ABSTRACT
To combat the growing threat of antibiotic resistance, environmental testing for antibiotic contamination is gaining an increasing role. This study aims to develop an easy-to-use assay for the detection of the fluoroquinolone antibiotic levofloxacin. Levofloxacin is used in human and veterinary medicine and has been detected in wastewater and river water. An RNA aptamer against levofloxacin was selected using RNA Capture-SELEX. The 73 nt long aptamer folds into three stems with a central three-way junction. It binds levofloxacin with a Kd of 6 µM and discriminates the closely related compound ciprofloxacin. Furthermore, the selection process was analyzed using a next-generation sequencing approach to better understand the sequence evolution throughout the selection. The aptamer was used as a bioreceptor for the development of a lateral flow assay. The biosensor exploited the innate characteristic of RNA Capture-SELEX to select aptamers that displace a complementary DNA oligonucleotide upon ligand binding. The lateral flow assay achieved a limit of visual detection of 100 µM. While the sensitivity of this assay constrains its immediate use in environmental testing, the present study can serve as a template for the selection of RNA aptamer-based biosensors.
Subject(s)
Aptamers, Nucleotide , Humans , Aptamers, Nucleotide/chemistry , Levofloxacin , SELEX Aptamer Technique , Anti-Bacterial Agents , RNAABSTRACT
Progress in the synthetic biology field is driven by the development of new tools for synthetic circuit engineering. Traditionally, the focus has relied on protein-based designs. In recent years, the use of RNA-based tools has tremendously increased, due to their versatile functionality and applicability. A promising class of molecules is RNA aptamers, small, single-stranded RNA molecules that bind to a target molecule with high affinity and specificity. When targeting bacterial repressors, RNA aptamers allow one to add a new layer to an established protein-based regulation. In the present study, we selected an RNA aptamer binding the bacterial repressor DasR, preventing its binding to its operator sequence and activating DasR-controlled transcription in vivo. This was made possible only by the combination of an in vitro selection and subsequent in vivo screening. Next-generation sequencing of the selection process proved the importance of the in vivo screening for the discovery of aptamers functioning in the cell. Mutational and biochemical studies led to the identification of the minimal necessary binding motif. Taken together, the resulting combination of bacterial repressor and RNA aptamer enlarges the synthetic biology toolbox by adding a new level of regulation.
Subject(s)
Aptamers, Nucleotide , Aptamers, Nucleotide/metabolism , SELEX Aptamer Technique/methods , RNAABSTRACT
Mg2+ ions play an essential part in stabilizing the tertiary structure of nucleic acids. While the importance of these ions is well documented, their localization and elucidation of their role in the structure and dynamics of nucleic acids are often challenging. In this work, pulsed electron-electron double resonance spectroscopy (PELDOR, also known as DEER) was used to localize two high affinity divalent metal ion binding sites in the tetracycline RNA aptamer with high accuracy. For this purpose, the aptamer was labeled at different positions with a semirigid nitroxide spin label and diamagnetic Mg2+ was replaced with paramagnetic Mn2+, which did not alter the folding process or ligand binding. Out of the several divalent metal ion binding sites that are known from the crystal structure, two binding sites with high affinity were detected: one that is located at the ligand binding center and another at the J1/2 junction of the RNA.
Subject(s)
Aptamers, Nucleotide , Nucleic Acids , Electron Spin Resonance Spectroscopy , Aptamers, Nucleotide/chemistry , Ligands , Spin Labels , Tetracycline , Binding Sites , Anti-Bacterial Agents , IonsABSTRACT
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.
Subject(s)
Aptamers, Nucleotide , Genetic Engineering , Riboswitch , Tobramycin , Aptamers, Nucleotide/chemistry , Ligands , Nucleic Acid Conformation , Protein Synthesis Inhibitors , RNA/chemistry , Tetracycline , Tobramycin/pharmacology , Saccharomyces cerevisiae/drug effects , Genetic Engineering/methodsABSTRACT
Engineering in vitro selected RNA aptamers into in vivo functional riboswitches represents a long-standing challenge in molecular biology. The highly specific aptamer domain of the riboswitch undergoes a conformational adjustment in response to ligand sensing, which in turn exerts the regulatory function. Besides essential factors like structural complexity and ligand binding kinetics, the active role of magnesium ions in stabilizing RNA tertiary structures and assisting in ligand binding can be a vital criterion. We present spectroscopic studies on the magnesium ion-driven folding of the Tetracycline binding aptamer. Using fluorescent labels, the aptamer pre-folding and subsequent ligand binding is monitored by magnesium titration experiments and time-resolved stopped-flow measurements. A minimum concentration of 0.5 mM magnesium is required to fold into a magnesium ion-stabilized binding-competent state with a preformed binding pocket. Tetracycline binding causes a pronounced conformational change that results in the establishment of the triple helix core motif, and that further propagates towards the closing stem. By a dynamic acquisition of magnesium ions, a kink motif is formed at the intersection of the triple helix and closing stem regions. This ultimately entails a stabilization of the closing stem which is discussed as a key element in the regulatory function of the Tetracycline aptamer.
