Plug-and-Play Fluorophores Extend the Spectral Properties of Spinach

Song, Weiguo; Strack, Rita; Svensen, Nina; Jaffrey, Samie R.

doi:10.1021/ja410819x

Cited by 224 publications

(228 citation statements)

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“…These sensors are based on Spinach, an "RNA mimic of green fluorescent protein" (6). Spinach is an RNA aptamer that binds fluorophores resembling the hydroxybenzylidene imidazolinone (HBI) fluorophore in GFP, including 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) (6,7). Similar to the manner by which GFP induces fluorescence in HBI, Spinach binds to the otherwise nonfluorescent DFHBI and switches it to a highly fluorescent state.…”

mentioning

confidence: 99%

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

You

Litke

Jaffrey

2015

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

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171

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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mentioning

confidence: 99%

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

You

Litke

Jaffrey

2015

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

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171

174

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“…The spectral properties of Spinach and Spinach2 are determined by their fluorophore, DFHBI, which exhibits an excitation maximum at 447 nm (55). However, filters commonly used for green fluorescence in fluorescence microscopes typically illuminate cells with ∼480-nm radiation.…”

Section: Directed Evolutionmentioning

confidence: 99%

“…As a result, Spinach-DFHBI complexes are inefficiently excited and exhibit suboptimal brightness in typical microscopy setups. This issue was addressed by designing novel DFHBI-like fluorophores that bind Spinach but exhibit altered excitation and emission maxima (55). A structure-activity relationship analysis of DFHBI showed that altering or adding halogen substituents on the benzylidene moiety did not markedly alter the fluorescence properties of altered compounds relative to those of DFHBI, although adding substituents on the imidazolinone moiety did result in Spinach-fluorophore complexes with altered spectral properties.…”

Section: Directed Evolutionmentioning

confidence: 99%

“…Similar spectral properties were also observed for Broccoli-DFHBI complexes (19). As a second example, substitution at the C-2 position of the imidazolinone ring also caused marked red shifts in the fluorescence emission spectra: DFHBI-2T [(Z)-4-(3,5-difluoro-4-hydroxybenzylidene)-1-methyl-2-(trifluoromethyl)-1H-imidazol-5(4H)-one)] bound to Spinach2 and exhibited a 53-nm red shift in the excitation maximum and a 22-nm red shift in the emission maximum, although the overall brightness was somewhat reduced owing to a decrease in the quantum yield (55). The increase in K D to ∼1.2 μM suggests that the bulky trifluoromethyl moiety in DFHBI-2T may exhibit steric hindrance with the Spinach2 aptamer.…”

Section: Directed Evolutionmentioning

confidence: 99%

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Structure and Mechanism of RNA Mimics of Green Fluorescent Protein

Meng

Jaffrey

2015

Annu. Rev. Biophys.

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112

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RNAs have highly complex and dynamic cellular localization patterns. Technologies for imaging RNA in living cells are important for uncovering their function and regulatory pathways. One approach for imaging RNA involves genetically encoding fluorescent RNAs using RNA mimics of green fluorescent protein (GFP). These mimics are RNA aptamers that bind fluorophores resembling those naturally found in GFP and activate their fluorescence. These RNA-fluorophore complexes, including Spinach, Spinach2, and Broccoli, can be used to tag RNAs and to image their localization in living cells. In this article, we describe the generation and optimization of these aptamers, along with strategies for expanding the spectral properties of their associated RNA-fluorophore complexes. We also discuss the structural basis for the fluorescence and photophysical properties of Spinach, and we describe future prospects for designing enhanced RNA-fluorophore complexes with enhanced photostability and increased sensitivity.

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“…Recently, Jaffrey and colleagues have engineered an RNA aptamer, denoted ''Spinach,'' that mimics the function of green fluorescent protein (GFP): when bound with 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI), Spinach becomes brightly fluorescent. It has been reported that Spinach/DFHBI displays fluorescence properties similar to GFP (*50 % brightness of EGFP), and therefore, this system represents a useful tool to examine RNA activities in cells (Paige et al 2012(Paige et al , 2011Song et al 2014;. The same group has also engineered another aptamer, named ''Broccoli,'' that can also bind and activate DFHBI (Filonov et al 2014).…”

Section: Introductionmentioning

confidence: 99%

Selection of Intracellularly Functional RNA Mimics of Green Fluorescent Protein Using Fluorescence-Activated Cell Sorting

Zou

Huang

et al. 2015

J Mol Evol

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Fluorescence-activated cell sorting (FACS) was exploited to isolate Escherichia coli cells that were highly fluorescent due to the expression of RNA aptamers that induce fluorescence of 3,5-difluoro-4-hydroxybenzylidene imidazolinone. Two different aptamers, named ZT-26 and ZT-324, were identified by this method and compared to the fluorescence-signaling properties of Spinach, a previously reported RNA aptamer. Aptamer ZT-26 exhibits significantly enhanced fluorescence over Spinach only in vitro. However, aptamer ZT-324 is 36 % brighter than Spinach when expressed in E. coli. The FACS-based selection strategy presented here is attractive for deriving fluorescent RNA aptamers that function in cells as it directly selects for cells with a high level of fluorescence due to the expression of the RNA aptamer.

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Plug-and-Play Fluorophores Extend the Spectral Properties of Spinach

Cited by 224 publications

References 16 publications

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

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

Structure and Mechanism of RNA Mimics of Green Fluorescent Protein

Selection of Intracellularly Functional RNA Mimics of Green Fluorescent Protein Using Fluorescence-Activated Cell Sorting

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