Riboswitch AND Sensor

A major revelation of genome-scale biological studies in the post-genomic era has been that two-thirds of human genes do not encode proteins. The majority of non-coding RNA transcripts in humans are long non-coding RNA (lncRNA) molecules, non-protein-coding regulatory transcripts with sizes greater than 500 nucleotides. LncRNAs are involved in nearly every aspect of cellular physiology, playing fundamental regulatory roles both in normal cells and in disease. As result, they are functionally linked to multiple human diseases, from cancer to autoimmune, inflammatory, and neurological disorders...

Genetically encoded sensor-actuator circuits aim at reprogramming cellular functions and are inspired by intracellular networks: from the input signal (sensor) to the desired output response (actuator). In the last years, circuits with posttranscriptional regulation of gene expression have aroused great interest for their potential in the biomedical space. Posttranscriptional modulation can be achieved with ribozymes, riboswitches (simple regulatory elements based on RNA secondary structures), noncoding RNAs, and RNA-binding proteins (RBPs)...

Leveraging riboswitches, non-coding mRNA fragments pivotal to gene regulation, poses a challenge in effectively selecting and enriching these functional genetic sensors, which can toggle between ON and OFF states in response to their cognate inducers. Here, we show our engineered phage T7, enabling the evolution of a theophylline riboswitch. We have replaced T7's DNA polymerase with a transcription factor controlled by a theophylline riboswitch and have created two types of host environments to propagate the engineered phage...

Detection of metabolites in real time and in whole cells requires effective molecular sensors. In this regard, fluorogenic light-up RNAs have recently become important tools for small-molecule detection in cells. However, the construction of light-up RNA sensors is an arduous task that requires structural knowledge of both the sensor and reporter RNA. De novo strategies for selecting sensors from RNA libraries are limited and are mostly restricted to known aptamers and riboswitches. Here, we provide a solution to this problem by developing a capture-SELEX variant that allows the obtained libraries and aptamers to be linked to fluorogenic RNAs in a modular and allosteric manner...

Engineered RNAs have applications in diverse fields from biomedical to environmental. In many cases, the folding of the RNA is critical to its function. Here we describe a strategy to improve the response time of a riboswitch-based fluorescent biosensor. Systematic mutagenesis was performed to either make transpose or transition base pair mutants or introduce orthogonal base pairs. Both natural and unnatural base pair mutants were found to improve the biosensor response time without compromising fold turn-on or ligand affinity...

Riboswitches sense various ions in bacteria and activate gene expression to synthesize proteins that help maintain ion homeostasis. The crystal structure of the aptamer domain (AD) of the fluoride riboswitch shows that the F- ion is encapsulated by three Mg2+ ions bound to the ligand-binding domain (LBD) located at the core of the AD. The assembly mechanism of this intricate structure is unknown. To this end, we performed computer simulations using coarse-grained and all-atom RNA models to bridge multiple time scales involved in riboswitch folding and ion binding...

Nucleic acids can undergo conformational changes upon binding small molecules. These conformational changes can be exploited to develop new therapeutic strategies through control of gene expression or triggering of cellular responses and can also be used to develop sensors for small molecules such as neurotransmitters. Many analytical approaches can detect dynamic conformational change of nucleic acids, but they need labeling, are expensive, and have limited time resolution. The nanopore approach can provide a conformational snapshot for each nucleic acid molecule detected, but has not been reported to detect dynamic nucleic acid conformational change in response to small -molecule binding...

Quantification of the concentration of particular cellular metabolites reports on the actual utilization of metabolic pathways in physiological and pathological conditions. Metabolite concentration also constitutes the readout for screening cell factories in metabolic engineering. However, there are no direct approaches that allow for real-time assessment of the levels of intracellular metabolites in single cells. In recent years, the modular architecture of natural bacterial RNA riboswitches has inspired the design of genetically encoded synthetic RNA devices that convert the intracellular concentration of a metabolite into a quantitative fluorescent signal...

We describe a sensitive and selective method for the determination of tetracycline content in foods using a riboswitch sensor. The sensor is based on a cell-free expression system that can be lyophilized to produce paper-based sensors or tube-based sensors for long-term storage. The riboswitch constructed using artificially screened tetracycline RNA aptamers was cloned into the pET-28a(+) vector of Escherichia coli TOP 10. The expression of the green fluorescent protein was positively correlated with the concentration of tetracyclines...

Fluorescent RNA-based biosensors are useful tools for real-time detection of molecules in living cells. These biosensors typically consist of a chromophore-binding aptamer and a target-binding aptamer, whereby the chromophore-binding aptamer is destabilized until a target is captured, which causes a conformational change to permit chromophore binding and an increase in fluorescence. The target-binding region is typically fabricated using known riboswitch motifs, which are already known to have target specificity and undergo structural changes upon binding...