Subject(s)
Anti-Bacterial Agents , Aptamers, Nucleotide , Magnesium , Riboswitch , Tetracycline , Anti-Bacterial Agents/chemistry , Aptamers, Nucleotide/chemistry , Ions , Ligands , Magnesium/chemistry , Nucleic Acid Conformation , Tetracycline/chemistryABSTRACT
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.
Subject(s)
Aptamers, Nucleotide , Riboswitch , Aptamers, Nucleotide/chemistry , Tetracycline/pharmacology , Anti-Bacterial Agents , LigandsABSTRACT
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.
Subject(s)
Escherichia coli , Escherichia coli/genetics , PrevalenceABSTRACT
SELEX has enabled the selection of aptamers, nucleic acids that can bind a defined ligand, in some cases with exceptionally high affinity and specificity. The SELEX protocol has been adapted many times to fit a variety of needs. This protocol describes such an adaptation, namely, RNA-Capture SELEX that we have used to successfully develop small molecule-binding RNA aptamers. Our proposed method specifically selects not only for excellent binding but also for conformational switching. In consequence, we found this SELEX method to be particularly suitable for identifying aptamers for further application in synthetic riboswitch engineering.
Subject(s)
Aptamers, Nucleotide , Riboswitch , Aptamers, Nucleotide/chemistry , Ligands , Magnetic Phenomena , RNA , SELEX Aptamer Technique/methods , Streptavidin/metabolismABSTRACT
Synthetic riboswitches are a promising tool for conditional gene expression. In vitro selected aptamers used as binding domains for the design of RNA-based switches have to exhibit excellent binding affinity as well as ligand binding-induced structural changes. Selection via Capture-SELEX favors the enrichment of aptamers which exhibit both characteristics. For the Capture-SELEX, an RNA pool is used that gets immobilized onto a capture oligonucleotide by hybridization. Addition of the ligand frees the aptamers by their binding to the ligand, resulting in the release from the capture oligonucleotide through structural changes. These sequences get reverse transcribed, PCR amplified, and used for the following selection rounds. In this publication, we present a detailed protocol for Capture-SELEX, followed by screening in yeast to identify aptamers suitable for the design of synthetic riboswitches.
Subject(s)
Aptamers, Nucleotide , Riboswitch , Aptamers, Nucleotide/chemistry , Ligands , RNA , Riboswitch/genetics , SELEX Aptamer Technique/methodsABSTRACT
Synthetic riboswitches are powerful tools in synthetic biology in which sensing and execution are consolidated in a single RNA molecule. By using SELEX to select aptamers in vitro, synthetic riboswitches can in theory be engineered against any ligand of choice. Surprisingly, very few in vitro selected aptamers have been used for the engineering of synthetic riboswitches. In-depth studies of these aptamers suggest that the key characteristics of such regulatory active RNAs are their structural switching abilities and their binding dynamics. Conventional SELEX approaches seem to be inadequate to select for these characteristics, which may explain the lack of in vitro selected aptamers suited for engineering of synthetic riboswitches. In this review, we explore the functional principles of synthetic riboswitches, identify key characteristics of regulatory active in vitro selected aptamers and integrate these findings in context with available in vitro selection methods. Based on these insights, we propose to use a combination of capture-SELEX and subsequent functional screening for a more successful in vitro selection of aptamers that can be applied for the engineering of synthetic riboswitches.
Subject(s)
Aptamers, Nucleotide , Riboswitch , SELEX Aptamer Technique , Aptamers, Nucleotide/chemistry , Ligands , RNA/genetics , Riboswitch/genetics , Synthetic BiologyABSTRACT
In recent years, synthetic riboswitches have become increasingly important to construct genetic circuits in all three domains of life. In bacteria, synthetic translational riboswitches are often employed that modulate gene expression by masking the Shine-Dalgarno (SD) sequence in the absence or presence of a cognate ligand. For (halo-)archaeal translation, a SD sequence is not strictly required. The application of synthetic riboswitches in haloarchaea is therefore limited so far, also because of the molar intracellular salt concentrations found in these microbes. In this study, we applied synthetic theophylline-dependent translational riboswitches in the archaeon Haloferax volcanii. The riboswitch variants A through E and E∗ were chosen since they not only mask the SD sequence but also the AUG start codon by forming a secondary structure in the absence of the ligand theophylline. Upon addition of the ligand, the ribosomal binding site and start codon become accessible for translation initiation. Riboswitch E mediated a dose-dependent, up to threefold activation of the bgaH reporter gene expression. Raising the salt concentration of the culture media from 3 to 4 M NaCl resulted in a 12-fold increase in the switching capacity of riboswitch E, and switching activity increased up to 26-fold when the cultivating temperature was reduced from 45 to 30°C. To construct a genetic circuit, riboswitch E was applied to regulate the synthesis of the transcriptional activator GvpE allowing a dose-dependent activation of the mgfp6 reporter gene under P pA promoter control.
