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.
Genetically encodable sensors have been widely used in the detection of intracellular molecules ranging from metal ions and metabolites to nucleic acids and proteins. These biosensors are capable of monitoring in real-time the cellular levels, locations, and cell-to-cell variations of the target compounds in living systems. Traditionally, the majority of these sensors have been developed based on fluorescent proteins. As an exciting alternative, genetically encoded RNA-based molecular sensors (GERMS) have emerged over the past few years for the intracellular imaging and detection of various biological targets. In view of their ability for the general detection of a wide range of target analytes, and the modular and simple design principle, GERMS are becoming a popular choice for intracellular analysis. In this review, we summarize different design principles of GERMS based on various RNA recognition modules, transducer modules, and reporting systems. Some recent advances in the application of GERMS for intracellular imaging are also discussed. With further improvement in biostability, sensitivity, and robustness, GERMS can potentially be widely used in cell biology and biotechnology.
A paper-based portable fluorogenic RNA sensor for the selective, sensitive, and rapid detection of target analytes.
Precisely determining the intracellular concentrations of metabolites and signaling molecules is critical in studying cell biology.F luorogenic RNA-based sensors have emerged to detect various targets in living cells.H owever,i ti s still challenging to apply these genetically encoded sensors to quantify the cellular concentrations and distributions of targets. Herein, using apair of orthogonal fluorogenic RNAaptamers, DNB and Broccoli, we engineered amodular 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,s ignaling molecules,a nd other synthetic compounds.Fluorescent probes that allow live-cell imaging of small molecules have enabled us to better understand cellular signaling and metabolite flux. Va rious fluorescent smallmolecule probes and genetically encoded fluorescent protein (FP)-based sensors have been developed to image metabolites and signaling molecules. [1] Thef unction of FP sensors requires at arget-binding domain that can both selectively recognize the target and result in sufficient conformational change to refold the FP or change the orientation between two FPs. [2] However,f or many physiologically important analytes,t hese adequate target-binding domains are not easily identified. Thelimited signal-to-noise ratio has further prevented their wide applications. [3] We and others have developed an ew class of genetically encoded sensors based on fluorogenic RNAa ptamers. [4] Aptamers are short single-stranded oligonucleotides that can bind to their targets with high affinity and specificity. [5] Fluorogenic RNAa ptamers,f or example,S pinach or Broccoli, can bind and activate the fluorescence of dyes such as 3,5-difluoro-4-hydroxybenzylidene-1-trifluoroethyl-imidazolinone (DFHBI-1T). [6] By fusing at arget-binding aptamer into Spinach/Broccoli, genetically encoded RNA-based sensors have been developed for live-cell imaging of metabolites, signaling molecules,proteins,a nd metal ions. [4,7] Almost all these fluorogenic RNAs ensors were developed based on aS pinach/Broccoli-dye complex (l ex /l em , approximately 480 nm/ 503 nm). With as ingle-wavelength readout, artifacts can easily arise from variations in the cellular RNAdistributions.F or quantitative and multiplexed imaging of cellular analytes, [8] it is critical to develop new RNAsensor pairs that have little spectra overlap and that can be orthogonally imaged.Here,w ed evelop ratiometric RNAs ensors to quantify the cellular concentrations and distributions of small molecules.T he sensor comprises Broccoli and ad initroaniline (DN)-binding aptamer,D NB. [6b,9] We have engineered novel red-colored RNAs ensors by fusing target-binding aptamers into DNB.U sing Broccoli as the reference,w ec an quantitatively image various small molecules in living cells.We first wondered if it is possible to develop DNB-based metabolite sensors.D initroaniline is ag eneral co...
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