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.

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.

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.

SICOR: Subgraph Isomorphism Comparison of RNA Secondary Structures.

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.

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.

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.

Characterization and Inkjet Printing of an RNA Aptamer for Paper-Based Biosensing of Ciprofloxacin.

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.

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.

Tuning the Performance of Synthetic Riboswitches using Machine Learning.

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.

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.

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.

TGFβ/SMAD signalling modulates MLL and MLL-AF4 mediated 5-lipoxygenase promoter activation.

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-β (TGFβ) 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 TGFβ/SMAD signalling in 5-LO promoter activation.

In vitro selection of antibiotic-binding aptamers.

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.

Water-soluble Py-BIPS spiropyrans as photoswitches for biological applications.

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.

RNA aptamers as genetic control devices: the potential of riboswitches as synthetic elements for regulating gene expression.

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.

Sequence elements distal to the ligand binding pocket modulate the efficiency of a synthetic riboswitch.

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.

Building a stable RNA U-turn with a protonated cytidine.

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).

Synthetic riboswitches - A tool comes of age.

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.

Gene expression of chicken gonads is sex- and side-specific.

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.