Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

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

ABSTRACT: 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.

SUBMITTER: Wu R

PROVIDER: S-EPMC6893093 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

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

Wu Rigumula R Karunanayake Mudiyanselage Aruni P K K APKK Shafiei Fatemeh F Zhao Bin B Bagheri Yousef Y Yu Qikun Q McAuliffe Kathleen K Ren Kewei K You Mingxu M You Mingxu M

Angewandte Chemie (International ed. in English) 20191024 50

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 ...[more]

PMID: 31591798

Similar Datasets

DNAzyme-Mediated Genetically Encoded Sensors for Ratiometric Imaging of Metal Ions in Living Cells.

Project description:Genetically encoded fluorescent proteins (FPs) have been used for metal ion detection. However, their applications are restricted to a limited number of metal ions owing to the lack of available metal-binding proteins or peptides that can be fused to FPs and the difficulty in transforming the binding of metal ions into a change of fluorescent signal. We report herein the use of Mg2+ -specific 10-23 or Zn2+ -specific 8-17 RNA-cleaving DNAzymes to regulate the expression of FPs as a new class of ratiometric fluorescent sensors for metal ions. Specifically, we demonstrate the use of DNAzymes to suppress the expression of Clover2, a variant of the green FP (GFP), by cleaving the mRNA of Clover2, while the expression of Ruby2, a mutant of the red FP (RFP), is not affected. The Mg2+ or Zn2+ in HeLa cells can be detected using both confocal imaging and flow cytometry. Since a wide variety of metal-specific DNAzymes can be obtained, this method can likely be applied to imaging many other metal ions, expanding the range of the current genetically encoded fluorescent protein-based sensors.

| S-EPMC6982602 | biostudies-literature

Imaging bacterial protein expression using genetically encoded RNA sensors.

Project description:The difficulties in imaging the dynamics of protein expression in live bacterial cells can be overcome by using fluorescent sensors based on Spinach, an RNA that activates the fluorescence of a small-molecule fluorophore. These RNAs selectively bind target proteins and exhibit fluorescence increases that enable protein expression to be imaged in living Escherichia coli. These sensors are key components of a generalizable strategy to image protein expression in a single bacterium in real time.

| S-EPMC3758421 | biostudies-literature

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

Project description: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+.

| S-EPMC6328328 | biostudies-literature

Imaging extracellular ATP with a genetically-encoded, ratiometric fluorescent sensor.

Project description:Extracellular adenosine triphosphate (ATP) is a key purinergic signal that mediates cell-to-cell communication both within and between organ systems. We address the need for a robust and minimally invasive approach to measuring extracellular ATP by re-engineering the ATeam ATP sensor to be expressed on the cell surface. Using this approach, we image real-time changes in extracellular ATP levels with a sensor that is fully genetically-encoded and does not require an exogenous substrate. In addition, the sensor is ratiometric to allow for reliable quantitation of extracellular ATP fluxes. Using live-cell microscopy, we characterize sensor performance when expressed on cultured Neuro2A cells, and we measure both stimulated release of ATP and its clearance by ectonucleotidases. Thus, this proof-of-principle demonstrates a first-generation sensor to report extracellular ATP dynamics that may be useful for studying purinergic signaling in living specimens.

| S-EPMC5679667 | biostudies-literature

High-speed imaging of glutamate release with genetically encoded sensors.

Project description:The strength of an excitatory synapse depends on its ability to release glutamate and on the density of postsynaptic receptors. Genetically encoded glutamate indicators (GEGIs) allow eavesdropping on synaptic transmission at the level of cleft glutamate to investigate properties of the release machinery in detail. Based on the sensor iGluSnFR, we recently developed accelerated versions of GEGIs that allow investigation of synaptic release during 100-Hz trains. Here, we describe the detailed procedures for design and characterization of fast iGluSnFR variants in vitro, transfection of pyramidal cells in organotypic hippocampal cultures, and imaging of evoked glutamate transients with two-photon laser-scanning microscopy. As the released glutamate spreads from a point source-the fusing vesicle-it is possible to localize the vesicle fusion site with a precision exceeding the optical resolution of the microscope. By using a spiral scan path, the temporal resolution can be increased to 1 kHz to capture the peak amplitude of fast iGluSnFR transients. The typical time frame for these experiments is 30 min per synapse.

| S-EPMC6751072 | biostudies-literature

Live Imaging with Genetically Encoded Physiologic Sensors and Optogenetic Tools.

