RNA aptamers targeting bacterial repressors show significant promise in synthetic biology. We determined the crystal structure of the SELEX-derived aptamer Dap1 in complex with its target, the GntR/HutC repressor family member DasR. In the complex, two monomers of Dap1 individually sequester the two DNA-binding domains (DBDs) of the dimeric repressor. The DBDs of DasR interact with Dap1 in a highly similar fashion to what is observed in GntR-dsDNA complexes. This also holds true at the level of single amino acids, and identical arginine residues play crucial roles in both RNA and DNA recognition. The structure shows how DasR binding to Dap1 competes with DasR binding to its operator dre sites and, hence, how Dap1 is able to induce DasR-controlled gene transcription in vivo. A structure-informed Dap1 aptamer pool randomized at only four positions allowed for the identification of the novel aptamer Dap1m1 that binds and de-represses the DasR homologous protein NagR instead of DasR. This shows that the complex structure can serve as a blueprint for the design of novel aptamer-protein interaction pairs, opening up new venues for the generation of aptamers directed against other GntR/HutC family members and possibly also against other repressor classes.

The design of large genetic circuits requires genetic regulatory devices capable of performing complex logic operations that place no excessive metabolic burden on the host cell. Hybrid riboswitches, synthetically enhanced compact RNA elements (<100 nucleotides) that form a tertiary structure with the ability to specifically bind two different target molecules, can be used to design genetic regulators that emulate Boolean logic. When inserted into the 5' UTR of a messenger RNA, these devices can regulate translation initiation upon specific binding of one or both ligands without the need for additional auxiliary factors. The goal of this study is to design hybrid riboswitches that emulate Boolean NAND logic in yeast. We propose a novel machine learning-based design framework combining high-throughput in vivo screening and deep Bayesian optimization. Through an initial screening, we discovered a hybrid riboswitch with NAND behavior. Using batch Bayesian optimization with an ensemble neural network as surrogate, we improved the NAND functionality of our hybrid riboswitch with respect to a performance score, thereby achieving near-digital NAND behavior. With its focus on model-based and score-driven design, our proposed method can complement experiment-driven approaches by allowing fine grained adaptation of functionality, including constructs sensitive to single nucleotide changes.

RNA devices, including riboswitches, aptazymes, and RNA-based biosensors, have become essential components in synthetic and molecular biology. These systems make use of RNA's modularity and structural diversity to build programmable tools for sensing, regulation, and cellular computation. To drive these systems, RNA aptamers usually need to undergo a conformational change upon ligand binding. However, the limited availability of such aptamers has restricted the development and application of RNA devices, particularly in mammalian systems. Traditional selection methods often prioritize high-affinity binding, resulting in a scarcity of conformation-switching aptamers. Here, we addressed this limitation by selecting a caffeine-binding aptamer with RNA Capture-SELEX and in vivo screenings. This aptamer functioned as a modular regulator of riboswitches and ribozymes in Saccharomyces cerevisiae and mammalian cells. Through new optofluidic screenings, we overcame throughput and sequence-function challenges inherent in ribozyme screening for mammalia. Additionally, a straightforward grafting method transformed the aptamer into a fluorogenic iSpinach aptasensor. This work demonstrates the successful selection of a modular and communicating aptamer for a challenging target like caffeine and establishes robust strategies for their modular integration into diverse RNA platforms. These strategies pave the way for broadening the repertoire of aptamers and expanding the potential of RNA-based synthetic biology.

Synthetic riboswitches have undergone great development in the past decade, evolving into valuable regulatory tools. Operating entirely at the RNA level and independently of auxiliary proteins, they offer a promising alternative to protein-based systems such as TetON/OFF or CRISPR-Cas. As compact, modular RNA elements they unite sensing and regulatory functions within a single molecule, giving them the advantages of high modularity, portability and low metabolic burden. Here, we explore the unique features of synthetic riboswitches, highlight key applications, assess current bottlenecks and limitations and put them in context with emerging solutions, to emphasise the potential of synthetic riboswitches.

