Synthetic riboswitches - A tool comes of age.

BBAGRM-00727; No. of pages: 10; 4C: 2, 4, 6, 7, 8 Biochimica et Biophysica Acta xxx (2014) xxx–xxx

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Review

Synthetic riboswitches — A tool comes of age☆ Florian Groher, Beatrix Suess ⁎ Department of Biology, Technical University Darmstadt, 64287 Darmstadt, Germany

a r t i c l e

i n f o

Article history: Received 11 February 2014 Received in revised form 29 April 2014 Accepted 8 May 2014 Available online xxxx Keywords: Synthetic biology Engineered riboswitch Aptamer Theophylline Tetracycline Ribozyme

a b s t r a c t Within the last decade, it has become obvious that RNA plays an important role in regulating gene expression. This has led to a plethora of approaches aiming at exploiting the outstanding chemical properties of RNA to develop synthetic RNA regulators for conditional gene expression systems. Consequently, many different regulators have been developed to act on various stages of gene expression. They can be engineered to respond to almost any ligand of choice and are, therefore, of great interest for applications in synthetic biology. This review presents an overview of such engineered riboswitches, discusses their applicability and points out recent trends in their development. This article is part of a Special Issue entitled: Riboswitches. © 2014 Elsevier B.V. All rights reserved.

1. Introduction The functional role of RNA had long been thought to be restricted to transferring genetic information from DNA to protein. However, the emerging awareness that RNA plays major roles in gene control and catalysis of chemical reactions has changed this perception fundamentally. In many ways, RNA is more akin to proteins than to the chemically closely-related DNA. Like proteins, RNA can adopt complex threedimensional structures which present chemical moieties precisely — an essential property for it to function as a biological catalyst, regulator or structural scaffold. The last decade has seen the discovery of many different types of regulatory RNA. Short and long non-coding RNAs have been shown to function as integral regulatory parts of many cellular processes in both bacteria and many eukaryotes. RNA silencing was first recognized as defensive mechanisms protecting plants against RNA viruses or preventing random integration of transposable elements [1]. Today it is known that miRNAs and other small non-coding RNAs have diverse expression patterns and regulate various developmental and physiological processes in plants and animals (reviewed in [2]). Small noncoding RNAs that act as regulators of translation and message stability have also been identified in bacteria. They are involved in the regulation of replication, the maintenance of prokaryotic extrachromosomal elements and in bacterial responses to changing environments

(reviewed in [3]). Although protein factors are often essential for these critical regulatory tasks, the initial trigger is always RNA. Riboswitches represent a further class of genetic regulatory elements that are widely distributed throughout the bacterial world. RNA sequence elements located in the 5′ untranslated region of an mRNA serve as a molecular switch that modulates transcription, translation or mRNA processing through conformational changes of the RNA structure prompted by direct interaction with a specific cellular metabolite. These riboswitches control numerous basic metabolic pathways in prokaryotes (reviewed in [4]). They consist solely of RNA, sense their ligand in a preformed binding pocket and undergo restructuring upon metabolite binding, whereby one of the two conformations efficiently interferes with gene expression. The specific characteristic of riboswitches is that RNA accomplishes both sensor and effector functions, demonstrating that a protein cofactor is not an obligate requirement for regulation. Based on the principles of riboswitch regulation, a versatile set of synthetic riboswitches has been engineered by combining RNA-based sensing domains with regulatory domains. In this review, we give an overview of these engineered riboswitches and their application in conditional gene expression. We provide insights into their mechanisms and consider problems and future perspectives. 2. Engineered riboswitches in bacteria 2.1. Control of ribosome binding

☆ This article is part of a Special Issue entitled: Riboswitches. ⁎ Corresponding author. E-mail address: [email protected] (B. Suess).

Controlling translation initiation by changing the accessibility of the ribosome binding site (RBS) is one of the two major mechanisms

http://dx.doi.org/10.1016/j.bbagrm.2014.05.005 1874-9399/© 2014 Elsevier B.V. All rights reserved.

Please cite this article as: F. Groher, B. Suess, Synthetic riboswitches — A tool comes of age, Biochim. Biophys. Acta (2014), http://dx.doi.org/ 10.1016/j.bbagrm.2014.05.005

