Development of a novel tobramycin dependent riboswitch.

We herein report the selection and characterization of a new riboswitch dependent on the aminoglycoside tobramycin. Its dynamic range rivals even the tetracycline dependent riboswitch to be the current best performing, synthetic riboswitch that controls translation initiation. The riboswitch was selected with RNA Capture-SELEX, a method that not only selects for binding but also for structural changes in aptamers on binding. This study demonstrates how this method can fundamentally reduce the labour required for the de novo identification of synthetic riboswitches. The initially selected riboswitch candidate harbours two distinct tobramycin binding sites with KDs of 1.1 nM and 2.4 µM, respectively, and can distinguish between tobramycin and the closely related compounds kanamycin A and B. Using detailed genetic and biochemical analyses and 1H NMR spectroscopy, the proposed secondary structure of the riboswitch was verified and the tobramycin binding sites were characterized. The two binding sites were found to be essentially non-overlapping, allowing for a separate investigation of their contribution to the activity of the riboswitch. We thereby found that only the high-affinity binding site was responsible for regulatory activity, which allowed us to engineer a riboswitch from only this site with a minimal sequence size of 33 nt and outstanding performance.

Inducible nuclear import by TetR aptamer-controlled 3' splice site selection.

Correct cellular localization is essential for the function of many eukaryotic proteins and hence cell physiology. Here, we present a synthetic genetic device that allows the control of nuclear and cytosolic localization based on controlled alternative splicing in human cells. The device is based on the fact that an alternative 3' splice site is located within a TetR aptamer that in turn is positioned between the branch point and the canonical splice site. The novel splice site is only recognized when the TetR repressor is bound. Addition of doxycycline prevents TetR aptamer binding and leads to recognition of the canonical 3' splice site. It is thus possible to produce two independent splice isoforms. Since the terminal loop of the aptamer may be replaced with any sequence of choice, one of the two isoforms may be extended by the respective sequence of choice depending on the presence of doxycycline. In a proof-of-concept study, we fused a nuclear localization sequence to a cytosolic target protein, thus directing the protein into the nucleus. However, the system is not limited to the control of nuclear localization. In principle, any target sequence can be integrated into the aptamer, allowing not only the production of a variety of different isoforms on demand, but also to study the function of mislocalized proteins. Moreover, it also provides a valuable tool for investigating the mechanism of alternative splicing in human cells.

Tapping the potential of synthetic riboswitches: reviewing the versatility of the tetracycline aptamer.

Synthetic riboswitches are a versatile class of regulatory elements that are becoming increasingly established in synthetic biology applications. They are characterized by their compact size and independence from auxiliary protein factors. While naturally occurring riboswitches were mostly discovered in bacteria, synthetic riboswitches have been designed for all domains of life. Published design strategies far exceed the number of riboswitches found in nature. A core element of any riboswitch is a binding domain, called an aptamer, which is characterized by high specificity and affinity for its ligand. Aptamers can be selected de novo, allowing the design of synthetic riboswitches against a broad spectrum of targets. The tetracycline aptamer has proven to be well suited for riboswitch engineering. Since its selection, it has been used in a variety of applications and is considered to be well established and characterized. Using the tetracycline aptamer as an example, we aim to discuss a large variety of design approaches for synthetic riboswitch engineering and their application. We aim to demonstrate the versatility of riboswitches in general and the high potential of synthetic RNA devices for creating new solutions in both the scientific and medical fields.

What defines a synthetic riboswitch? - Conformational dynamics of ciprofloxacin aptamers with similar binding affinities but varying regulatory potentials.

Among the many in vitro-selected aptamers derived from SELEX protocols, only a small fraction has the potential to be applied for synthetic riboswitch engineering. Here, we present a comparative study of the binding properties of three different aptamers that bind to ciprofloxacin with similar KD values, yet only two of them can be applied as riboswitches. We used the inherent ligand fluorescence that is quenched upon binding as the reporter signal in fluorescence titration and in time-resolved stopped-flow experiments. Thus, we were able to demonstrate differences in the binding kinetics of regulating and non-regulating aptamers. All aptamers studied underwent a two-step binding mechanism that suggests an initial association step followed by a reorganization of the aptamer to accommodate the ligand. We show that increasing regulatory potential is correlated with a decreasing back-reaction rate of the second binding step, thus resulting in a virtually irreversible last binding step of regulating aptamers. We suggest that a highly favoured structural adaption of the RNA to the ligand during the final binding step is essential for turning an aptamer into a riboswitch. In addition, our results provide an explanation for the fact that so few aptamers with regulating capacity have been found to date. Based on our data, we propose an adjustment of the selection protocol for efficient riboswitch detection.

