An engineered small RNA-mediated genetic switch based on a ribozyme expression platform

Abstract

An important requirement for achieving many goals of synthetic biology is the availability of a large repertoire of reprogrammable genetic switches and appropriate transmitter molecules. In addition to engineering genetic switches, the interconnection of individual switches becomes increasingly important for the construction of more complex genetic networks. In particular, RNA-based switches of gene expression have become a powerful tool to post-transcriptionally program genetic circuits. RNAs used for regulatory purposes have the advantage to transmit, sense, process and execute information. We have recently used the hammerhead ribozyme to control translation initiation in a small molecule-dependent fashion. In addition, riboregulators have been constructed in which a small RNA acts as transmitter molecule to control translation of a target mRNA. In this study, we combine both concepts and redesign the hammerhead ribozyme to sense small trans-acting RNAs (taRNAs) as input molecules resulting in repression of translation initiation in Escherichia coli. Importantly, our ribozyme-based expression platform is compatible with previously reported artificial taRNAs, which were reported to act as inducers of gene expression. In addition, we provide several insights into key requirements of riboregulatory systems, including the influences of varying transcriptional induction of the taRNA and mRNA transcripts, 5′-processing of taRNAs, as well as altering the secondary structure of the taRNA. In conclusion, we introduce an RNA-responsive ribozyme-based expression system to the field of artificial riboregulators that can serve as reprogrammable platform for engineering higher-order genetic circuits.

A Matter of Location: Influence of G-Quadruplexes on Escherichia coli Gene Expression

“…Different systems have been described using engineered devices as sensitive switches of gene expression in prokaryotic organisms (Klauser and Hartig, 2013;Wieland et al, 2009;Wieland and Hartig, 2007;Winkler et al, 2002). In this study, we successfully constructed a system where the SD region was masked via a hairpin structure.…”

Computational Selection and Experimental Validation of Allosteric Ribozymes That Sense a Specific Sequence of Human Telomerase Reverse Transcriptase mRNAs as Universal Anticancer Therapy Agents

“…All these findings made the presented designer ribozyme a promising tool for metabolic engineering of exogenous control of gene expression. In addition, RNA-sensing ribozymes based on hammerhead ribozyme from S. mansoni have been shown to work in vivo (Klauser and Hartig, 2013). Such approach may be used for reprogramming the cell fate by exogenous control of p53 expression and its potential applications in novel anticancer gene therapies.…”

Synthetic small RNAs: Current status, challenges, and opportunities

“…Thus, a ribozyme-based riboregulator could be utilized to address these problems, by regulating the ability of a catalytic RNA molecule to cleave a target nucleic acid sequence. 72 32 In an experimental analysis, the hammerhead-shaped ribozyme RNA molecule is activated or inactivated depending upon changes in the secondary structure, induced by hybridizing a signal molecule.…”