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F. Groher, B. Suess / Biochimica et Biophysica Acta xxx (2014) xxx–xxx

employed by bacterial riboswitches. Ligand-induced conformational changes lead to either sequestration or release of the RBS. Based on this mechanism, Gallivan and coworkers developed theophyllinedependent riboswitches (Fig. 1A). They placed a theophylline aptamer [5] close to the RBS of a reporter gene and flanked the aptamer with a limited number of randomized positions. In a subsequent screen of 106 different clones, using the cat gene which mediates resistance against the antibiotic chloramphenicol, they looked for candidates that can adopt two conformations: one in which the RBS is sequestered and an alternative conformation in which aptamer formation frees the RBS. [6]. This approach resulted in riboswitch sequences which displayed an up to 11-fold increase in reporter activity upon addition of ligand. The riboswitches were then applied to control chemotaxis in Escherichia coli. The flagellum can rotate clockwise or counterclockwise, thus causing pausing or movement, respectively. The duration of the migration phase is controlled by the dephosphorylation of the CheY-P protein through CheZ [7]. Topp & Gallivan inserted the theophylline riboswitch upstream of the RBS of cheZ gene. Addition of theophylline led not only to ligand-dependent cell migration, but also to a functioning chemotaxis allowing the cell to migrate toward the ligand [8]. Using rational design, the Gallivan group then developed a set of riboswitches adjusted to different RBS sequences, enabling them to control gene expression in various Gram-positive and Gramnegative bacteria. The general applicability of these switches was then widely demonstrated [9]. For example, in Mycobacterium tuberculosis the theophylline riboswitch was used either to control reporter gene expression or to create a conditional gene knockdown of katG, which encodes a catalase-peroxidase required for converting the prodrug isoniazid into its active form. Control of gene expression was demonstrated in a macrophage infection model [10]. Furthermore, riboswitchcontrolled virulence factors were analyzed in Francisella novicida [11, 12]. Using reporter gene assays, about 200-fold induction of gene expression was obtained in Synechococcus elongatus, a model organism for photosynthesis [13], and in Streptomyces coelicolor, a model organism for GC-rich Gram-positive bacteria [14]. With regard to all currently known examples, these riboswitches from the Gallivan lab can be considered to be among the most robust and universally applicable. We used a slightly different approach to control ribosomal access in Bacillus subtilis. The theophylline aptamer was fused to a helical slippage element and inserted close to the RBS of the xylR gene. Ligand binding

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leads to a slippage of the helical element, removing the aptamer from the vicinity of the RBS. In a dose-dependent manner, reporter gene activity increased up to 8-fold [15]. Yokobayashi and coworkers used a dual genetic selection system to screen for riboswitches in which a pseudo-Shine-Dalgarno sequence was placed near or integrated into the aptamer. The switches they obtained reduced gene expression 10fold in E. coli [16]. 2.2. Control of antitermination Some of the most remarkable work in the last years came from the Mörl lab [17]. They engineered a riboswitch to regulate transcription termination in E. coli. It is based on the theophylline aptamer as sensor domain followed by a spacer sequence complementary to the 3′-part of the aptamer and a poly-U stretch. Calculating length and sequence of the spacer and the complementary stretch by using inverse folding algorithms [18], they generated a construct in which the stem-loop is formed and transcription is terminated in the absence of theophylline, whereas the presence of the ligand stabilized the aptamer so that no terminator was formed, allowing transcription to continue past the termination site. This approach made it possible to regulate gene expression of two different reporter genes in E. coli roughly 6.5-fold. The only drawback of the method is the need for a well-characterized aptamer domain to be able to perform the necessary calculations for construct design (Fig. 1B). Another exciting approach came from the Batey lab. They designed new riboswitches by using the expression platform of a natural transcriptional riboswitch, coupling it with a diverse set of aptamers, derived either from natural riboswitches, or from in vitro selected ones, such as the theophylline and tetracycline aptamers [19,20]. Their switches display high modularity and should therefore be easily and widely applicable. 2.3. Control of gene expression by RNA expressed in trans One of the earliest examples of an engineered riboswitch is a simple and elegant genetic device that relies on the interaction of a “cis-repressor” with a “trans-activator” RNA (crRNA and taRNA, respectively) [21]. The crRNA is an RNA sequence located upstream of the RBS that sequesters it through Watson–Crick base pairing, thereby inhibiting protein translation. The stem-loop structure is

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Fig. 1. Common mechanisms of engineered riboswitches in bacteria. (A) Regulation of translation initiation: In the absence of ligand, a stem-loop structure is formed between the aptamer domain (depicted in blue) and a sequence element complementary to the Shine-Dalgarno (SD) sequence (red). Thus, the SD sequence (green) is accessible for 30S binding and translation initiation occurs. As a consequence of ligand binding (orange pentagon) and folding of the aptamer domain, an alternative stem-loop is formed which sequesters the SD sequence, blocking the binding of the 30S ribosomal subunit. (B) Regulation of transcription termination: The aptamer domain is fused to a short spacer region (gray), followed by a sequence complementary to the 3′ part of the aptamer (red) and a U stretch. In the absence of ligand, the complementary 3′ part is base-paired with the aptamer forming a terminator structure; thus, RNA polymerase (RNAP) dissociates and transcription are blocked. Upon ligand binding, terminator structure formation is inhibited and transcription can proceed, resulting in expression of the reporter gene. (C) Regulation of translation initiation with trans-expressed RNAs: A small sequence (red, “cis repressor” crRNA), complementary to the SD sequence, is inserted upstream of the SD sequence via a short linker (gray). After transcription, a stem-loop is formed at the 5′ end of the mRNA, which blocks ribosome binding and translation initiation. Overexpression of a trans-activating RNA (taRNA) which targets the crRNA releases the SD and translation initiation can occur. (D) Similar to the regulation mechanism presented in (B), a heterologously expressed taRNA, complementary to the terminator sequence, leads to the formation of an antiterminator and transcription can occur.



