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ABSTRACT: In cellular systems environmental and metabolic signals are integrated for the conditional control of gene expression. On the other hand, artificial manipulation of gene expression is of high interest for metabolic and genetic engineering. Especially the reprogramming of gene expression patterns to orchestrate cellular responses in a predictable fashion is considered to be of great importance. Here we introduce a highly modular RNA-based system for performing Boolean logic computation at a post-transcriptional level in Escherichia coli. We have previously shown that artificial riboswitches can be constructed by utilizing ligand-dependent Hammerhead ribozymes (aptazymes). Employing RNA self-cleavage as the expression platform-mechanism of an artificial riboswitch has the advantage that it can be applied to control several classes of RNAs such as mRNAs, tRNAs, and rRNAs. Due to the highly modular and orthogonal nature of these switches it is possible to combine aptazyme regulation of activating a suppressor tRNA with the regulation of mRNA translation initiation. The different RNA classes can be controlled individually by using distinct aptamers for individual RNA switches. Boolean logic devices are assembled by combining such switches in order to act on the expression of a single mRNA. In order to demonstrate the high modularity, a series of two-input Boolean logic operators were constructed. For this purpose, we expanded our aptazyme toolbox with switches comprising novel behaviours with respect to the small molecule triggers thiamine pyrophosphate (TPP) and theophylline. Then, individual switches were combined to yield AND, NOR, and ANDNOT gates. This study demonstrates that post-transcriptional aptazyme-based switches represent versatile tools for engineering advanced genetic devices and circuits without the need for regulatory protein cofactors.
Molecular BioSystems 07/2012; 8(9):2242-8. · 3.53 Impact Factor
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ABSTRACT: The utilization of toehold-containing DNA strands allows for the assembly of complex nanostructures via kinetically driven hybridization reactions. Here, we have rendered this strategy ligand-dependent, resulting in small-molecule-inducible DNA nanoarchitectures.
Chemical Communications 03/2010; 46(11):1866-8. · 6.17 Impact Factor
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ABSTRACT: Developing artificial genetic switches in order to control gene expression via an external stimulus is an important aim in chemical and synthetic biology. Here, we expand the application range of RNA switches to the regulation of 16S rRNA function in Escherichia coli. For this purpose, we incorporated hammerhead ribozymes at several positions into orthogonalized 16S rRNA. We observed that ribosomal function is remarkably tolerant toward the incorporation of large additional RNA fragments at certain sites of the 16S rRNA. However, ribozyme-mediated cleavage results in severe reduction of 16S rRNA stability. We carried out an in vivo screen for the identification of sequences acting as ligand-responsive RNA switches, enabling thiamine-dependent switching of 16S rRNA function. In addition to expanding the regulatory toolbox, the presented artificial riboswitches should prove valuable to study aspects of rRNA folding and stability in bacteria.
Chemistry & biology 03/2010; 17(3):236-42. · 6.52 Impact Factor
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Angewandte Chemie International Edition 10/2009; 48(41):7564-7. · 13.45 Impact Factor
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ABSTRACT: Four-stranded DNA and RNA quadruplexes or G4 motifs are non-B DNA conformations that are presumed to form in vivo, although only few explicit evidence has been reported. Using bioinformatics the presence of putative DNA G-quadruplexes within critical promoter regions has been demonstrated and a regulatory role in transcription has been suspected. However, in genomic DNA the presence of the complementary strand interferes with the potential to form a quadruplex motif. Contrarily RNA G4 motifs have no such limitation and consequently strong interference with gene expression is suspected. Nevertheless, experimental evidence is scarce. Here we show a well-defined structure-function relationship of synthetic quadruplex sequences in 5'-UTRs in multiple mammalian cell-lines. We establish a universal 'translational suppressor' effect of these motifs on gene expression at the translational level and show for the first time that specific features such as loop-length and the number of 'GGG'-repeats further determine the suppressive impact. Moreover, a consistent and predictable repression of gene expression is observed for naturally occurring RNA G4 motifs, augmenting the functional relevance of these unusual nucleic acid structures.
