Selection of tetracycline inducible self-cleaving ribozymes as synthetic devices for gene regulation in yeast

Wittmann, Alexander; Suess, Beatrix

doi:10.1039/c1mb05070b

Cited by 71 publications

(94 citation statements)

References 50 publications

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“…In this way, we designed several ribozyme constructs that differ in sequence, length, and stability of P1 (K3−K20, Figure 2A). Note, the strategy we used slightly differed from a design of a previous study in which we selected for tetracycline-dependent OFF switches (Supporting Information Figure S1, 4 ). However, we were not able to isolate any candidates with ON switch function using the old design (data not shown).…”

Section: ■ Results and Discussionmentioning

confidence: 98%

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

et al. 2014

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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.

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Section: ■ Results and Discussionmentioning

confidence: 98%

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

et al. 2014

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“…A recent bioinformatics inventory of HHR sequences revealed widespread abundance of HHRs in all kingdoms of life 25 . Naturally occurring ribozyme sequences have been successfully used for the design of ribozymes with novel properties [26][27][28][29][30] . Integration of small molecule-sensing aptamer structures rendered the self-cleavage activity of such ribozymes and corresponding target-gene translation trigger responsive [26][27][28][29][30] .…”

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confidence: 99%

“…Naturally occurring ribozyme sequences have been successfully used for the design of ribozymes with novel properties [26][27][28][29][30] . Integration of small molecule-sensing aptamer structures rendered the self-cleavage activity of such ribozymes and corresponding target-gene translation trigger responsive [26][27][28][29][30] . However, protein-responsive ribozymes have been validated only in vitro and could not be used to control target-gene expression in living cells 31,32 .…”

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A general design strategy for protein-responsive riboswitches in mammalian cells

et al. 2014

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RNAs are ideal for the design of gene switches that can monitor and program cellular behavior because of their high modularity and predictable structure-function relationship. We have assembled an expression platform with an embedded modular ribozyme scaffold that correlates self-cleavage activity of designer ribozymes with transgene translation in bacteria and mammalian cells. A design approach devised to screen ribozyme libraries in bacteria and validate variants with functional tertiary stem-loop structures in mammalian cells resulted in a designer ribozyme with a protein-binding nutR-boxB stem II and a selected matching stem I. In a mammalian expression context, this designer ribozyme exhibited dose-dependent translation control by the N-peptide, had rapid induction kinetics and could be combined with classic small molecule-responsive transcription control modalities to construct complex, programmable genetic circuits.

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“…Their application in mammalian systems, however, has been challenging. This is mostly because aptazymes selected in vitro, in bacteria, or in yeast showed loss or strong reduction of regulation when applied in mammalian cells (20,(22)(23)(24). Previous reports on aptazyme applications in mammalian cells were reporter gene studies or studies on regulation of therapeutic gene expression (17,23,25,26).…”

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confidence: 99%

“…Aptazymes have been extensively developed and studied in vitro, in bacteria, and in yeast (6,12,13,(19)(20)(21)(22). Their application in mammalian systems, however, has been challenging.…”

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Artificial riboswitches for gene expression and replication control of DNA and RNA viruses

Ketzer

Kaufmann

Engelhardt

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

100

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Aptazymes are small, ligand-dependent self-cleaving ribozymes that function independently of transcription factors and can be customized for induction by various small molecules. Here, we introduce these artificial riboswitches for regulation of DNA and RNA viruses. We hypothesize that they represent universally applicable tools for studying viral gene functions and for applications as a safety switch for oncolytic and live vaccine viruses. Our study shows that the insertion of artificial aptazymes into the adenoviral immediate early gene E1A enables small-molecule-triggered, dosedependent inhibition of gene expression. Aptazyme-mediated shutdown of E1A expression translates into inhibition of adenoviral genome replication, infectious particle production, and cytotoxicity/ oncolysis. These results provide proof of concept for the aptazyme approach for effective control of biological outcomes in eukaryotic systems, specifically in virus infections. Importantly, we also demonstrate aptazyme-dependent regulation of measles virus fusion protein expression, translating into potent reduction of progeny infectivity and virus spread. This not only establishes functionality of aptazymes in fully cytoplasmic genetic systems, but also implicates general feasibility of this strategy for application in viruses with either DNA or RNA genomes. Our study implies that gene regulation by artificial riboswitches may be an appealing alternative to Tet-and other protein-dependent gene regulation systems, based on their small size, RNA-intrinsic mode of action, and flexibility of the inducing molecule. Future applications range from gene analysis in basic research to medicine, for example as a safety switch for new generations of efficiency-enhanced oncolytic viruses.

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Selection of tetracycline inducible self-cleaving ribozymes as synthetic devices for gene regulation in yeast

Cited by 71 publications

References 50 publications

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

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

A general design strategy for protein-responsive riboswitches in mammalian cells

Artificial riboswitches for gene expression and replication control of DNA and RNA viruses

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