RESUMO
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.
Assuntos
Aptâmeros de Nucleotídeos/química , Ciprofloxacina/química , RNA/química , Riboswitch , Técnica de Seleção de Aptâmeros/métodos , Ligantes , Conformação de Ácido NucleicoRESUMO
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.
Assuntos
Aptâmeros de Nucleotídeos/química , Proteínas de Ligação a DNA/ultraestrutura , Proteínas de Escherichia coli/ultraestrutura , Conformação Proteica , Aptâmeros de Nucleotídeos/genética , Cristalografia por Raios X , DNA de Forma B/química , DNA de Forma B/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Escherichia coli/ultraestrutura , Proteínas de Escherichia coli/química , Regulação da Expressão Gênica/genética , Substâncias Macromoleculares/química , Substâncias Macromoleculares/ultraestrutura , Modelos Moleculares , Ligação Proteica/genética , RNA/química , RNA/genéticaRESUMO
RNA aptamer selection during SELEX experiments builds on secondary structural diversity. Advanced structural comparison methods can focus this diversity. We develop SICOR, which uses probabilistic subgraph isomorphisms for graph distances between RNA secondary structure graphs. SICOR outperforms other comparison methods and is applicable to many structural comparisons in experimental design.
Assuntos
Aptâmeros de Nucleotídeos/química , Biologia Computacional/métodos , RNA/química , Técnica de Seleção de Aptâmeros/métodos , Algoritmos , Bases de Dados de Ácidos Nucleicos , Bases de Dados de Produtos Farmacêuticos , Conformação de Ácido NucleicoRESUMO
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.
Assuntos
Aptâmeros de Nucleotídeos/química , Engenharia Genética/métodos , Splicing de RNA , Proteínas Repressoras/metabolismo , Células HEK293 , Células HeLa , Humanos , Íntrons , Ligação Proteica , Sítios de Splice de RNA , Biologia Sintética/métodosRESUMO
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.
Assuntos
Aptâmeros de Nucleotídeos/química , RNA/química , Riboswitch/genética , Técnica de Seleção de Aptâmeros , Aptâmeros de Nucleotídeos/genética , Ligantes , Neomicina/química , Conformação de Ácido Nucleico/efeitos dos fármacos , Paromomicina/química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , Teofilina/químicaRESUMO
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.
Assuntos
Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/metabolismo , RNA/genética , RNA/metabolismo , Técnica de Seleção de Aptâmeros/métodos , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Transcrição Reversa/fisiologiaRESUMO
The excessive use of antibiotics in food-producing animals causes a steady rise of multiple antibiotic resistance in foodborne bacteria. Next to sulfonamides, the most common antibiotics groups are fluoroquinolones, aminoglycosides, and ß-lactams. Therefore, there is a need for a quick, efficient, and low-cost detection procedure for antibiotics. In this study, we propose an inkjet-printed aptamer-based biosensor developed for the detection of the fluoroquinolone ciprofloxacin. Due to their extraordinary high affinity and specificity, aptamers are already widely used in various applications. Here we present a ciprofloxacin-binding RNA aptamer developed by systematic evolution of ligands by exponential enrichment (SELEX). We characterized the secondary structure of the aptamer and determined the KD to 36 nM that allow detection of antibiotic contamination in a relevant range. We demonstrate that RNA aptamers can be inkjet-printed, dried, and resolved while keeping their functionality consistently intact. With this proof of concept, we are paving the way for a potential range of additional aptamer-based, printable biosensors.
Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , Ciprofloxacina/análise , Papel , Animais , Aptâmeros de Nucleotídeos/metabolismo , Técnicas Biossensoriais/instrumentação , Ciprofloxacina/metabolismo , Fluoroquinolonas/análise , Fluoroquinolonas/metabolismo , Análise de Alimentos , Conformação de Ácido Nucleico , Técnica de Seleção de AptâmerosRESUMO
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.
Assuntos
Aptâmeros de Nucleotídeos/química , Compostos Azo/química , Conformação de Ácido Nucleico/efeitos da radiação , RNA/química , Aptâmeros de Nucleotídeos/efeitos da radiação , Fenômenos Biofísicos , Ligantes , Luz , RNA/efeitos da radiação , Riboswitch/efeitos da radiação , Bibliotecas de Moléculas Pequenas/químicaRESUMO
Riboswitch development for clinical, technological, and synthetic biology applications constantly seeks to optimize regulatory behavior. Here, we present a machine learning approach to improve the regulation of a tetracycline (tc)-dependent riboswitch device composed of two individual tc aptamers. We developed a bioinformatics model that combines random forest analysis with a convolutional neural network to predict the switching behavior of such tandem riboswitches. We found that both biophysical parameters and the hydrogen bond pattern influence regulation. Our new design pipeline led to significant improvement of the tc riboswitch device with a dynamic range extension from 8.5 to 40-fold. We are confident that our novel method not only results in an excellent tc-dependent riboswitch device but further holds great promise and potential for the optimization of other riboswitches.
