RNA-based Sensors Research Articles

RNA-Based Sensor Systems for Affordable Diagnostics in the Age of Pandemics.

In the era of the COVID-19 pandemic, the significance of point-of-care (POC) diagnostic tools has become increasingly vital, driven by the need for quick and precise virus identification. RNA-based sensors, particularly toehold sensors, have emerged as promising candidates for POC detection systems due to their selectivity and sensitivity. Toehold sensors operate by employing an RNA switch that changes the conformation when it binds to a target RNA molecule, resulting in a detectable signal. This review focuses on the development and deployment of RNA-based sensors for POC viral RNA detection with a particular emphasis on toehold sensors. The benefits and limits of toehold sensors are explored, and obstacles and future directions for improving their performance within POC detection systems are presented. The use of RNA-based sensors as a technology for rapid and sensitive detection of viral RNA holds great potential for effectively managing (dealing/coping) with present and future pandemics in resource-constrained settings.

Modular Approach for Rapid Identification of RNA-Based Sensors.

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. The approach is generally applicable and allows for rapid modular allosteric assembly with green- or red-shifted fluorogenic RNAs.

Genetically encoded RNA-based sensors with Pepper fluorogenic aptamer.

Sensors to measure the abundance and signaling of intracellular molecules are crucial for understanding their physiological functions. Although conventional fluorescent protein-based sensors have been designed, RNA-based sensors are promising imaging tools. Numerous RNA-based sensors have been developed. These sensors typically contain RNA G-quadruplex (RG4) motifs and thus may be suboptimal in living cells. Here we describe RNA-based sensors based on Pepper, a fluorogenic RNA without an RG4 motif. With Pepper, we engineered various sensors for metabolites, synthetic compounds, proteins and metal ions in vitro and in living cells. In addition, these sensors show high activation and selectivity, demonstrating their universality and robustness. In the case of sensors responding to S-adenosylmethionine (SAM), a metabolite produced by methionine adenosyltransferase (MATase), we showed that our sensors exhibited positively correlated fluorescence responding to different SAM levels. Importantly, we revealed the SAM biosynthesis pathway and monitored MATase activity and gene expression spatiotemporally in living individual human cells. Additionally, we constructed a ratiometric SAM sensor to determine the inhibition efficacy of a MATase inhibitor in living cells. Together, these sensors comprising Pepper provide a useful platform for imaging diverse cellular targets and their signaling pathway.

Real-Time Assessment of Intracellular Metabolites in Single Cells through RNA-Based Sensors.

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. These so-called RNA-based sensors are composed of a metabolite-binding RNA aptamer as the sensor domain, connected through an actuator segment to a signal-generating reporter domain. However, at present, the variety of available RNA-based sensors for intracellular metabolites is still very limited. Here, we go through natural mechanisms for metabolite sensing and regulation in cells across all kingdoms, focusing on those mediated by riboswitches. We review the design principles underlying currently developed RNA-based sensors and discuss the challenges that hindered the development of novel sensors and recent strategies to address them. We finish by introducing the current and potential applicability of synthetic RNA-based sensors for intracellular metabolites.

Reversible Modulation of Aptamer-Ligand Binding in RNA Light-Up Aptamers Containing G-Quadruplex Using Chemical Stimuli.

Regulating a system in equilibrium transiently to out-of-equilibrium by using certain stimuli is the strategy used by natural biomolecules to function. Herein, we showed that the interaction of synthetic RNA aptamers, having a G-quadruplex core structure, with their corresponding ligands could be regulated from their equilibrium state to non-equilibrium state in a reversible manner using simple chemical stimuli (Ag+ and cysteine). The approach would be useful for designing aptamer regulators that work in a dynamic nucleic acid network, where a strict control on aptamer-ligand interaction is needed. In addition, to the best of our knowledge, this is the first report which shows that RNA G-quadruplexes can be disrupted by the addition of silver ions. This would be useful not only in designing RNA-based sensors or regulators but would also be useful for understanding the role of metal ions in RNA folding and catalysis.