Dicer up-regulation by inhibition of specific proteolysis in differentiating monocytic cells.

Dicer is a ribonuclease III enzyme in biosynthesis of micro-RNAs (miRNAs). Here we describe a regulation of Dicer expression in monocytic cells, based on proteolysis. In undifferentiated Mono Mac 6 (MM6) cells, full-length Dicer was undetectable; only an ∼50-kDa fragment appeared in Western blots. However, when MM6 cells were treated with zymosan or LPS during differentiation with TGF-ß and 1,25diOHvitD3, full-length Dicer became abundant together with varying amounts of ∼170- and ∼50-kDa Dicer fragments. Mass spectrometry identified the Dicer fragments and showed cleavage about 450 residues upstream from the C terminus. Also, PGE2 (prostaglandin E2) added to differentiating MM6 cells up-regulated full-length Dicer, through EP2/EP4 and cAMP. The TLR stimuli strongly induced miR-146a-5p, while PGE2 increased miR-99a-5p and miR-125a-5p, both implicated in down-regulation of TNFα. The Ser protease inhibitor AEBSF (4-[2-aminoethyl] benzene sulfonyl fluoride) up-regulated full-length Dicer, both in MM6 cells and in primary human blood monocytes, indicating a specific proteolytic degradation. However, AEBSF alone did not lead to a general increase in miR expression, indicating that additional mechanisms are required to increase miRNA biosynthesis. Finally, differentiation of monocytes to macrophages with M-CSF or GM-CSF strongly up-regulated full-length Dicer. Our results suggest that differentiation regimens, both in the MM6 cell line and of peripheral blood monocytes, inhibit an apparently constitutive Dicer proteolysis, allowing for increased formation of miRNAs.

Investigating the Prevalence of RNA-Binding Metabolic Enzymes in E. coli.

An open research field in cellular regulation is the assumed crosstalk between RNAs, metabolic enzymes, and metabolites, also known as the REM hypothesis. High-throughput assays have produced extensive interactome data with metabolic enzymes frequently found as hits, but only a few examples have been biochemically validated, with deficits especially in prokaryotes. Therefore, we rationally selected nineteen Escherichia coli enzymes from such datasets and examined their ability to bind RNAs using two complementary methods, iCLIP and SELEX. Found interactions were validated by EMSA and other methods. For most of the candidates, we observed no RNA binding (12/19) or a rather unspecific binding (5/19). Two of the candidates, namely glutamate-5-kinase (ProB) and quinone oxidoreductase (QorA), displayed specific and previously unknown binding to distinct RNAs. We concentrated on the interaction of QorA to the mRNA of yffO, a grounded prophage gene, which could be validated by EMSA and MST. Because the physiological function of both partners is not known, the biological relevance of this interaction remains elusive. Furthermore, we found novel RNA targets for the MS2 phage coat protein that served us as control. Our results indicate that RNA binding of metabolic enzymes in procaryotes is less frequent than suggested by the results of high-throughput studies, but does occur.

Modulation of microRNA processing by 5-lipoxygenase.

The miRNA biogenesis is tightly regulated to avoid dysfunction and consequent disease development. Here, we describe modulation of miRNA processing as a novel noncanonical function of the 5-lipoxygenase (5-LO) enzyme in monocytic cells. In differentiated Mono Mac 6 (MM6) cells, we found an in situ interaction of 5-LO with Dicer, a key enzyme in miRNA biogenesis. RNA sequencing of small noncoding RNAs revealed a functional impact, knockout of 5-LO altered the expression profile of several miRNAs. Effects of 5-LO could be observed at two levels. qPCR analyses thus indicated that (a) 5-LO promotes the transcription of the evolutionarily conserved miR-99b/let-7e/miR-125a cluster and (b) the 5-LO-Dicer interaction downregulates the processing of pre-let-7e, resulting in an increase in miR-125a and miR-99b levels by 5-LO without concomitant changes in let-7e levels in differentiated MM6 cells. Our observations suggest that 5-LO regulates the miRNA profile by modulating the Dicer-mediated processing of distinct pre-miRNAs. 5-LO inhibits the formation of let-7e which is a well-known inducer of cell differentiation, but promotes the generation of miR-99b and miR-125a known to induce cell proliferation and the maintenance of leukemic stem cell functions.

The complex formed between a synthetic RNA aptamer and the transcription repressor TetR is a structural and functional twin of the operator DNA-TetR regulator complex.