then unwound by the trans expressed taRNA, which targets the crRNA with high specificity. This disrupts the stem-loop structure, releasing the RBS and, thus, activating gene expression (Fig. 1C). This riboswitch has several features favorable to system implementation, including a very simple design, tunability of the quantitative regulatory response through the modulation of the thermodynamic binding properties of the cr and taRNAs, and the ability to simultaneously regulate multiple genes with the same taRNA. These benefits have supported the use of these devices in more complex genetic systems, such as a genetically-encoded counter in E. coli [22]. This counter is able to integrate up to three induction events and is based on either a riboregulated transcriptional cascade or a recombinase-based cascade of memory units. Further applications include a GFP fusion protein tracking system, and a bacterial kill switch that integrates multiple orthogonal inputs before triggering [23]. Isambert and coworkers designed a set of trans-expressed antisense RNAs that activate and repress transcription by having their association interfere with an antiterminator or a terminator structure, respectively (Fig. 1D) [24]. Based on this approach, the Arkin group developed another series of synthetic trans-expressed riboregulators [25]. However, these exploited a natural system from the Staphylococcus aureus plasmid pT181 operating by transcriptional attenuation. Two orthogonal antisense RNAs expressed in trans were designed to bind to the attenuator sequence, so that the interaction between attenuator and trans acting RNA controls transcription. In one approach, the devices were implemented simultaneously to independently control the expression of two fluorescent reporter genes in the same cell. In a second approach, they were daisy-chained on the same transcript to have transcription control of this gene respond to two inputs. This resulted in a NORgate expression pattern, in which the presence of any antisense RNA signal leads to downregulation of target gene expression. In a third approach, the authors constructed a transcriptional cascade containing two attenuator-antisense pairs. The transcription of the first antisense RNA, regulating expression of the reporter gene, was placed under the control of a second, orthogonal antisenseattenuator pair, so that the input of the second antisense regulatory signal was propagated through a double inverter, resulting in the transcription of the reporter gene. In a follow-up study, Takahashi and Luchs created an even larger set of mutually orthogonal regulators [26]. An interesting improvement of the system involved coupling the theophylline aptamer to one of the trans-expressed RNAs in a pseudoknot-like manner to downregulate gene expression upon ligand binding [27]. This strategy features high modularity between ligandsensing aptamer and the antisense RNA target-recognition motif. It allows the easy engineering of orthogonal pairs, so that the same cell can regulate different target genes in response to different ligands. The authors connected their antisense RNA to different sensing domains, resulting in a NOR gate that downregulates gene expression in the presence of any of the specific ligands. Finally, the Jaramillo group developed a computational algorithm based on theoretical principles and combinatorial optimization to design trans-activating RNA sequences [28]. They concluded that only the formation energy and the activation energy are sufficient to engineer such antisense based regulators, making them an easily applicable tool. Their trans-activating RNAs worked independently from each other and also in combination with transcription factors, permitting the design of even more complex logic gates. Taken together, like their natural counterparts the majority of riboswitches engineered for use in bacteria control either translation initiation (by sequestering the RBS) or transcription termination. Signal input can occur either by a cis element (integration of a small molecule-binding aptamer within the mRNA) or by expressing an antisense RNA in trans. Most of the currently published molecular devices have proven their orthogonality and universal applicability, allowing the integration of manifold user-defined inputs. Therefore,