The Biochemical Landscape of Riboswitch Ligands.

More than 55 distinct classes of riboswitches that respond to small metabolites or elemental ions have been experimentally validated to date. The ligands sensed by these riboswitches are biased in favor of fundamental compounds or ions that are likely to have been relevant to ancient forms of life, including those that might have populated the "RNA World", which is a proposed biochemical era that predates the evolutionary emergence of DNA and proteins. In the following text, I discuss the various types of ligands sensed by some of the most common riboswitches present in modern bacterial cells and consider implications for ancient biological processes centered on the proven capabilities of these RNA-based sensors. Although most major biochemical aspects of metabolism are represented by known riboswitch classes, there are striking sensory gaps in some key areas. These gaps could reveal weaknesses in the performance capabilities of RNA that might have hampered RNA World evolution, or these could highlight opportunities to discover additional riboswitch classes that sense essential metabolites.

Live-Cell Imaging of Guanosine Tetra- and Pentaphosphate (p)ppGpp with RNA-based Fluorescent Sensors*.

Guanosine tetra- and pentaphosphate, (p)ppGpp, are important alarmone nucleotides that regulate bacterial survival in stressful environment. A direct detection of (p)ppGpp in living cells is critical for our understanding of the mechanism of bacterial stringent response. However, it is still challenging to image cellular (p)ppGpp. Here, we report RNA-based fluorescent sensors for the live-cell imaging of (p)ppGpp. Our sensors are engineered by conjugating a recently identified (p)ppGpp-specific riboswitch with a fluorogenic RNA aptamer, Broccoli. These sensors can be genetically encoded and enable direct monitoring of cellular (p)ppGpp accumulation. Unprecedented information on cell-to-cell variation and cellular dynamics of (p)ppGpp levels is now obtained under different nutritional conditions. These RNA-based sensors can be broadly adapted to study bacterial stringent response.

Engineered RNA biosensors enable ultrasensitive SARS-CoV-2 detection in a simple color and luminescence assay.

The continued resurgence of the COVID-19 pandemic with multiple variants underlines the need for diagnostics that are adaptable to the virus. We have developed toehold RNA-based sensors across the SARS-CoV-2 genome for direct and ultrasensitive detection of the virus and its prominent variants. Here, isothermal amplification of a fragment of SARS-CoV-2 RNA coupled with activation of our biosensors leads to a conformational switch in the sensor. This leads to translation of a reporter protein, for example, LacZ or nano-lantern that is easily detected using color/luminescence. By optimizing RNA amplification and biosensor design, we have generated a highly sensitive diagnostic assay that is capable of detecting as low as 100 copies of viral RNA with development of bright color. This is easily visualized by the human eye and quantifiable using spectrophotometry. Finally, this PHAsed NASBA-Translation Optical Method (PHANTOM) using our engineered RNA biosensors efficiently detects viral RNA in patient samples. This work presents a powerful and universally accessible strategy for detecting COVID-19 and variants. This strategy is adaptable to further viral evolution and brings RNA bioengineering center-stage.

Naturally occurring three-way junctions can be repurposed as genetically encoded RNA-based sensors

Small molecules can be imaged in living cells using biosensors composed of RNA. However, RNA-based devices are difficult to design. Here, we describe a versatile platform for designing RNA-based fluorescent small-molecule sensors using naturally occurring highly stable three-way junction RNAs. We show that ligand-binding aptamers and fluorogenic aptamers can be inserted into three-way junctions and connected in a way that enables the three-way junction to function as a small-molecule-regulated fluorescent sensor invitro and in cells. The sensors are designed so that the interhelical stabilizing interactions in the three-way junction are only induced upon ligand binding. We use these RNA-based devices to measure the dynamics of S-adenosylmethionine levels in mammalian cells in real time. We show that this strategy is compatible with diverse metabolite-binding RNA aptamers, fluorogenic aptamers, and three-way junctions. Overall, these data demonstrate a versatile method for readily generating RNA devices that function in living cells.