Assuntos
Aprendizado de Máquina , Riboswitch/genética , Aptâmeros de Nucleotídeos/genética , Biologia Computacional/métodos , Conformação de Ácido Nucleico/efeitos dos fármacos , Biologia Sintética , Tetraciclina/farmacologiaRESUMO
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.
Assuntos
Ciprofloxacina/química , Riboswitch , Bioengenharia , Regulação da Expressão Gênica , Células HeLa , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Ligantes , Mutação , Conformação de Ácido Nucleico , Técnica de Seleção de Aptâmeros , Saccharomyces cerevisiae/genéticaRESUMO
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.
Assuntos
Regulação da Expressão Gênica , Mamíferos/genética , MicroRNAs/genética , Precursores de RNA/genética , Animais , Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/metabolismo , Sequência de Bases , RNA Helicases DEAD-box/metabolismo , Células HeLa , Humanos , Mamíferos/metabolismo , MicroRNAs/biossíntese , Modelos Genéticos , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Ribonuclease III/metabolismoRESUMO
5-Lipoxygenase (5-LO) catalyzes the initial two steps of the conversion of arachidonic acid to leukotrienes which represent a group of pro-inflammatory lipid mediators involved in immune defense reactions as well as inflammation, allergy and cancer. Transforming growth factor-ß (TGFß) and calcitriol strongly upregulate 5-LO expression during myeloid cell differentiation and MLL-AF4 has been shown to strongly activate the 5-LO promoter. Here, we investigated the role of TGFß/SMAD signalling in 5-LO promoter activation. We identified two functional SMAD binding elements in the proximal part of the 5-LO promoter which significantly induce 5-LO promoter activity via TGFß and SMAD3/4. Since aberrant 5-LO gene expression has been linked with mixed lineage leukemia (MLL) which is characterized by the presence of MLL fusion proteins (e.g. MLL-AF4), we also investigated the influence of TGFß/SMADs on MLL- and MLL-AF4-mediated 5-LO promoter activation. Our data show that induction of 5-LO promoter activity by SMAD3/4 is MLL-dependent and that knockdown of the MLL complex component MEN1 attenuates the SMAD effect. Our data suggest that induction of 5-LO gene expression by TGFß is at least in part due to stimulation of transcript initiation.
Assuntos
Araquidonato 5-Lipoxigenase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Regiões Promotoras Genéticas/genética , Transdução de Sinais , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Araquidonato 5-Lipoxigenase/biossíntese , Sequência de Bases , Sítios de Ligação , Indução Enzimática , Técnicas de Silenciamento de Genes , Células HeLa , HumanosRESUMO
Despite its wide applicability the selection of small molecule-binding RNA aptamers with high affinity binding and specificity is still challenging. We will present here a protocol which allows the in vitro selection of antibiotic-binding aptamers which turned out to be important building blocks for the design process of synthetic riboswitches. The presented methods will be compared with alternative in vitro selection protocols. A detailed note section will point out useful tips and pitfalls.
Assuntos
Antibacterianos/química , Aptâmeros de Nucleotídeos/genética , Técnica de Seleção de Aptâmeros/métodos , Aptâmeros de Nucleotídeos/química , Riboswitch/genéticaRESUMO
The ultrafast photochemistry of a new spiropyran photoswitch (Py-BIPS) has been investigated, revealing many advantages in the application in water over the previously studied spiropyrans. Functionalized Py-BIPS derivatives are presented for the study of pH dependence, stability, toxicity, and the thermal and photochemical behavior on longer time scales in aqueous media using several spectroscopic methods. These investigations pave the way for the practical use of Py-BIPS derivatives as photoswitchable ligands of biomolecules.