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in bacteria engineered riboswitches can now be considered a serious working alternative to the established protein-based conditional gene expression systems. 3. Controlling gene expression in eukaryotes 3.1. Control of cap-dependent translation initiation That small molecule binding aptamers can be used to regulate gene expression in eukaryotic cells has also been demonstrated. Inserted into the 5′UTR of an mRNA, they can interfere with the initial steps of translation. This was first shown by Werstuck and Green who selected aptamers which specifically bound a Hoechst dye. Two copies of the aptamer were inserted into the 5′UTR of a luciferase reporter gene [29]. In the presence of 10 mM dye, luciferase activity was 10-fold reduced in Chinese hamster ovary cells. Despite the rather high ligand concentration, this convincingly provided the proof of concept for small molecule-aptamer controlled gene expression (Fig. 2A). A similar approach for inhibiting translation initiation in the yeast Saccharomyces cerevisiae was reported by Grate and Wilson [30]. They inserted an in vitro selected aptamer against malachite green into the 5′UTR of the CLB2 cyclin gene directly upstream of the start codon. Gene expression was inhibited 10-fold after the addition of the malachite green analog tetramethylrosamine (TMR). As a result, progression through the cell cycle was slowed down and the cell morphology was affected. These findings confirm that this method can interfere with cellular processes and therefore serve as a genetic tool for investigating cellular pathways. We identified a tetracycline-binding aptamer capable of controlling translation in yeast [31,32]. When inserted into the 5′UTR of several reporter genes, the aptamer led to reversible and dose-dependent repression of reporter gene expression [33]. Insertion of the aptamer directly downstream of the cap structure prevents binding of the small ribosomal subunit to the cap structure once the tetracyclineaptamer-complex is formed. If the aptamer is instead located close to the AUG start codon, in vitro translation and subsequent sucrose gradient analysis demonstrated that the ligand-bound form of the aptamer interfered with the formation of the 80S ribosome, presumably by blocking scanning (Fig. 2A, B). [33]. The efficiency of regulation was position-dependent, with cap proximal aptamers being less active than cap distal ones. Inserting three copies of the aptamer resulted in up to 40-fold regulation. Starting with this construct, we developed a simple and powerful PCR-based strategy which allows the easy tagging of any target gene in yeast. The target gene expression window can be further adjusted by using promoters with different expression strength [34]. The flexibility of regulation was further increased after a neomycin binding aptamer which mediates conditional gene expression in yeast with comparable efficiency to the tetracycline aptamer was identified by a combined approach of in vitro selection and in vivo screening [35]. A detailed structure-function analyses of both tetracycline [36,37] and neomycin aptamers [38–40] gave first insight into why these aptamers can be used for engineering riboswitches, but many other aptamers failed. We have included our thoughts regarding the functionality of these aptamers later in this review. Pelletier and coworkers present another example for aptamer-based control of gene expression in eukaryotes [41]. They obtained 10-fold inhibition of a cat reporter construct both in vitro in wheat germ extracts and in vivo in Xenopus oocytes by inserting one to three copies of the biotin [42] or theophylline aptamer into the 5′UTR [5]. Here too, regulatory efficiency increased with the aptamer copy number. Interestingly, the cap proximal insertions were more active than the cap distal ones, in contrast to the results obtained in yeast [33]. The results are consistent with data obtained from inserting stem-loop structures, which inhibit translation, into the 5′UTR. In higher eukaryotes, stem-loop structures are more inhibitory when located in a cap-proximal position


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[43], whereas in yeast there are only minor differences in the inhibitory capacity of the stem-loop structure at either position [44,45], suggesting mechanistic differences between yeast and other eukaryotes. Smolke and coworkers showed that translation initiation could also be regulated by small trans-acting RNAs, similar to the bacterial systems described above [46]. They attached the theophylline aptamer to a small RNA which was complementary to a sequence element from the 5′UTR of an mRNA. Upon ligand binding, the sequence complementary to the 5′UTR of this so called “antiswitch” was set free, and its subsequent annealing to the mRNA resulted in antisense-mediated inhibition of reporter gene expression in S. cerevisiae. 3.2. Control of IRES-mediated translation initiation Translation initiation mediated by an internal ribosome entry site (IRES) can also be controlled externally (Fig. 2C). A fully rational design strategy was applied to engineer an synthetic riboswitch, once again making use of the theophylline aptamer [47]. In vitro translation via the IRES element was promoted only in the presence of the ligand, with 10-fold induction of reporter gene activity. Interestingly in terms of application, the OFF-state of the system was extremely low. The theophylline aptamer could be replaced by several other aptamers including the FMN, the sulforhodamine and the tetracycline aptamer, which all worked with comparable efficiency. 3.3. Control of ribosomal shunting A completely new approach to control gene expression was presented by Ogawa in 2013 [48]. He placed the theophylline aptamer between a short open reading frame (sORF) and a downstream ORF (dORF) to control ribosomal shunting, rather then scanning, to turn on downstream gene expression. Ligand binding led to stabilization of the aptamer, allowing ribosomal shunting, and resulting in a 10-fold increase in reporter gene activity in an in vitro translation assay. Similar efficiencies were achieved using the TMR aptamer (Fig. 2D). 3.4. Control of splicing The programmed removal of intronic sequences or the influence on alternative splicing by synthetic riboswitches offers an additional layer of controlling gene expression. By modulating the accessibility of essential splicing elements, such as the 5′ splice site, the branch point or the 3′ splice site, engineered riboswitches have been shown to control both constitutive and alternative splicing. Gaur and coworkers placed the theophylline aptamer close to the 3′ splice site and observed a 4-fold reduction of gene expression upon the addition of theophylline in an in vitro splicing assay [49]. Their data indicated that theophylline specifically blocked recognition of the 3′ splice site. The resulting intron retention, however, led to rapid mRNA degradation. The aptamer was also used to modulate splicing efficiency in HeLa cells by inclusion of the branch point sequence into the aptamer [50]. In the presence of theophylline, the downstream exon was skipped two-fold more often than in its absence, indicating that engineered riboswitches can also be used to modulate and thereby help to investigate the impact of alternative splicing.