Nucleic Acids Research 10/2009; 37(20):6811-7. · 8.03 Impact Factor
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ABSTRACT: Recently, hammerhead ribozyme (HHR) motifs have been utilized as powerful tools for gene regulation. Here we present a novel design of expanded full-length HHRs that allows attaching additional functionalities to the ribozyme. These features allowed us to construct a very efficient artificial riboswitch in bacteria. Following the design of naturally occurring three-way junctions we attached an additional helix (IV) to stem I of the HHR while maintaining very fast cleavage rates. We found that the cleavage activity strongly depends on the exact design of the junction site. Incorporation of the novel ribozyme scaffold into a bacterial mRNA allowed the control of gene expression mediated by autocatalytic cleavage of the ribozyme. Appending an aptamer to the newly introduced stem enabled the identification of very powerful theophylline-inducible RNA switches by in vivo screening. Further investigations revealed a cascading system operating beyond the ribozyme-dependent mechanism. In conclusion, we extended the hammerhead toolbox for synthetic biology applications by providing an additional position for the attachment of regulatory modules for in vivo control of gene expression.
RNA 04/2009; 15(5):968-76. · 5.09 Impact Factor
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ABSTRACT: RNA Lego: The use of natural riboswitch aptamers in synthetic RNA switches (see picture) should broaden the scope of artificial RNA regulators dramatically. It is shown that thiamine pyrophosphate (TPP) aptamers can be used in engineered devices as very sensitive switches of gene expression in unmodified organisms. The approach demonstrates that intrinsic metabolites can be utilized as external effectors of cellular functions.
Angewandte Chemie International Edition 02/2009; 48(15):2715-8. · 13.45 Impact Factor
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ABSTRACT: The protocol presented here allows for the investigation of the formation of unusual nucleic acid structures in the 5'-untranslated region (UTR) of bacteria by correlating gene expression levels to the in vitro stability of the respective structure. In particular, we describe the introduction of G-quadruplex forming sequences close to the ribosome-binding site (RBS) on the mRNA of a reporter gene and the subsequent read-out of the expression levels. Insertion of a stable secondary structure results in the cloaking of RBS and eventually reduced gene expression levels. The structures and stability of the introduced sequences are further characterized by circular dichroism (CD) spectroscopy and thermal melting experiments. The extent of inhibition is then correlated to the stability of the respective quadruplex structure, allowing judgement of whether factors other than thermodynamic stability affect the formation of a given quadruplex sequence in vivo. Measuring gene expression levels takes 2 d including cloning; CD experiments take 5 hours per experiment.
Nature Protocol 01/2009; 4(11):1632-40. · 8.36 Impact Factor
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ChemBioChem 08/2008; 9(12):1873-8. · 3.94 Impact Factor
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ChemBioChem 06/2008; 9(7):1061-4. · 3.94 Impact Factor
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Angewandte Chemie International Edition 02/2008; 47(14):2604-7. · 13.45 Impact Factor
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ABSTRACT: RNA-based modules such as riboswitches represent straightforward and simplified approaches for the regulation of gene expression, as no additional proteins are needed. G-rich sequences are known to adopt stable four-stranded structures, and such quadruplexes have been suspected to play important roles in key functions such as the control of gene expression. Here we demonstrate that RNA quadruplexes readily form in vivo. We have constructed mRNA-based G-rich elements that mask the ribosome binding site by folding into four-stranded structures. The suppression of gene expression correlates with the stability of inserted G quadruplexes. Moreover, quadruplexes with moderate stability respond to changes in temperature, thus behaving as artificial RNA thermometers. In conclusion, we introduce tuneable mRNA-based devices that enable modulation of gene expression by a predictable but thus far unknown mechanism.
Chemistry & Biology 08/2007; 14(7):757-63. · 5.83 Impact Factor
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Angewandte Chemie International Edition 10/2006; 45(35):5875-8. · 13.45 Impact Factor
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Angewandte Chemie 07/2006; 118(35):6007 - 6010.
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First publ. in: ChemBioChem 9 (2008), 9, pp. 1061-1064.
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Publ. in: Angewandte Chemie International Edition 47 (2008), 14, pp. 2604-2607.
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ABSTRACT: Genetic RNA switches: An overview of the different concepts that are based on the insertion of ligand-sensing elements into mRNAs that enable the regulation of expression of the respective message is given.
Publ. in: ChemBioChem 9 (2008), 12, pp. 1873-1878.