Cell-free genetically encoded biosensors have been developed to detect small molecules and nucleic acids, but they have yet to be reliably engineered to detect proteins. Here we develop an automated platform to convert protein-binding RNA aptamers into riboswitch sensors that operate within low-cost cell-free assays. We demonstrate the platform by engineering 35 protein-sensing riboswitches for human monomeric C-reactive protein, human interleukin-32γ, and phage MS2 coat protein. The riboswitch sensors regulate output expression levels by up to 16-fold with input protein concentrations within the human serum range...

Riboswitches are conserved structural ribonucleic acid (RNA) sensors that are mainly found to regulate a large number of genes/operons in bacteria. Presently, >50 bacterial riboswitch classes have been discovered, but only the thiamine pyrophosphate riboswitch class is detected in a few eukaryotes like fungi, plants and algae. One of the most important challenges in riboswitch research is to discover existing riboswitch classes in eukaryotes and to understand the evolution of bacterial riboswitches. However, traditional search methods for riboswitch detection have failed to detect eukaryotic riboswitches besides just one class and any distant structural homologs of riboswitches...

Pseudomonas aeruginosa is intrinsically resistant to many classes of antibiotics, reflecting the restrictive nature of its outer membrane and the action of its numerous efflux systems. However, the dynamics of compound uptake, retention, and efflux in this bacterium remain incompletely understood. Here, we exploited the sensor capabilities of a Z-nucleotide-sensing riboswitch to create an experimental system able to identify physicochemical and structural properties of compounds that permeate the bacterial cell, avoid efflux, and perturb the folate cycle or de novo purine synthesis...

UNLABELLED: Pseudomonas aeruginosa is intrinsically resistant to many classes of antibiotics, reflecting the restrictive nature of its outer membrane and the action of its numerous efflux systems. However, the dynamics of compound uptake, retention and efflux in this bacterium remain incompletely understood. Here, we exploited the sensor capabilities of a Z-nucleotide sensing riboswitch to create an experimental system able to identify physicochemical and structural properties of compounds that permeate the bacterial cell, avoid efflux, and perturb the folate cycle or de novo purine synthesis...

Flavin mononucleotide (FMN) is the active form of riboflavin. It has a wide range of application scenarios in the pharmaceutical and food additives. However, there are limitations in selecting generic high-throughput screening platforms that improve the properties of enzymes. First, the biosensor in response to FMN concentration was constructed using the FMN riboswitch and confirmed the function of this sensor. Next, the FMN binding site of the sensor was saturated with a mutation that increased its fluorescence range by approximately 127%...

Ligand-binding RNAs (RNA aptamers) are widespread in the three domains of life, serving as sensors of metabolites and other small molecules. When aptamers are embedded within RNA transcripts as components of riboswitches, they can regulate gene expression upon binding their ligands. Previous methods for biochemical validation of computationally predicted aptamers are not well-suited for rapid screening of large numbers of RNA aptamers. Therefore, we utilized DRaCALA (Differential Radial Capillary Action of Ligand Assay), a technique designed originally to study protein-ligand interactions, to examine RNA-ligand binding, permitting rapid screening of dozens of RNA aptamer candidates concurrently...

Cell-free systems have enabled the development of genetically encoded biosensors to detect a range of environmental and biological targets. Encapsulation of these systems in synthetic membranes to form artificial cells can reintroduce features of the cellular membrane, including molecular containment and selective permeability, to modulate cell-free sensing capabilities. Here, we demonstrate robust and tunable performance of a transcriptionally regulated, cell-free riboswitch encapsulated in lipid membranes, allowing the detection of fluoride, an environmentally important molecule...

Food safety has always been a hot issue of social concern, and biosensing has been widely used in the field of food safety detection. Compared with traditional aptamer-based biosensors, aptamer-based riboswitch biosensing represents higher precision and programmability. A riboswitch is an elegant example of controlling gene expression, where the target is coupled to the aptamer domain, resulting in a conformational change in the downstream expression domain and determining the signal output. Riboswitch-based biosensing can be extensively applied to the portable real-time detection of food samples...

Nucleic acids are not only essential actors of cell life but also extremely appealing molecular objects in the development of synthetic molecules for biotechnological application, such as biosensors to report on the presence and concentration of a target ligand by emission of a measurable signal. In this work, FluorMango, a fluorogenic ribonucleic acid (RNA)-based biosensor specific for fluoride is introduced. The molecule consists of two RNA aptamer modules, a fluoride-specific sensor derived from the crcB riboswitch which changes its structure upon interaction with the target ion, and the light-up RNA Mango-III that emits fluorescence when complexed with a fluorogen...

To synthesize a chirally inverted ribosome with the goal of building mirror-image biology systems requires the preparation of kilobase-long mirror-image ribosomal RNAs that make up the structural and catalytic core and about two-thirds of the molecular mass of the mirror-image ribosome. Here, we chemically synthesized a 100-kilodalton mirror-image T7 RNA polymerase, which enabled efficient and faithful transcription of the full-length mirror-image 5 S , 16 S , and 23 S ribosomal RNAs from enzymatically assembled long mirror-image genes...