A synthetic RNA-based biosensor for fructose-1,6-bisphosphate that reports glycolytic flux

RNA-based sensors for intracellular metabolites are a promising solution to the emerging issue of metabolic heterogeneity. However, their development, i.e., the conversion of an aptamer into an invivo-functional intracellular metabolite sensor, still harbors challenges. Here, we accomplished this for the glycolytic flux-signaling metabolite, fructose-1,6-bisphosphate (FBP). Starting from invitro selection of an aptamer, we constructed device libraries with a hammerhead ribozyme as actuator. Using high-throughput screening in yeast with fluorescence-activated cell sorting (FACS), next-generation sequencing, and genetic-environmental perturbations to modulate the intracellular FBP levels, we identified a sensor that generates ratiometric fluorescent readout. An abrogated response in sensor mutants and occurrence of two sensor conformations-revealed by RNA structural probing-indicated invivo riboswitching activity. Microscopy showed that the sensor can differentiate cells with different glycolytic fluxes within yeast populations, opening research avenues into metabolic heterogeneity. We demonstrate the possibility to generate RNA-based sensors for intracellular metabolites for which no natural metabolite-binding RNA element exits.

Engineering Allosteric Ribozymes to Detect Thiamine Pyrophosphate in Whole Blood.

Thiamine deficiency contributes to several human diseases including Alzheimer's. As its biologically active form, thiamine pyrophosphate (TPP) has been considered as a potential biomarker for Alzheimer's disease (AD) based on several clinical reports that apparently lower blood TPP levels were found in patients with mild cognitive impairment to AD. However, highly sensitive and high-throughput detection of TPP in biological fluids remains an analytical challenge. Here, we report engineering RNA-based sensors to quantitatively measure TPP concentrations in whole blood samples with a detection limit down to a few nM. By fusing a TPP-specific aptamer with the hammerhead ribozyme for in vitro selection, we isolated an allosteric ribozyme with an EC50 value (68 nM) similar to the aptamer's KD value (50 nM) for TPP, which for the first time demonstrates the possibility to maintain the effector binding affinity of the aptamer in such engineered allosteric RNA constructs. Meanwhile, we developed a new blood sample preparation protocol to be compatible with RNA. By coupling the TPP-induced ribozyme cleavage event with isothermal amplification, we achieved fluorescence monitoring of whole blood TPP levels through the "mix-and-read" operation with high-throughput potential. We expect that the engineered TPP-sensing RNAs will facilitate clinical research on AD as well as other thiamine-related diseases.

Rational Design of Allosteric Fluorogenic RNA Sensors for Cellular Imaging.

Fluorescence-based tools are invaluable in studying cellular functions. Traditional small molecule or protein-based fluorescent sensors have been widely used for the cellular imaging, but the choice of targets is still limited. Recently, fluorogenic RNA-based sensors gained lots of attention. This novel sensor system can function as a general platform for various cellular targets. Here, we describe the steps to rationally design, optimize, and apply fluorogenic RNA-based sensors, using the intracellular imaging of tetracycline in living E. coli cells as an example.

Imaging Intracellular S-Adenosyl Methionine Dynamics in Live Mammalian Cells with a Genetically Encoded Red Fluorescent RNA-Based Sensor.

