Conditional Control of Mammalian Gene Expression by Tetracycline-Dependent Hammerhead Ribozymes

Abstract

Robust synthetic devices are requisite for the construction of synthetic genetic circuits and important scientific and technological tools to control cellular processes. We developed tetracycline-dependent ribozymes, which can switch on gene expression up to 8.7-fold upon addition of tetracycline. A tetracycline aptamer was grafted onto the hammerhead ribozyme in such a way that ligand binding to the aptamers destroys a loop-loop interaction within the ribozyme thereby inhibiting ribozyme cleavage and allowing gene expression. The advantage of the presented regulatory system is its independence of any regulatory proteins. The stable integration of the ribozyme into the genome of HeLa cells indicates a low background activity in the absence of ligand. Furthermore, the ligand concentration required to robustly flip the switch does not affect cell viability and therefore allows a long-term application of the system. These properties turn the tetracycline-dependent ribozymes into a very promising tool for conditional gene expression in mammalian cells.

The Expression of Antibiotic Resistance Methyltransferase Correlates with mRNA Stability Independently of Ribosome Stalling

“…It is possible that ERY prevents mRNA decay by altering the mRNA conformation through direct RNA binding. Although macrolide binding to other structured RNAs has not been reported, the direct binding of aminoglycosides to other viral RNAs, ribozymes, and synthetic riboswitches has been described (88–92). Alternatively, ERY might indirectly stabilize mRNA by influencing the expression of protein factors and regulatory sRNAs that are responsible for the activity and expression of RNase.…”

Rational design of aptazyme riboswitches for efficient control of gene expression in mammalian cells

“…Tc-P1 aptazyme Tc12 and Tc-P2 aptazyme Tc40 are compared to 9.19 (Wittmann and Suess, 2011) ( K ); Theo5 is compared to P1-F5 (Auslander et al, 2010) ( L ); and Gua3 aptazyme is compared to RzGuaM5 (Nomura et al, 2012) ( M ). ( N ) Tc40 is compared to a previously described aptazyme on-switch, 3’K19 (Beilstein et al, 2015). Data shown are representative of two or three independent experiments.…”

“…We evaluated three more validation panels: (1) aptazymes using the same tetracycline aptamer, but fused at a different aptamer stem (Tc-P2 aptazymes), (2) aptazymes constructed using a theophylline aptamer (Theo aptazymes), and (3) aptazymes constructed with a guanine aptamer (Gua aptazymes). To date, all reported Tc aptazymes are fused at the aptamer P1 stem (Win and Smolke, 2007; Wittmann and Suess, 2011; Beilstein et al, 2015), but the structure of the Tc aptamer suggests that Tc-binding might have a larger effect on the P2 stem than the P1 stem (Xiao et al, 2008). We therefore explored variants of our Tc aptazymes in which the ribozyme was fused to the P2 stem of the Tc aptamer (Tc-P2 aptazymes; Figure 3C) rather than the P1 stem (Tc-P1 aptazymes; Figure 3A).…”

“…In each case, our rationally designed aptazymes substantially outperformed these previously described aptazymes (Figure 3K–M, and Figure 3—figure supplement 2A–C). We also directly compared our Tc40 off-switch with a previously described Tc aptazyme on-switch, 3’K19 (Beilstein et al, 2015). Again Tc40 showed significantly higher CDR (19.6-fold at 100 µM Tc) than the CDR (6.6-fold at 100 µM Tc) of this previously described aptazyme (Figure 3N, and Figure 3—figure supplement 2D).…”

“…This aptazyme was functional only in cell-free systems. The discovery of tertiary interaction between stems I and II of the full-length hammerhead ribozyme (De la Peña et al, 2003; Khvorova et al, 2003; Martick and Scott, 2006) and the development of optimized hammerhead ribozymes efficient in mammalian cell culture and in vivo (Yen et al, 2004) led to the development of hammerhead ribozyme-based aptazymes functional in yeast (Win and Smolke, 2007; Wittmann and Suess, 2011; Klauser et al, 2015; Townshend et al, 2015), or mammalian cells (Kumar et al, 2009; Auslander et al, 2010; Nomura et al, 2012; Wei et al, 2013; Beilstein et al, 2015). These aptazymes have been tested for a range of applications, including construction of synthetic gene networks in yeast (Win and Smolke, 2008), and regulation of transgene expression (Ketzer et al, 2012) or virus replication (Ketzer et al, 2014) in mammalian cell culture.…”