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We used the tetracycline aptamer to regulate pre-mRNA splicing in yeast [51] (Fig. 2e). The aptamer was inserted into a yeast intron in close proximity to either the 5′ splice site or the branch point. Maximal regulation was observed with a construct in which the 5′ splice site was masked by intramolecular base pairing with the closing stem of the aptamer. This probably blocked the U1-snRNP from accessing the 5′ splice site. The dynamic range of regulation was increased up to 16fold when a stabilized minimal version of the aptamer was used instead [52]. Insertion of a second aptamer-containing intron further increased the dynamic range up to 32-fold. Interestingly, no regulatory activity was observed when the aptamer was inserted close to the branch point, which is in contrast to the results obtained in HeLa cells [50]. 4. Conditional control of gene expression by allosterically controlled ribozymes Research on natural riboswitches has shown that direct interaction of intracellular metabolites with RNA sequences can affect gene expression through RNA self-cleavage [53]. The first example of an allosterically regulated self-cleaving ribozyme, also sometimes referred to as aptazyme, being controlled by a small molecule was developed by the Breaker group [54]. They fused an ATP aptamer to a minimal hammerhead ribozyme by rational design. Depending on the linker sequence between aptamer and ribozyme (the so called communication module), they observed that hammerhead cleavage could be induced or inhibited allosterically. The combination of rational design and selection yielded two classes of communication modules, one able to trigger activation and the other for the inhibition of hammerhead cleavage [55]. In further studies, they used their communication modules and selected for new ligand specificities using a randomized aptamer domain [56]. Regardless of these and other promising studies, ribozymes based on the minimal hammerhead ribozyme turned out to be unsuitable for cellular application. The minimal variant lacks sequences mediating tertiary loop–loop interactions, an absolute requirement for proper folding and, hence, catalysis at physiological magnesium concentrations [57, 58] (Fig. 3A). Using an optimized, full-length hammerhead ribozyme from Schistosoma mansonii, Mulligan and co-worker were able to reduce reporter gene expression in mice. Mutagenesis studies and correct positioning of the ribozyme directly upstream of the translational start site resulted in a remarkable decrease in gene expression [59]. A highthroughput approach screening over 50,000 compounds identified several nucleoside analogs which led to reduced gene expression. Mechanistic studies, however, indicated that the compounds were incorporated into the RNA, thereby inhibiting RNA self-cleaving [60]. The resulting cytotoxic effects render the selected inhibitors inapplicable. Most attempts to subsequently develop allosteric hammerhead ribozymes, however, have made use of the full-length hammerhead variant N79 from this initial study by Yen and Mulligan. Several independent approaches were undertaken to regulate gene expression by ribozymes both in bacteria and eukaryotes. Hartig and co-workers first focused their efforts on bacteria. They sequestered the bacterial RBS by integrating it into the hammerhead fold. Ribozyme cleavage releases the SD sequence and translation initiation can occur. Coupling the theophylline aptamer to stem III of the ribozyme via a randomized linker (Fig. 3A, B), followed by in vivo screening, resulted in an optimized communication module that triggered hammerhead

Fig. 2. Common mechanisms of engineered riboswitches in eukaryotes. (A) Regulation of small ribosomal subunit binding: In the absence of ligand, the tertiary structure of the aptamer domain, which is located in the 5′UTR of an mRNA, is not formed, allowing the 40S subunit to bind the cap of the mRNA (black circle). In the presence of ligand, the aptamer domain is correctly folded and ribosomal subunit binding and translation initiation is blocked. (B) Regulation of ribosome scanning: After insertion of the aptamer domain in a cap-distal position, ligand binding prevents ribosomal scanning and, consequently, translation initiation. (C) Regulation of IRES-mediated translation: In the absence of a ligand, a modulator sequence (MS, light gray) hybridizes with the 3′ part of an anti-anti-IRES sequence (aaIRES, dark gray) and with a 5′ part of the aptamer domain to form a stable stem-loop structure, inducing anti-IRES/IRES duplex formation and thus inhibiting IRES-mediated translation. Upon ligand binding, the MS sequence is released from the aptamer and the aaIRES, and the aaIRES/aIRES duplex forms, allowing PK-III (green) formation to occur, thus promoting IRES-mediated translation. (D) Regulation of ribosomal shunting: An aptamer domain is inserted between a short open reading frame (sORF) and a downstream ORF (dORF). Ligand binding induces the formation of a rigid stem structure that allows the ribosomal subunit, after translating the sORF, to bypass, i.e., shunt over, this stem to the landing site (LS) and reinitiate translation of the downstream ORF. (E) Regulation of pre-mRNA splicing: An aptamer is integrated into an intron of a eukaryotic mRNA to control the accessibility of essential splice elements, like the 5′ splice site (5′SS), the branch point (BP) or the 3′ splice site (3′SS). Ligand binding either inhibits splicing or enhances exon skipping.


