Abstract
Organisms have different circuitries that allow converting signal molecule levels to changes in gene expression. An important challenge in synthetic biology involves the de novo design of RNA modules enabling dynamic signal processing in live cells. This requires a scalable methodology for sensing, transmission, and actuation, which could be assembled into larger signaling networks. Here, we present a biochemical strategy to design RNA-mediated signal transduction cascades able to sense small molecules and small RNAs. 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.
Highlights
Natural signal transduction systems allow organisms to adapt to fluctuating environments, often by exploiting subcellular localization, molecular cascades and protein allostericity [1,2]
This way, we propose to engineer synthetic signal transduction systems relying on RNA by using a transcriptional fusion strategy, exploiting sequence fragments with definite interaction and catalytic properties
We considered a system composed of two transcriptional units: regazyme and messenger RNAs (mRNAs) of a reporter gene, but our methodology could be generalized to an arbitrary number of transcriptional units containing switchable functional elements
Summary
Natural signal transduction systems allow organisms to adapt to fluctuating environments, often by exploiting subcellular localization, molecular cascades and protein allostericity [1,2]. Most engineering efforts have relied on the translational fusion of known protein domains with specific interaction or catalytic functionalities [2]. This approach is limited by the availability of known natural interaction domains that are specific enough to avoid cross-talk with other molecules in the cellular context. The use of RNA as programmable molecules would allow engineering an unlimited number of interaction partners [3,4]. This way, we propose to engineer synthetic signal transduction systems relying on RNA by using a transcriptional fusion strategy, exploiting sequence fragments with definite interaction and catalytic properties. In the following we refer to this multifunctional RNA molecule as regazyme
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