to control gene expression.
Bottom Line: We design switchable functional RNA domains by using strand-displacement techniques.We experimentally characterize the molecular mechanism underlying our synthetic RNA signaling cascades, show the ability to regulate gene expression with transduced RNA signals, and describe the signal processing response of our systems to periodic forcing in single live cells.The engineered systems integrate RNA-RNA interaction with available ribozyme and aptamer elements, providing new ways to engineer arbitrary complex gene circuits.
Affiliation: Institute of Systems and Synthetic Biology, Université d'Évry-Val-d'Essonne, CNRS, F-91000 Évry, France.Show MeSH
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Mentions: In this work, we considered three possible sensor domains, two sensing a small molecule (theophylline − Theo − and thiamine pyrophosphate − TPP, Figure 2 and Supplementary Figure S7), and another sensing a specific sRNA (Break1, Figure 3). This sRNA is induced with anhydrotetracycline (aTc) in our system. More specifically, each sensor is composed of a binding domain (e.g., an aptamer) and a catalytic domain (e.g. a hammerhead ribozyme). Our ligand-induced ribozymes (aptazymes) are theoHHAz and tppHHAz for sensing small molecules (11,31), and breakHHRz for sensing sRNA (32) (Supplementary Figure S3). For the mediator domain, we considered three synthetic riboregulators known to activate the initiation of translation, two engineered in Rodrigo et al. (RAJ11 and RAJ12) (18) and one in Isaacs et al. (RR12) (19) (Supplementary Figure S8). We then designed the rest of the regazyme sequence according to the specifications required to generate the RNA signaling cascade. Exploiting the modularity of this system, we engineered the following regazymes: theoHHAzRAJ11, theoHHAzRAJ12, theoHHAzRR12, tppHHAzRAJ12 and breakHHAzRAJ12. The regazyme produces a mediator molecule (riboregulator) that is independent of the signal and sensor molecules. In the following, we investigate, on the one hand, how different signal molecules (Theo, TPP and Break1) activate a common mediator (RAJ12), and, on the other hand, how different implementations of the wire (RAJ11, RAJ12 and RR12) transduce the information from a common signal molecule (Theo).
Affiliation: Institute of Systems and Synthetic Biology, Université d'Évry-Val-d'Essonne, CNRS, F-91000 Évry, France.