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Fig. 3. Allosterically controlled ribozymes. (A) Secondary structure of the Schistosoma mansonii hammerhead ribozyme. It consists of three stems connected by joining regions. Conserved nucleotides are represented in red and the cleavage site is marked by a red arrow. Loop I (yellow) and loop II (green) influence folding and cleavage by loop-loop interactions. Every stem in this ribozmye has been exchanged by different research groups with an aptamer domain to control gene expression in a ligand-dependent manner (see text). (B) Control of gene expression with catalytic riboswitches: A self-cleaving aptazyme is inserted into the 5′- or 3′-UTR of a eukaryotic mRNA. Ligand-induced self-cleavage reduces mRNA stability and triggers RNA degradation via exoribonucleases (gray pacman). In bacteria, catalytic riboswitches are used to liberate a sequestered SD sequence to induce translation initiation.

cleavage allosterically [61]. Functional switches were also obtained by exchanging the theophylline aptamer against the aptamer domain of the TPP riboswitch demonstrating the modularity of these switches. Furthermore, hammerhead-based riboswitches have been used to control not only mRNA, but also tRNA [62], rRNA [63] and RNAi (see below). Fortunately, the allosteric ribozymes selected for bacterial application turned out to regulate gene expression in mammalian reporter gene systems, too [64,65]. Using a similar design, the Fussenegger group performed fluorescence activated cell sorting (FACS) and identified further ribozymes active in bacteria and mammalian cells [66]. Coupling of the theophylline aptamer via a randomized linker to stem I was performed in the Breaker lab (Fig. 3A). The underlying idea was to destroy the loop–loop interaction with stem II upon ligand binding. In vitro selection with physiological magnesium concentrations resulted in high speed allosteric ribozymes whose cleavage activity was induced by the ligand [67]. We used the same design strategy and selected tetracycline inducible hammerhead ribozymes. The switches that were isolated showed remarkable cleavage characteristics in vitro, but only moderate activity in yeast [68]. By rational design, we recently developed a second set of ribozymes. Cleavage is inhibited in the presence of only 50 μM tetracycline, resulting in 5-fold induction of gene expression (unpublished data). Using this approach, we combined fast hammerhead cleavage and a ligand concentration with no toxic effects on cell viability. Smolke and co-workers combined the theophylline aptamer by rational design with the tobacco ringspot virus hammerhead ribozyme via stem II and obtained both ligand-dependent induction and inhibition of reporter gene expression in yeast [69] (Fig. 3A). The modularity of the system was shown by exchanging the theophylline against the tetracycline aptamer. Additionally, coupling two aptamers to a hammerhead ribozyme or combining two ribozymes allowed the construction of simple logical gates that converted two input signals into one output signal [70]. Proliferation control in primary human T-cells was effective if several copies of the same aptazyme were incorporated into the 3′UTR of the transgene. The moderate switching properties of the aptazyme were compensated by signal amplification controlling IL-2 expression [71].

An alternative approach was reported by the Yokobayashi group who used the Hepatitis Delta Virus ribozyme. They replaced the P4-L4 stem-loop by the theophylline aptamer resulting in moderate regulation. However, the use of the aptamer domain from a guanine riboswitch increased regulation dramatically up to 30-fold [72]. Another highly interesting example is the temperature-sensitive hammerhead ribozymes called thermozymes [73]. There, the hammerhead ribozyme was fused to a FourU-type RNA thermometer from Salmonella enterica. Both elements were linked by a short randomized region functioning as communication module. Using in vivo selection, thermozymes with switching capabilities of about 5-fold and good in vitro cleavage kinetics were obtained. Most remarkable is the inversed temperature behavior in contrast to the wildtype RNA thermometer [74]. 5. Control of the RNAi pathway by engineered riboswitches In 1990, the discovery of the RNA-silencing mechanism known as RNAi drew attention to the importance of postranscriptional gene regulation in mammals [1]. The RNAi pathway processes RNA transcripts which are either endogenously expressed (miRNA) or supplied as exogenous substrates (siRNA) in two enzymatic steps. These steps are catalyzed by Drosha (cleavage of pri-miRNA in the nucleus), followed by cytoplasmatic cleavage through Dicer, resulting in small regulatory RNAs, 21–23 nt in length. The small RNAs are then incorporated into the RISC complex to silence gene expression by either mRNA degradation or translational repression. The degree of sequence complementarity between the small RNA and its target mRNA determines whether the mRNA is degraded or translation is repressed (reviewed in [2]). Attempts to control the RNAi pathway led to the development of synthetic riboswitches regulating both processing steps — Drosha cleavage of pri-miRNAs and cleavage of exogenously applied small hairpin RNAs (shRNAs) by Dicer (Fig. 4A). Yokobayashi and coworkers incorporated the theophylline binding aptamer into the terminal region of an shRNA [75]. Binding of theophylline to the aptamer may then interfere with recognition or cleavage by Dicer. In the absence of ligand, shRNA processing resulted in low levels of reporter gene expression,



