Engineering Genetically Encoded FRET Sensors

Lindenburg, LH Laurens; Merkx, Maarten

doi:10.3390/s140711691

Cited by 90 publications

(71 citation statements)

References 113 publications

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“…Although FRET-based or other split-protein sensors in principle can be designed for measurements of most PIs, proper detection of FRET can be technically challenging and is often associated with poor signal-to-noise ratio due to inefficient energy transfer between the fluorophores. However, this methodology is rapidly advancing with the development on improved FRET sensors and fluorescent FRET pairs [48]. …”

Section: Measuring Pi Levelsmentioning

confidence: 99%

Detection and manipulation of phosphoinositides

Idevall‐Hagren

Camilli

2015

Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biolog

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Phosphoinositides (PIs) are minor components of cell membranes, but play key roles in cell function. Recent refinements in techniques for their detection, together with imaging methods to study their distribution and changes, have greatly facilitated the study of these lipids. Such methods have been complemented by the parallel development of techniques for the acute manipulation of their levels, which in turn allow bypassing the long-term adaptive changes implicit in genetic perturbations. Collectively, these advancements have helped elucidate the role of PIs in physiology and the impact of the dysfunction of their metabolism in disease. Combining methods for detection and manipulation enables the identification of specific roles played by each of the PIs and may eventually lead to the complete deconstruction of the PI signaling network. Here, we review current techniques used for the study and manipulation of cellular PIs and also discuss advantages and disadvantages associated with the various methods. This article is part of a Special Issue entitled Phosphoinositides.

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Section: Measuring Pi Levelsmentioning

confidence: 99%

Detection and manipulation of phosphoinositides

Idevall‐Hagren

Camilli

2015

Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biolog

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“…5). 75 The design of intermolecular FRET-based biosensors for protein-protein interaction detection is similar to that of FP complementation. However, rather than having the interacting proteins of interest fused to the FP fragments, they are fused to the donor FP and acceptor FP.…”

Section: Rfp Biosensors Based On Fretmentioning

confidence: 99%

Red fluorescent proteins (RFPs) and RFP-based biosensors for neuronal imaging applications

2015

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“…This repositioning results in changes in FRET between the fluorescent proteins, which can be imaged by microscopy in living cells (3). These sensors have provided fundamentally novel insights into the dynamic changes in calcium, cyclic nucleotides, glucose, and other molecules during cellular signaling and disease states (4).…”

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confidence: 99%

Imaging metabolite dynamics in living cells using a Spinach-based riboswitch

You

Litke

Jaffrey

2015

Proc. Natl. Acad. Sci. U.S.A.

181

174

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Riboswitches are natural ligand-sensing RNAs typically that are found in the 5′ UTRs of mRNA. Numerous classes of riboswitches have been discovered, enabling mRNA to be regulated by diverse and physiologically important cellular metabolites and small molecules. Here we describe Spinach riboswitches, a new class of genetically encoded metabolite sensor derived from naturally occurring riboswitches. Drawing upon the structural switching mechanism of natural riboswitches, we show that Spinach can be swapped for the expression platform of various riboswitches, allowing metabolite binding to induce Spinach fluorescence directly. In the case of the thiamine 5′-pyrophosphate (TPP) riboswitch from the Escherichia coli thiM gene encoding hydroxyethylthiazole kinase, we show that insertion of Spinach results in an RNA sensor that exhibits fluorescence upon binding TPP. This TPP Spinach riboswitch binds TPP with affinity and selectivity similar to that of the endogenous riboswitch and enables the discovery of agonists and antagonists of the TPP riboswitch using simple fluorescence readouts. Furthermore, expression of the TPP Spinach riboswitch in Escherichia coli enables live imaging of dynamic changes in intracellular TPP concentrations in individual cells. Additionally, we show that other riboswitches that use a structural mechanism similar to that of the TPP riboswitch, including the guanine and adenine riboswitches from the Bacillus subtilis xpt gene encoding xanthine phosphoribosyltransferase, and the S-adenosyl-methionine-I riboswitch from the B. subtilis yitJ gene encoding methionine synthase, can be converted into Spinach riboswitches. Thus, Spinach riboswitches constitute a novel class of RNA-based fluorescent metabolite sensors that exploit the diversity of naturally occurring ligand-binding riboswitches. 2). Binding of the target molecule induces conformational changes that reposition the fluorescent proteins. This repositioning results in changes in FRET between the fluorescent proteins, which can be imaged by microscopy in living cells (3). These sensors have provided fundamentally novel insights into the dynamic changes in calcium, cyclic nucleotides, glucose, and other molecules during cellular signaling and disease states (4).Although genetically encoded sensors are powerful tools for imaging cellular metabolites, only a small number of sensors have been created because these sensors require a protein that binds the target molecule of interest and which undergoes a targetinduced conformational change that is sufficient to induce a change in FRET. However, proteins with these properties are not readily available for most physiologically important metabolites. As a result, sensors have not been generated for many important metabolites and signaling molecules.Recently we described an alternative class of genetically encoded sensors composed of RNA (5). These sensors are based on Spinach, an "RNA mimic of green fluorescent protein" (6). Spinach is an RNA aptamer that binds fluorophores resembling the hydroxybe...

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Engineering Genetically Encoded FRET Sensors

Cited by 90 publications

References 113 publications

Detection and manipulation of phosphoinositides

Detection and manipulation of phosphoinositides

Red fluorescent proteins (RFPs) and RFP-based biosensors for neuronal imaging applications

Imaging metabolite dynamics in living cells using a Spinach-based riboswitch

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