RNAs play major roles in the regulation of gene expression. Hence, designer RNA molecules are increasingly explored as regulatory switches in synthetic biology. Among these, the TetR-binding RNA aptamer was selected by its ability to compete with operator DNA for binding to the bacterial repressor TetR. A fortuitous finding was that induction of TetR by tetracycline abolishes both RNA aptamer and operator DNA binding in TetR. This enabled numerous applications exploiting both the specificity of the RNA aptamer and the efficient gene repressor properties of TetR. Here, we present the crystal structure of the TetR-RNA aptamer complex at 2.7 Å resolution together with a comprehensive characterization of the TetR-RNA aptamer versus TetR-operator DNA interaction using site-directed mutagenesis, size exclusion chromatography, electrophoretic mobility shift assays and isothermal titration calorimetry. The fold of the RNA aptamer bears no resemblance to regular B-DNA, and neither does the thermodynamic characterization of the complex formation reaction. Nevertheless, the functional aptamer-binding epitope of TetR is fully contained within its DNA-binding epitope. In the RNA aptamer complex, TetR adopts the well-characterized DNA-binding-competent conformation of TetR, thus revealing how the synthetic TetR-binding aptamer strikes the chords of the bimodal allosteric behaviour of TetR to function as a synthetic regulator.

Influence of Mg2+ on the conformational flexibility of a tetracycline aptamer.

The tetracycline-binding RNA aptamer (TC-aptamer) is a synthetic riboswitch that binds the antibiotic tetracycline (TC) with exceptionally high affinity. Although a crystal structure exists of the TC-bound state, little is known about the conformational dynamics and changes upon ligand binding. In this study, pulsed electron paramagnetic resonance techniques for measuring distances (PELDOR) in combination with rigid nitroxide spin labels (Çm spin label) were used to investigate the conformational flexibility of the TC-aptamer in the presence and absence of TC at different Mg2+ concentrations. TC was found to be the essential factor for stabilizing the tertiary structure at intermediate Mg2+ concentrations. At higher Mg2+ concentrations, Mg2+ alone is sufficient to stabilize the tertiary structure. In addition, the orientation of the two spin-labeled RNA helices with respect to each other was analyzed with orientation-selective PELDOR and compared to the crystal structure. These results demonstrate for the first time the unique value of the Çm spin label in combination with PELDOR to provide information about conformational flexibilities and orientations of secondary structure elements of biologically relevant RNAs.

Robust gene expression control in human cells with a novel universal TetR aptamer splicing module.

Fine-tuning of gene expression is desirable for a wide range of applications in synthetic biology. In this context, RNA regulatory devices provide a powerful and highly functional tool. We developed a versatile, robust and reversible device to control gene expression by splicing regulation in human cells using an aptamer that is recognized by the Tet repressor TetR. Upon insertion in proximity to the 5' splice site, intron retention can be controlled via the binding of TetR to the aptamer. Although we were able to demonstrate regulation for different introns, the genomic context had a major impact on regulation. In consequence, we advanced the aptamer to develop a splice device. Our novel device contains the aptamer integrated into a context of exonic and intronic sequences that create and maintain an environment allowing a reliable and robust splicing event. The exon-born, additional amino acids will then be cleaved off by a self-cleaving peptide. This design allows portability of the splicing device, which we confirmed by demonstrating its functionality in different gene contexts. Intriguingly, our splicing device shows a high dynamic range and low basal activity, i.e. desirable features that often prove a major challenge when implementing synthetic biology in mammalian cell lines.

Next-level riboswitch development-implementation of Capture-SELEX facilitates identification of a new synthetic riboswitch.

The development of synthetic riboswitches has always been a challenge. Although a number of interesting proof-of-concept studies have been published, almost all of these were performed with the theophylline aptamer. There is no shortage of small molecule-binding aptamers; however, only a small fraction of them are suitable for RNA engineering since a classical SELEX protocol selects only for high-affinity binding but not for conformational switching. We now implemented RNA Capture-SELEX in our riboswitch developmental pipeline to integrate the required selection for high-affinity binding with the equally necessary RNA conformational switching. Thus, we successfully developed a new paromomycin-binding synthetic riboswitch. It binds paromomycin with a KD of 20 nM and can discriminate between closely related molecules both in vitro and in vivo. A detailed structure-function analysis confirmed the predicted secondary structure and identified nucleotides involved in ligand binding. The riboswitch was further engineered in combination with the neomycin riboswitch for the assembly of an orthogonal Boolean NOR logic gate. In sum, our work not only broadens the spectrum of existing RNA regulators, but also signifies a breakthrough in riboswitch development, as the effort required for the design of sensor domains for RNA-based devices will in many cases be much reduced.