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Fig. 4. Control of the RNAi pathway by engineered riboswitches. (A) Schematic representation of a pri-miRNA, cleavage sites for Dicer and Drosha and the respective research groups using the upper or lower part for aptamer integration. (B) Regulation of pre-miRNA processing: The integrated aptamer domain interferes in its ligand bound state with the enzymatic activity of Dicer, thus, leading to a reduced RISC-activity which correlates with increased gene expression.

and addition of theophylline increased gene expression. In an initial study, the targeted proteins were all exogenously introduced fluorescent reporter proteins. A follow-up study by the same lab demonstrated the regulation of endogenous mRNA [76] (Fig. 4B). In a further study, Yokobayashi et al. chose to control the RNAi pathway by rendering precursor processing dependent on the selfcleavage of an attached ribozyme [77]. To achieve this, they fused the theophylline-dependent hammerhead ribozyme from the Hartig group [61] to the 5′-end of the precursor, thereby masking the Drosha recognition site. The ribozyme removes itself from the pri-miRNA through theophylline induced self-cleavage, and Drosha processing can then occur. Rational design to further improve the regulation factor however was not successful [77]. Smolke and coworkers controlled an siRNA which was directed against GFP [78] by incorporating a theophylline aptamer into the basal stem region of a precursor, thereby targeting Drosha cleavage. The RNA sensor can adopt two different conformations. In its unbound state, the shRNA can be processed properly, whereas binding of theophylline to the aptamer locks the precursor RNA in an inactive state. The construct shows modularity, since the aptamer can be replaced by other aptamers without changing the target. All in all, using ribozymes to regulate RNAi seems to be a promising approach. However, so far the current attempts suffer from extremely high ligand concentration necessary to achieve efficient regulation. Furthermore, they were performed exclusively with siRNA, which may be easier to control because of its strong signal amplification. In addition, processing is not strictly dependent on a defined cleavage site. Whether riboswitch regulation also has the potential to control endogenous miRNA still has to be proven and is, without doubt, one of the very interesting challenges in the field.

The following examples do not really count as riboswitches, because they also include a protein factor in the regulatory device. Nevertheless, they are interesting examples of synthetic RNA-based devices and are therefore included in this review. One example of an RNP switch makes use of the interaction between the archaeal ribosomal protein L7Ae and the box C/D kink-turn [79]. The authors placed the kink-turn element directly downstream of the EGFP start codon. Binding of L7Ae leads to decreased reporter gene activity. The OFF-switch was converted into an ON-switch by replacement of the box C/D kink-turn with its complementary sequence. The expression of a trans-expressed RNA containing the box C/D-sequence shuts down translation by binding to the mRNA. Overexpressed L7Ae binds the regulatory trans-RNA, setting the start codon free so that translation can occur. In further studies, these switches were used to control apoptosis by blocking Bcl-xL translation in a Bcl-xL/Bim apoptosis switch, and to control shRNA maturation by interfering with Dicer cleavage [80]. In a recent follow up study, the L7Ae-kink-turn RNP switch was employed to regulate gene expression through nonsense-mediated mRNA decay (NMD) [81]. The OFF-switch was expanded by adding a switch-inverting module consisting of a bait ORF with two premature stop codons, and the beta-globin intron followed by an IRES element to drive translation of the output gene. In the parental OFF-switch, L7Ae normally inhibits translation. Due to the inclusion of the switchinverting module, only translation of the bait ORF was blocked. Consequently, the mRNA does not represent an NMD target and the output gene could be translated via the IRES. Without the ligand L7Ae, translation occurred and the entire mRNA was degraded in the NMD pathway. Another type of RNP switch was developed to control alternative splicing [82]. Here, protein-binding aptamers were inserted at different positions in an intron, close to an alternatively spliced exon. The cassette exon carries a stop codon, which leads to a reduced gene expression upon exon inclusion. Protein binding then alters the splicing pattern, probably through sterical hindrance or the recruitment of factors involved in splice site recognition. By inserting multiple aptamers into different intronic integration sites, the authors demonstrated combinatorial integrative processing of multiple protein inputs. Our lab generated another RNA switch using a different aptamer [83]. It binds the bacterial tetracycline repressor TetR and was found by combining in vitro selection with in vivo screening in E. coli. The aptamer can be used to activate TetR-controlled transcription in E. coli by displacing TetR from its DNA-binding site, the tet operator sequences [84]. Niles and coworkers, who selected the same aptamer in an independent approach, used it to control translation [85]. Binding of TetR to an aptamer inserted into the 5′UTR of a reporter mRNA reduced gene expression [86]. Addition of doxycycline, which leads to conformational changes within TetR, results in aptamer release, once again permitting gene expression. Recent data from our lab indicate that this RNP switch can also be used to efficiently control alternative splicing (unpublished data). The Fussenegger group joined the TetR aptamer to the theophylline aptamer and triggered the Tet OFF system in a tetracycline-independent manner. Addition of theophylline resulted in enhanced binding of the TetR aptamer to tTA in vitro and also in inhibition of reporter gene expression in HEK cells [87]. This dual aptamer construct now allows the control of gene expression by two input signals, demonstrating the potential of the TetR aptamer as a regulatory device. 7. Aptamers which can be used to engineer riboswitches A major advantage of riboswitches is the possibility to combine sensing, transmitting and regulating domains within one molecule, enabling a straightforward approach to engineering synthetic molecules with desired and defined functions. In principle, aptamers can be generated for any ligand of choice. However, it has so far turned out that only