The human 5-lipoxygenase (5-LOX), which is encoded by the arachidonate 5-lipoxygenase (ALOX5) gene, has its canonical function in leukotriene (LT) biosynthesis, which controls inflammatory and allergic responses. Besides oxylipin formation from polyunsaturated fatty acids, 5-LOX has several noncanonical functions. It acts as transcriptional regulator in the nucleus but also interacts with Dicer and modulates microRNA expression and processing. In this study, we employed a tetracycline riboswitch-controlled cassette-exon system to conditionally control ALOX5 expression in the monocytic leukemic cell line MonoMac6. Synthetic riboswitches are gaining increasing interest as a means of controlling transgene expression, with applications in functional genomics and potential therapeutic strategies. We designed an artificial ALOX5 gene that contains two cassette exons with premature termination codons (PTCs), thus only being expressed when both synthetic exons are skipped. The switchable ALOX5 gene was transduced into MonoMac6 5-LOX knock-out (KO) cells, thereby enabling the tetracycline-dependent re-expression of 5-LOX proteins. The newly established cell line was characterized in terms of tetracycline dose dependency and switching kinetics. Induction of ALOX5 exerted the non-canonical 5-LOX effects on prostaglandin-endoperoxide synthase 2 (PTGS2) and L-kynureninase (KYNU) gene expression. This allowed us to demonstrate the outstanding advantages of a riboswitch-controlled system in terms of time dependency and gene function. The novel MonoMac6 cell line now provides a perfect tool for further research into the non-canonical functions of 5-LOX.

Synthetic riboswitches are attracting increasing interest for a diverse range of applications, including synthetic biology, functional genomics, and prospective therapeutic strategies. This study demonstrates that controlling alternative splicing with synthetic riboswitches represents a promising approach to effectively regulating transgene expression in mammalian cells. However, the function of synthetic riboswitches in the eukaryotic system in controlling gene expression is often limited to certain genes or cell types. So far, strategies to increase the dynamic range of regulation have been focused on adapting and modifying the riboswitch sequence itself without taking into account the context in which the riboswitch was inserted. In the present study, the tetracycline riboswitch was chosen to investigate the effects of the context and insertion site of a cassette exon within the gene to control the expression of an artificial arachidonate 5-lipoxygenase gene (ALOX5) in HEK293 cells. We demonstrate here that the use of riboswitch-controlled cassette exons for the control of gene expression via alternative splicing can be easily transferred to another gene through the process of contextual sequence adaptation. This was achieved through the introduction of gene-specific intronic and exonic sequences with different intron lengths and positions being tested. In contrast, the introduction of nonadapted constructs resulted in an unanticipated functionality outcome of the gene switch. Furthermore, we demonstrate that the combination of two cassette exons into a single gene resulted in a notable enhancement in the dynamic range. Finally, we generated a novel riboswitch-controlled splicing concept that enabled us to switch 5-LO wild-type to expression of an ALOX5 isoform that lacks exon 13 (5-LOΔ13). Taken together, this study demonstrates that synthetic riboswitches that control alternative splicing are a powerful tool to regulate gene expression when applied in combination with gene-specific intronic and exonic sequences.

Synthetic riboswitches, genetic regulatory elements composed entirely of RNA, have been engineered to control a variety of mechanisms at the level of both transcription and translation in all domains of life. The efficiency of riboswitch regulation can be increased by inserting two of them into an mRNA sequence in close proximity, resulting in a tandem riboswitch. The tandem state results in improved regulation beyond that of a single riboswitch by allowing both binding pockets to contribute to a higher dynamic range. The focus of this study was to create a novel tandem riboswitch design by integrating the binding pockets of two different riboswitches into one continuous structure, thereby creating a dual-input hybrid riboswitch. These hybrids remain compact in size with a shorter sequence length compared to a tandem riboswitch, while taking advantage of the binding pockets and scaffold sequences provided by both parental riboswitches. Through rational design, hybrid constructs derived from the combination of tetracycline-, tobramycin-, neomycin-, and paromomycin-binding riboswitches were engineered that significantly increase the dynamic range (e.g., from 14- to 36-fold for tobramycin) while increasing their expression levels in the absence of ligand (e.g., 28% to 68% expression for tetracycline). This study expands the toolbox of synthetic riboswitches and establishes general design guidelines applicable to similar riboswitches. Additionally, the dual-input state makes hybrid riboswitches an interesting target for the design of genetic regulators following Boolean logic.

Recently, a novel tobramycin-responsive riboswitch was developed by a combination of Capture-SELEX and in vivo screening. This riboswitch regulates translation initiation in eukaryotes with a high dynamic range and remarkable ligand affinity and selectivity. Its secondary structure differs from all previously described aminoglycoside-binding RNA motifs, suggesting a novel mode of ligand recognition. To provide a structural basis for the remarkable regulatory efficiency and ligand selectivity of this riboswitch, we investigated its structure in complex with its cognate ligand tobramycin by high-resolution solution nuclear magnetic resonance spectroscopy. The structure of the complex reveals a novel structural organization for an aminoglycoside binding motif with a unique pattern of intermolecular hydrogen bonds and electrostatic interactions between the RNA and functional groups of all three rings of the ligand. In contrast to other aminoglycoside binding motifs, ligand binding of the tobramycin riboswitch is coupled with the formation of an extensive network of noncanonical RNA-RNA interactions, rationalizing the high ligand affinity of this small hairpin RNA. Comparison with the free form of the RNA shows that the latter is much less compact, lacking many RNA-RNA interactions, in particular in the bulge regions, thereby immediately providing a rationale for the exceptional switching efficiency of this synthetic riboswitch.