A light-responsive RNA aptamer for an azobenzene derivative.

Regulation of complex biological networks has proven to be a key bottleneck in synthetic biology. Interactions between the structurally flexible RNA and various other molecules in the form of riboswitches have shown a high-regulation specificity and efficiency and synthetic riboswitches have filled the toolbox of devices in many synthetic biology applications. Here we report the development of a novel, small molecule binding RNA aptamer, whose binding is dependent on light-induced change of conformation of its small molecule ligand. As ligand we chose an azobenzene because of its reliable photoswitchability and modified it with chloramphenicol for a better interaction with RNA. The synthesis of the ligand 'azoCm' was followed by extensive biophysical analysis regarding its stability and photoswitchability. RNA aptamers were identified after several cycles of in vitro selection and then studied regarding their binding specificity and affinity toward the ligand. We show the successful development of an RNA aptamer that selectively binds to only the trans photoisomer of azoCm with a KD of 545 nM. As the aptamer cannot bind to the irradiated ligand (λ = 365 nm), a light-selective RNA binding system is provided. Further studies may now result in the engineering of a reliable, light-responsible riboswitch.

Altered stoichiometry of an evolved RNA aptamer.

Inhibitory aptamers against a protein are promising as antagonistic reagents and repressive genetic components. Typically, improvement of such aptamers is achieved by acquiring higher binding affinity. Here, we report an alternative mechanism for the improvement of aptamer activity. Recently, we reported a transcriptional activator based on an inhibitory RNA aptamer against lambda cI repressor. We improved the aptamer through in vitro selection (SELEX) from a randomly mutagenized aptamer pool, followed by in vivo screening and truncation. Biochemical analyses indicated that the activity improvement was achieved by alteration of the complex formation stoichiometry, rather than by higher affinity or expression. Our results suggest an alternative strategy for improving aptamer activity.

miR-574-5p as RNA decoy for CUGBP1 stimulates human lung tumor growth by mPGES-1 induction.

MicroRNAs (miRs) are important posttranscriptional regulators of gene expression. Besides their well-characterized inhibitory effects on mRNA stability and translation, miRs can also activate gene expression. In this study, we identified a novel noncanonical function of miR-574-5p. We found that miR-574-5p acts as an RNA decoy to CUG RNA-binding protein 1 (CUGBP1) and antagonizes its function. MiR-574-5p induces microsomal prostaglandin E synthase-1 (mPGES-1) expression by preventing CUGBP1 binding to its 3'UTR, leading to an enhanced alternative splicing and generation of an mPGES-1 3'UTR isoform, increased mPGES-1 protein expression, PGE2 formation, and tumor growth in vivo. miR-574-5p-induced tumor growth in mice could be completely inhibited with the mPGES-1 inhibitor CIII. Moreover, miR-574-5p is induced by IL-1ß and is strongly overexpressed in human nonsmall cell lung cancer where high mPGES-1 expression correlates with a low survival rate. The discovered function of miR-574-5p as a CUGBP1 decoy opens up new therapeutic opportunities. It might serve as a stratification marker to select lung tumor patients who respond to the pharmacological inhibition of PGE2 formation.-Saul, M. J., Baumann, I., Bruno, A., Emmerich, A. C., Wellstein, J., Ottinger, S. M., Contursi, A., Dovizio, M., Donnini, S., Tacconelli, S., Raouf, J., Idborg, H., Stein, S., Korotkova, M., Savai, R., Terzuoli, E., Sala, G., Seeger, W., Jakobsson, P.-J., Patrignani, P., Suess, B., Steinhilber, D. miR-574-5p as RNA decoy for CUGBP1 stimulates human lung tumor growth by mPGES-1 induction.

RNA-based Capture-SELEX for the selection of small molecule-binding aptamers.

Despite their wide applicability, the selection of small molecule-binding RNA aptamers with both high affinity binding and specificity is still challenging. Aptamers that excel at both binding and structure switching are particularly rare and difficult to find. Here, we present the protocol of a Capture-SELEX that specifically allows the in vitro selection of small-molecule binding aptamers, which are essential building blocks for the design process of synthetic riboswitches and biosensors. Moreover, we provide a comparative overview of our proposed methodology versus alternative in vitro selection protocols with a special focus on the design of the pool. Finally, we have included detailed notes to point out useful tips and pitfalls for future application.

A small, portable RNA device for the control of exon skipping in mammalian cells.