8


Another yet unsolved question is the fact that, in most studies, regulatory activity required high ligand concentrations which often affected cell viability to at least some extent, effectively rendering most engineered riboswitches nearly inapplicable. De facto, only the theophylline and the tetracycline aptamers are widely used, followed by some applications employing the neomycin and TMR aptamers (Fig. 5). Speculations why this is the case include proposals that the accessibility and correct folding of the aptamers are affected by nonspecific RNA binding proteins, that cotranscriptional folding can interfere with proper riboswitch assembly and functionality, that the ionic conditions in the cell are different from those used for in vitro selection, or that molecular crowding affects the aptamers under cellular conditions. All of these parameters can and may influence the performance of an aptamer expressed in a cellular environment and further systematic mechanistic studies will be necessary, not only to further improve these regulators, but also to distill the parameters essential for in vivo activity.

a few aptamers have the properties that allow their exploitation as sensing domains of riboswitches. Despite the fact that several dozen small molecule binding aptamers have been selected [88], only a handful of them can be used in riboswitch applications [89]. Therefore, the question arises — why do so many aptamers fail in engineering and, in turn, what makes an aptamer part of a functional riboswitch? During the last years, detailed genetic, biochemical and structural analyses have led to a much deeper understanding of the structure and ligand binding properties of some aptamers and may help shed some light on this conundrum. The analysis of neomycin aptamers indicated that high affinity binding of the ligand is not the only requirement for successful regulation. NMR spectroscopic analyses showed that large conformational changes accompany ligand binding in regulatory active aptamers [39]. In contrast, other neomycin aptamers from the same in vitro selection [90] bind their ligand with similar high affinity, but failed in regulation [38]. In these cases, spectroscopic analyses revealed no change at all in their conformations upon ligand binding. An important observation was that the candidate which could be used for riboswitch engineering was underrepresented in the enriched aptamer pool. Only an additional GFP-based screening step in yeast allowed its identification [35]. These data clearly indicate that in vitro selection may indeed result in aptamers with perfect binding properties, but not necessarily in candidates suitable for engineering, so that additional screening procedures may be necessary to identify the subpopulation of aptamers active in regulation.

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8. Conclusion and further perspectives Engineered riboswitches are finally coming of age. They have mostly left behind the stage of being only a proof-of-concept. Robust and orthogonal systems have been developed both for bacteria and eukaryotes. These systems make use of distinct regulatory mechanisms, some of which are close to their prototypes in nature. The fact that they are (mostly) composed of RNA only with no further need of protein

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Fig. 5. Aptamers used for engineered riboswitches. Shown are the 2D and 3D structures of the (A) tetracycline, (B) theophylline, (C) neomycin and (D) malachite green aptamers. Stems, bulges and loops are indicated with P, B and L, respectively, and labeled with the same color code in every illustration. Nucleotides involved in ligand binding are circled with dots in the 2D structure and specified in the 3D structure. A magnesium ion is indicated as a yellow ball and ligand molecules are colored in green. 3D structures were created with UCSF Chimera using pdb IDs 3EGZ, 1EHT, 2KXM and 1F1T [39,91–93].



co-factors makes them excellent tools for synthetic biology. Two extraordinary advantages are the possibility to select their sensing domains in vitro against virtually any ligand and the development of methods that improve their subsequent integration into functional riboswitches. At the moment, several restrictions remain. It has turned out that only a few aptamers can be used to control gene expression in vivo. One of the major challenges in the field will be the development of new and suitable aptamer domains that work in the various model organisms. Another weakness is the high ligand concentration needed for many applications, which unfortunately causes toxicity for most types of cells. The growing field of computational tools to design RNA devices still needs to be expanded, but there are good examples that this field will offer enormous opportunities in the future. Despite the current limitations, synthetic biology offers a vast potential to create new biological components and organisms, and to improve the properties of the already existing ones. We can expect that future achievements will bring improved, more complex and even novel engineered regulatory and metabolic pathways, new classes of therapeutics, and a deeper insight into cellular regulation.

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A family of synthetic riboswitches adopts a kinetic trapping mechanism.

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Design criteria for synthetic riboswitches acting on transcription.

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