The application of synthetic riboswitches or aptamer-based biosensors for the monitoring of engineered metabolic pathways greatly depends on a high degree of target molecule specificity. Since metabolic pathways include close derivatives that often differ only in single moieties, the binding specificity of aptamers utilized for these systems has to be high. In the present study, we selected an RNA aptamer that is highly specific in its binding to homoeriodictyol while discriminating its close derivatives eriodictyol and naringenin. This high degree in specificity was achieved through three consecutive SELEX approaches while the selection parameters were adjusted and refined from one to the next. The adjustments along the process, with the selection outcome and next-generation sequencing analysis of the selection rounds, led to valuable insights into the stringency necessary to facilitate target specificity in aptamers obtained from SELEX. From the third selection, we obtained a highly binding specific aptamer and examined its structure and binding properties. Overall, our results connect the importance of selection stringency with SELEX outcome and aptamer specificity while providing a highly selective, homoeriodictyol-binding RNA aptamer.

The design of large genetic circuits requires genetic regulatory devices capable of performing complex logic operations. Hybrid riboswitches, synthetically enhanced compact RNA elements (<100 nucleotides) that form a tertiary structure with the ability to specifically bind two different target molecules, can be used to design genetic regulators that emulate Boolean logic. When inserted into the 5' UTR of an mRNA, these devices can regulate translation initiation upon specific binding of one or both ligands. The goal of this study is to design hybrid riboswitches that emulate Boolean NAND logic in yeast. We propose a novel machine learning-based design framework combining high-throughput in vivo screening and deep Bayesian optimization. Through an initial screening, we discovered a hybrid riboswitch with NAND behavior. Using batch Bayesian optimization with an ensemble neural network as surrogate, we further improve the NAND functionality of our hybrid riboswitch with respect to a performance score, thereby achieving near digital NAND behavior. With its focus on model-based and score-driven design, our proposed method can complement experiment driven approaches by allowing fine grained adaptation of functionality, including constructs sensitive to single nucleotide changes. ### Competing Interest Statement The authors have declared no competing interest.

Synthetic elements made entirely of RNA are suitable as regulatory elements in genetically modified systems and as biosensors. Such RNA aptamers are highly structured nucleotide sequences capable of specifically binding a target molecule. De novo selection of aptamers against a wide variety of potential targets is possible. By integrating RNA aptamers as binding domains into natural or synthetically designed regulatory circuits in the form of so-called riboswitches, new regulatory mechanisms can be generated that do not require additional regulatory elements. In addition, these binding domains can be used in cell-free systems to perform highly specific and affine molecular detection assays. By presenting two well-established aptamer designs, we aim to demonstrate the potential of RNA aptamer-based riboswitches and biosensors in various applications.

Progress in synthetic biology relies on developing new tools for circuit engineering. RNA aptamers, single stranded small RNA molecules, show promise by binding targets specifically. Aptamers targeting bacterial repressors offer new opportunity for conditional gene regulation. We develop aptamers that bind bacterial repressor proteins thereby activating repressor-controlled gene expression. These repressor-aptamer pairs significantly expand the synthetic biology tool box.

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.

Enzymes are instrumental to life and key actors of pathologies, making them relevant drug targets. Most enzyme inhibitors consist of small molecules. Although efficient, their development is long, costly and can come with unwanted off-targeting. Substantial gain in specificity and discovery efficiency is possible using biologicals. Best exemplified by antibodies, these drugs derived from living systems display high specificity and their development is eased by harnessing natural evolution. Aptamers are nucleic acids sharing functional similarities with antibodies while being deprived of many of their limitations. Yet, the success rate of inhibitory aptamer discovery remained hampered by the lack of an efficient discovery pipeline. In this work, we addressed this issue by introducing an ultrahigh-throughput strategy combining in vitro selection, microfluidic screening and bioinformatics. We demonstrate its efficiency by discovering a modified aptamer that specifically and strongly inhibits SPM-1, a beta-lactamase that remained recalcitrant to the development of potent inhibitors. ### Competing Interest Statement The authors have declared no competing interest.

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.

Abstract 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.

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.

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.

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.