Splicing is an essential and highly regulated process in mammalian cells. We developed a synthetic riboswitch that efficiently controls alternative splicing of a cassette exon in response to the small molecule ligand tetracycline. The riboswitch was designed to control the accessibility of the 3' splice site by placing the latter inside the closing stem of a conformationally controlled tetracycline aptamer. In the presence of tetracycline, the cassette exon is skipped, whereas it is included in the ligand's absence. The design allows for an easy, context-independent integration of the regulatory device into any gene of interest. Portability of the device was shown through its functionality in four different systems: a synthetic minigene, a reporter gene and two endogenous genes. Furthermore, riboswitch functionality to control cellular signaling cascades was demonstrated by using it to specifically induce cell death through the conditionally controlled expression of CD20, which is a target in cancer therapy.

Riboswitching with ciprofloxacin-development and characterization of a novel RNA regulator.

RNA molecules play important and diverse regulatory roles in the cell. Inspired by this natural versatility, RNA devices are increasingly important for many synthetic biology applications, e.g. optimizing engineered metabolic pathways, gene therapeutics or building up complex logical units. A major advantage of RNA is the possibility of de novo design of RNA-based sensing domains via an in vitro selection process (SELEX). Here, we describe development of a novel ciprofloxacin-responsive riboswitch by in vitro selection and next-generation sequencing-guided cellular screening. The riboswitch recognizes the small molecule drug ciprofloxacin with a KD in the low nanomolar range and adopts a pseudoknot fold stabilized by ligand binding. It efficiently interferes with gene expression both in lower and higher eukaryotes. By controlling an auxotrophy marker and a resistance gene, respectively, we demonstrate efficient, scalable and programmable control of cellular survival in yeast. The applied strategy for the development of the ciprofloxacin riboswitch is easily transferrable to any small molecule target of choice and will thus broaden the spectrum of RNA regulators considerably.

Design and implementation of a synthetic pre-miR switch for controlling miRNA biogenesis in mammals.

Synthetic RNA-based systems have increasingly been used for the regulation of eukaryotic gene expression. Due to their structural properties, riboregulators provide a convenient basis for the development of ligand-dependent controllable systems. Here, we demonstrate reversible conditional control of miRNA biogenesis with an aptamer domain as a sensing unit connected to a natural miRNA precursor for the first time. For the design of the pre-miR switch, we replaced the natural terminal loop with the TetR aptamer. Thus, the TetR aptamer was positioned close to the Dicer cleavage sites, which allowed sterical control over pre-miR processing by Dicer. Our design proved to be highly versatile, allowing us to regulate the biogenesis of three structurally different miRNAs: miR-126, -34a and -199a. Dicer cleavage was inhibited up to 143-fold via co-expression of the TetR protein, yet could be completely restored upon addition of doxycycline. Moreover, we showed the functionality of the pre-miR switches for gene regulation through the interaction of the respective miRNA with its specific target sequence. Our designed device is capable of robust and reversible control of miRNA abundance. Thus, we offer a novel investigational tool for functional miRNA analysis.

Ligand binding to 2΄-deoxyguanosine sensing riboswitch in metabolic context.

The mfl-riboswitch is a transcriptional off-switch, which down-regulates expression of subunit ß of ribonucleotide reductase in Mesoplasma florum upon 2΄-deoxyguanosine binding. We characterized binding of 2΄-deoxyguanosine to the mfl-aptamer domain (WT aptamer) and a sequence-stabilized aptamer (MT aptamer) under in vitro and 'in-cell-like' conditions by isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR) spectroscopy. 'In-cell-like' environment was simulated by Bacillus subtilis cell extract, in which both aptamers remained sufficiently stable to detect the resonances of structural elements and ligand binding in 2D NMR experiments. Under 'in-cell-like'-environment, (i) the WT aptamer bound the endogenous metabolite guanosine and (ii) 2΄-deoxyguanosine efficiently displaced guanosine from the WT aptamer. In contrast, MT aptamer exhibited moderate binding to 2΄-deoxyguanosine and weak binding to guanosine. NMR experiments indicated that binding of guanosine was not limited to the aptamer domain of the riboswitch but also the full-length mfl-riboswitch bound guanosine, impacting on the regulation efficiency of the riboswitch and hinting that, in addition to 2΄-deoxyguanosine, guanosine plays a role in riboswitch function in vivo. Reporter gene assays in B. subtilis demonstrated the regulation capacity of the WT aptamer, whereas the MT aptamer with lower affinity to 2΄-deoxyguanosine was not able to regulate gene expression.