To understand the role of intracellular metabolites in cellular processes, it is important to measure the dynamics and fluxes of small molecules in living cells. Although conventional metabolite sensors composed of fluorescent proteins have been made to detect some metabolites, an emerging approach is to use genetically encoded sensors composed of RNA. Because of the ability to rapidly generate metabolite-binding RNA aptamers, RNA-based sensors have the potential to be designed more readily than protein-based sensors. Numerous strategies have been developed to convert the green-fluorescent Spinach or Broccoli fluorogenic RNA aptamers into metabolite-regulated sensors. Nevertheless, red fluorescence is particularly desirable because of the low level of red background fluorescence in cells. However, the red fluorescent variant of the Broccoli aptamer, Red Broccoli, does not exhibit red fluorescence in cells when imaged with its cognate fluorophore. It is not known why Red Broccoli is fluorescent in vitro but not in live mammalian cells. Here, we develop a new fluorophore, OBI (3,5-difluoro-4-hydroxybenzylidene-imidazolinone-2-oxime-1-benzoimidazole), which binds Red Broccoli with high affinity and makes Red Broccoli resistant to thermal unfolding. We show that OBI enables Red Broccoli to be readily detected in live mammalian cells. Furthermore, we show that Red Broccoli can be fused to a S-adenosyl methionine (SAM)-binding aptamer to generate a red fluorescent RNA-based sensor that enables imaging of SAM in live mammalian cells. These results reveal a red fluorescent fluorogenic aptamer that functions in mammalian cells and that can be readily developed into red fluorescent RNA-based sensors.

Ratiometric Fluorogenic RNA-Based Sensors for Imaging Live-Cell Dynamics of Small Molecules.

Imaging the cellular dynamics of metabolites and signaling molecules is critical for understanding various metabolism and signal transduction pathways. Genetically encoded RNA-based sensors are emerging powerful tools for this purpose. However, it was challenging to use these sensors to precisely determine the intracellular concentrations of target analytes. To solve this problem, we have recently developed ratiometric sensors using an orthogonal pair of RNA/fluorophore conjugates: Broccoli/DFHBI-1T (3,5-difluoro-4-hydroxybenzylidene-1-trifluoroethyl-imidazolinone) and DNB (dinitroaniline-binding aptamer)/SR-DN (sulforhodamine B-dinitroaniline). The cellular DNB-to-Broccoli fluorescence intensity ratio can be directly applied to quantify the target concentrations at the single-cell level. Unfortunately, due to the instability of the SR-DN dye, this ratiometric sensor is difficult to use for monitoring target dynamics. Herein, by replacing SR-DN with a stable TMR (tetramethylrhodamine)-DN dye, we developed a ratiometric sensor system based on Broccoli/DFHBI-1T and DNB/TMR-DN, which can be used for dynamic imaging in living cells. We believe these advanced genetically encoded ratiometric sensors can be widely used for intracellular studies of various target analytes.

Genetically Encoded Ratiometric RNA-Based Sensors for Quantitative Imaging of Small Molecules in Living Cells.

Precisely determining the intracellular concentrations of metabolites and signaling molecules is critical in studying cell biology. Fluorogenic RNA-based sensors have emerged to detect various targets in living cells. However, it is still challenging to apply these genetically encoded sensors to quantify the cellular concentrations and distributions of targets. Herein, using a pair of orthogonal fluorogenic RNA aptamers, DNB and Broccoli, we engineered a modular sensor system to apply the DNB-to-Broccoli fluorescence ratio to quantify the cell-to-cell variations of target concentrations. These ratiometric sensors can be broadly applied for live-cell imaging and quantification of metabolites, signaling molecules, and other synthetic compounds.

A Fluorogenic RNA-Based Sensor Activated by Metabolite-Induced RNA Dimerization

Corn is a fluorogenic RNA aptamer that forms a high-affinity quasi-symmetric homodimer. The Corn dimer interface binds DFHO, resulting in highly photostable yellow fluorescence. Because of its photostability, Corn would be useful in RNA-based small-molecule biosensors, where quantitative accuracy would be affected by photobleaching. Here we describe a strategy for converting the constitutive Corn dimer into a small-molecule-regulated fluorescent biosensor that detects S-adenosylmethionine (SAM) invitro and in living cells. We fused the Corn aptamer into a helical stem that was engineered by circularly permuting the SAM aptamer from the SAM-III riboswitch. In the absence of SAM, the Corn portion of this fusion RNA is unable to dimerize. However, upon binding SAM, the RNA dimerizes and binds DFHO. This RNA-based biosensor enables detection of SAM dynamics in living mammalian cells. Together, these data describe a class of RNA-based biosensor based on small-molecule-regulated dimerization of Corn.