Assuntos
Benzopiranos/química , Indóis/química , Nitrocompostos/química , Processos Fotoquímicos , Água/química , Ligantes , Estrutura Molecular , Ressonância Magnética Nuclear BiomolecularRESUMO
RNA utilizes many different mechanisms to control gene expression. Among the regulatory elements that respond to external stimuli, riboswitches are a prominent and elegant example. They consist solely of RNA and couple binding of a small molecule ligand to the so-called "aptamer domain" with a conformational change in the downstream "expression platform" which then determines system output. The modular organization of riboswitches and the relative ease with which ligand-binding RNA aptamers can be selected in vitro against almost any molecule have led to the rapid and widespread adoption of engineered riboswitches as artificial genetic control devices in biotechnology and synthetic biology over the past decade. This review highlights proof-of-principle applications to demonstrate the versatility and robustness of engineered riboswitches in regulating gene expression in pro- and eukaryotes. It then focuses on strategies and parameters to identify aptamers that can be integrated into synthetic riboswitches that are functional in vivo, before finishing with a reflection on how to improve the regulatory properties of engineered riboswitches, so that we can not only further expand riboswitch applicability, but also finally fully exploit their potential as control elements in regulating gene expression.
Assuntos
Aptâmeros de Nucleotídeos/genética , Riboswitch , Aptâmeros de Nucleotídeos/química , Regulação da Expressão Gênica , Biologia Sintética/métodosRESUMO
The U-turn is a classical three-dimensional RNA folding motif first identified in the anticodon and T-loops of tRNAs. It also occurs frequently as a building block in other functional RNA structures in many different sequence and structural contexts. U-turns induce sharp changes in the direction of the RNA backbone and often conform to the 3-nt consensus sequence 5'-UNR-3' (N = any nucleotide, R = purine). The canonical U-turn motif is stabilized by a hydrogen bond between the N3 imino group of the U residue and the 3' phosphate group of the R residue as well as a hydrogen bond between the 2'-hydroxyl group of the uridine and the N7 nitrogen of the R residue. Here, we demonstrate that a protonated cytidine can functionally and structurally replace the uridine at the first position of the canonical U-turn motif in the apical loop of the neomycin riboswitch. Using NMR spectroscopy, we directly show that the N3 imino group of the protonated cytidine forms a hydrogen bond with the backbone phosphate 3' from the third nucleotide of the U-turn analogously to the imino group of the uridine in the canonical motif. In addition, we compare the stability of the hydrogen bonds in the mutant U-turn motif to the wild type and describe the NMR signature of the C+-phosphate interaction. Our results have implications for the prediction of RNA structural motifs and suggest simple approaches for the experimental identification of hydrogen bonds between protonated C-imino groups and the phosphate backbone.
Assuntos
Citidina , Conformação de Ácido Nucleico , Motivos de Nucleotídeos , RNA/química , Sequência de Bases , Ligação de Hidrogênio , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Fosfatos/química , RNA/genética , Riboswitch , UridinaRESUMO
Synthetic riboswitches can serve as sophisticated genetic control devices in synthetic biology, regulating gene expression through direct RNA-ligand interactions. We analyzed a synthetic neomycin riboswitch, which folds into a stem loop structure with an internal loop important for ligand binding and regulation. It is closed by a terminal hexaloop containing a U-turn and a looped-out adenine. We investigated the relationship between sequence, structure, and biological activity in the terminal loop by saturating mutagenesis, ITC, and NMR. Mutants corresponding to the canonical U-turn fold retained biological activity. An improvement of stacking interactions in the U-turn led to an RNA element with slightly enhanced regulatory activity. For the first position of the U-turn motif and the looped out base, sequence-activity relationships that could not initially be explained on the basis of the structure of the aptamer-ligand complex were observed. However, NMR studies of these mutants revealed subtle relationships between structure and dynamics of the aptamer in its free or bound state and biological activity.
Assuntos
Riboswitch/genética , Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/metabolismo , Sítios de Ligação , Ligantes , Modelos Moleculares , Mutação , Neomicina/metabolismoRESUMO
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.
RESUMO
In chicken, the left and right female gonads undergo a completely different program during development. To learn more about the molecular factors underlying side-specific development and to identify potential sex- and side-specific genes in developing gonads, we separately performed next-generation sequencing-based deepSuperSAGE transcription profiling from left and right, female and male gonads of 19-day-old chicken embryos. A total of 836 transcript variants were significantly differentially expressed (p < 10(-5)) between combined male and female gonads. Left-right comparison revealed 1,056 and 822 differentially (p < 10(-5)) expressed transcript variants for male and female gonads, respectively, of which 72 are side-specific in both sexes. At least some of these may represent key players for lateral development in birds. Additionally, several genes with laterally differential expression in the ovaries seem to determine female gonads for growth or regression, whereas right-left differences in testes are mostly limited to the differentially expressed genes present in both sexes. With a few exceptions, side-specific genes are not located on the sex chromosomes. The large differences in lateral gene expression in the ovaries in almost all metabolic pathways suggest that the regressing right gonad might have undergone a change of function during evolution.