Project description:Barrier tissues such as the epidermis employ complex signal transduction systems to execute morphogenetic programs and to rapidly respond to environmental cues to promote homeostasis. Recent advances in live-imaging techniques and tools allow precise spatial and temporal monitoring and manipulation of intracellular signaling cascades. Leveraging the chemistry of naturally occurring light-sensitive proteins, genetically encoded fluorescent biosensors have emerged as robust tools for visualizing dynamic signaling events. In contrast, optogenetic protein constructs permit laser-mediated control of signal receptors and effectors within live cells, organoids, and even model organisms. In this paper, we review the basic principles underlying novel biosensors and optogenetic tools and highlight how recent studies in cutaneous biology have leveraged these imaging strategies to illuminate the spatiotemporal signals regulating epidermal development, barrier formation, and tissue homeostasis.

| S-EPMC9972253 | biostudies-literature

Genetically Encoded RNA-Based Bioluminescence Resonance Energy Transfer (BRET) Sensors.

Project description:RNA-based nanostructures and molecular devices have become popular for developing biosensors and genetic regulators. These programmable RNA nanodevices can be genetically encoded and modularly engineered to detect various cellular targets and then induce output signals, most often a fluorescence readout. Although powerful, the high reliance of fluorescence on the external excitation light raises concerns about its high background, photobleaching, and phototoxicity. Bioluminescence signals can be an ideal complementary readout for these genetically encoded RNA nanodevices. However, RNA-based real-time bioluminescent reporters have been rarely developed. In this study, we reported the first type of genetically encoded RNA-based bioluminescence resonance energy transfer (BRET) sensors that can be used for real-time target detection in living cells. By coupling a luciferase bioluminescence donor with a fluorogenic RNA-based acceptor, our BRET system can be modularly designed to image and detect various cellular analytes. We expect that this novel RNA-based bioluminescent system can be potentially used broadly in bioanalysis and nanomedicine for engineering biosensors, characterizing cellular RNA-protein interactions, and high-throughput screening or in vivo imaging.

| S-EPMC10630924 | biostudies-literature

Genetically Encoded Ratiometric pH Sensors for the Measurement of Intra- and Extracellular pH and Internalization Rates.

Project description:pH-sensitive fluorescent proteins as genetically encoded pH sensors are promising tools for monitoring intra- and extracellular pH. However, there is a lack of ratiometric pH sensors, which offer a good dynamic range and can be purified and applied extracellularly to investigate uptake. In our study, the bright fluorescent protein CoGFP_V0 was C-terminally fused to the ligand epidermal growth factor (EGF) and retained its dual-excitation and dual-emission properties as a purified protein. The tandem fluorescent variants EGF-CoGFP-mTagBFP2 (pK' = 6.6) and EGF-CoGFP-mCRISPRed (pK' = 6.1) revealed high dynamic ranges between pH 4.0 and 7.5. Using live-cell fluorescence microscopy, both pH sensor molecules permitted the conversion of fluorescence intensity ratios to detailed intracellular pH maps, which revealed pH gradients within endocytic vesicles. Additionally, extracellular binding of the pH sensors to cells expressing the EGF receptor (EGFR) enabled the tracking of pH shifts inside cultivation chambers of a microfluidic device. Furthermore, the dual-emission properties of EGF-CoGFP-mCRISPRed upon 488 nm excitation make this pH sensor a valuable tool for ratiometric flow cytometry. This high-throughput method allowed for the determination of internalization rates, which represents a promising kinetic parameter for the in vitro characterization of protein-drug conjugates in cancer therapy.

| S-EPMC9138566 | biostudies-literature

Ultrafast neuronal imaging of dopamine dynamics with designed genetically encoded sensors.

Project description:Neuromodulatory systems exert profound influences on brain function. Understanding how these systems modify the operating mode of target circuits requires spatiotemporally precise measurement of neuromodulator release. We developed dLight1, an intensity-based genetically encoded dopamine indicator, to enable optical recording of dopamine dynamics with high spatiotemporal resolution in behaving mice. We demonstrated the utility of dLight1 by imaging dopamine dynamics simultaneously with pharmacological manipulation, electrophysiological or optogenetic stimulation, and calcium imaging of local neuronal activity. dLight1 enabled chronic tracking of learning-induced changes in millisecond dopamine transients in mouse striatum. Further, we used dLight1 to image spatially distinct, functionally heterogeneous dopamine transients relevant to learning and motor control in mouse cortex. We also validated our sensor design platform for developing norepinephrine, serotonin, melatonin, and opioid neuropeptide indicators.

| S-EPMC6287765 | biostudies-literature

Engineering genetically encoded FRET sensors.

Project description:Förster Resonance Energy Transfer (FRET) between two fluorescent proteins can be exploited to create fully genetically encoded and thus subcellularly targetable sensors. FRET sensors report changes in energy transfer between a donor and an acceptor fluorescent protein that occur when an attached sensor domain undergoes a change in conformation in response to ligand binding. The design of sensitive FRET sensors remains challenging as there are few generally applicable design rules and each sensor must be optimized anew. In this review we discuss various strategies that address this shortcoming, including rational design approaches that exploit self-associating fluorescent domains and the directed evolution of FRET sensors using high-throughput screening.

| S-EPMC4168480 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data