ABSTRACT
BACKGROUND: Alternative polyadenylation (APA) refers to the regulated selection of polyadenylation sites (PASs) in transcripts, which determines the length of their 3' untranslated regions (3'UTRs). We have recently shown that SRSF3 and SRSF7, two closely related SR proteins, connect APA with mRNA export. The mechanism underlying APA regulation by SRSF3 and SRSF7 remained unknown. RESULTS: Here we combine iCLIP and 3'-end sequencing and find that SRSF3 and SRSF7 bind upstream of proximal PASs (pPASs), but they exert opposite effects on 3'UTR length. SRSF7 enhances pPAS usage in a concentration-dependent but splicing-independent manner by recruiting the cleavage factor FIP1, generating short 3'UTRs. Protein domains unique to SRSF7, which are absent from SRSF3, contribute to FIP1 recruitment. In contrast, SRSF3 promotes distal PAS (dPAS) usage and hence long 3'UTRs directly by counteracting SRSF7, but also indirectly by maintaining high levels of cleavage factor Im (CFIm) via alternative splicing. Upon SRSF3 depletion, CFIm levels decrease and 3'UTRs are shortened. The indirect SRSF3 targets are particularly sensitive to low CFIm levels, because here CFIm serves a dual function; it enhances dPAS and inhibits pPAS usage by binding immediately downstream and assembling unproductive cleavage complexes, which together promotes long 3'UTRs. CONCLUSIONS: We demonstrate that SRSF3 and SRSF7 are direct modulators of pPAS usage and show how small differences in the domain architecture of SR proteins can confer opposite effects on pPAS regulation.
Subject(s)
3' Untranslated Regions , Cleavage And Polyadenylation Specificity Factor/genetics , Gene Expression Regulation , Poly A , Serine-Arginine Splicing Factors/metabolism , Alternative Splicing , Animals , Base Sequence , Mice , Models, Biological , Monomeric GTP-Binding Proteins/metabolism , Neurons , Phosphorylation , Poly(A)-Binding Proteins/metabolism , Polyadenylation , Protein Binding , Protein Interaction Domains and Motifs , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
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.
Subject(s)
Aptamers, Nucleotide/chemistry , Ciprofloxacin/chemistry , RNA/chemistry , Riboswitch , SELEX Aptamer Technique/methods , Ligands , Nucleic Acid ConformationABSTRACT
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.
Subject(s)
Alternative Splicing , Aptamers, Nucleotide/metabolism , Nuclear Localization Signals/metabolism , RNA Splice Sites , Repressor Proteins/metabolism , Active Transport, Cell Nucleus/drug effects , Alternative Splicing/drug effects , Aptamers, Nucleotide/chemical synthesis , Aptamers, Nucleotide/genetics , Base Pairing , Base Sequence , Binding Sites , Cell Nucleus/chemistry , Cell Nucleus/metabolism , Cytosol/chemistry , Cytosol/metabolism , Doxycycline/pharmacology , Exons , HeLa Cells , Humans , Introns , Models, Molecular , Nuclear Localization Signals/chemistry , Nuclear Localization Signals/genetics , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Repressor Proteins/chemistry , Repressor Proteins/geneticsABSTRACT
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.
Subject(s)
Arachidonate 5-Lipoxygenase/metabolism , MicroRNAs/metabolism , RNA Processing, Post-Transcriptional , Arachidonate 5-Lipoxygenase/genetics , Cell Line, Tumor , Down-Regulation , Humans , MicroRNAs/genetics , Ribonuclease III/metabolism , TranscriptomeABSTRACT
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 â¼50-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 â¼170- and â¼50-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.
Subject(s)
Cell Differentiation , DEAD-box RNA Helicases/metabolism , MicroRNAs/metabolism , Monocytes/metabolism , Prostaglandin-E Synthases/metabolism , Proteolysis , Ribonuclease III/metabolism , Cells, Cultured , DEAD-box RNA Helicases/genetics , Dinoprostone/pharmacology , Hematopoiesis , Humans , Lipopolysaccharides/pharmacology , MicroRNAs/genetics , Monocytes/cytology , Monocytes/drug effects , Prostaglandin-E Synthases/genetics , Receptors, Prostaglandin E, EP2 Subtype/metabolism , Receptors, Prostaglandin E, EP4 Subtype/metabolism , Ribonuclease III/genetics , Zymosan/pharmacologyABSTRACT
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.
Subject(s)
Aptamers, Nucleotide/chemistry , DNA-Binding Proteins/ultrastructure , Escherichia coli Proteins/ultrastructure , Protein Conformation , Aptamers, Nucleotide/genetics , Crystallography, X-Ray , DNA, B-Form/chemistry , DNA, B-Form/genetics , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Escherichia coli/genetics , Escherichia coli/ultrastructure , Escherichia coli Proteins/chemistry , Gene Expression Regulation/genetics , Macromolecular Substances/chemistry , Macromolecular Substances/ultrastructure , Models, Molecular , Protein Binding/genetics , RNA/chemistry , RNA/geneticsABSTRACT
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.