"Second-generation" fluorogenic RNA-based sensors.

A fluorogenic aptamer can specifically interact with a fluorophore to activate its fluorescence. These nucleic acid-based fluorogenic modules have been dramatically developed over the past decade, and have been used as versatile reporters in the sensor development and for intracellular imaging. In this review, we summarize the design principles, applications, and challenges of the first-generation fluorogenic RNA-based sensors. Moreover, we discuss some strategies to develop next-generation biosensors with improved sensitivity, selectivity, quantification property, and eukaryotic robustness. Using genetically encoded catalytic hairpin assembly strategy as an example, we further introduce a standard protocol to design, characterize, and apply these fluorogenic RNA-based sensors for in vitro detection and cellular imaging of target biomolecules. By incorporating natural RNA machineries, nucleic acid nanotechnology, and systematic evolution approaches, next-generation fluorogenic RNA-based devices can be potentially engineered to be widely applied in cell biology and biomedicine.

Genetically encoded RNA-based sensors for intracellular imaging of silver ions.

Silver has been widely used for disinfection. The cellular accumulation of silver ions (Ag+) is critical in these antibacterial effects. The direct cellular measurement of Ag+ is useful for the study of disinfection mechanisms. Herein, we reported a novel genetically encoded RNA-based sensor to image Ag+ in live bacterial cells. The sensor is designed by introducing a cytosine-Ag+-cytosine metallo base pair into a fluorogenic RNA aptamer, Broccoli. The binding of Ag+ induces the folding of Broccoli and activates a fluorescence signal. This sensor can be genetically encoded to measure the cellular flux and antibacterial effect of Ag+.

RNA-Based Fluorescent Biosensors for Detecting Metabolites in vitro and in Living Cells.

Genetically encoded sensors are important tools for measuring metabolites and other small molecules in vitro and in live cells. Until recently, genetically encoded sensors exclusively comprised fluorescent proteins that undergo changes in Förster resonance energy transfer upon binding a target analyte. However, recently a new class of fluorescent sensor has been developed composed of RNA. These RNA-based sensors rely on Spinach and other RNA mimics of green fluorescent protein. In each case, the RNA-based sensors contain an analyte-binding aptamer domain which transduces binding of the analyte into a conformational change in Spinach. Two types of sensors have been developed: allosteric Spinach sensors and Spinach riboswitches. Allosteric Spinach sensors exhibit metabolite-induced folding and subsequent fluorescence. Spinach riboswitches are naturally occurring riboswitches that have been modified to contain the Spinach aptamer. The resulting RNA is a fluorogenic riboswitch, and produces fluorescence upon binding its cognate analyte. We describe the development of this new technology, its uses, and future directions to facilitate the use of this assay technology in mammalian cells and in high-throughput applications.

FASTmiR: an RNA-based sensor for in vitro quantification and live-cell localization of small RNAs.

Small RNAs, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), play a variety of important regulatory roles in many eukaryotes. Their small size has made it challenging to study them directly in live cells. Here we describe an RNA-based fluorescent sensor for small RNA detection both in vitro and in vivo, adaptable for any small RNA. It utilizes an sxRNA switch for detection of miRNA–mRNA interactions combined with a fluorophore-binding sequence ‘Spinach’, a GFP-like RNA aptamer for which the RNA–fluorophore complex exhibits strong and consistent fluorescence under an excitation wavelength. Two example sensors, FASTmiR171 and FASTmiR122, can rapidly detect and quantify the levels of miR171 and miR122 in vitro. The sensors can determine relative levels of miRNAs in total RNA extracts with sensitivity similar to small RNA sequencing and northern blots. FASTmiR sensors were also used to estimate the copy number range of miRNAs in total RNA extracts. To localize and analyze the spatial distribution of small RNAs in live, single cells, tandem copies of FASTmiR122 were expressed in different cell lines. FASTmiR122 was able to quantitatively detect the differences in miR122 levels in Huh7 and HEK293T cells demonstrating its potential for tracking miRNA expression and localization in vivo.