Article

Fluorescence Imaging of Cellular Metabolites with RNA

Department of Pharmacology, Weill Medical College, Cornell University, New York, NY 10065, USA.
Science (Impact Factor: 33.61). 03/2012; 335(6073):1194. DOI: 10.1126/science.1218298
Source: PubMed

ABSTRACT

Genetically encoded sensors are powerful tools for imaging intracellular metabolites and signaling molecules. However, developing
sensors is challenging because they require proteins that undergo conformational changes upon binding the desired target molecule.
We describe an approach for generating fluorescent sensors based on Spinach, an RNA sequence that binds and activates the
fluorescence of a small-molecule fluorophore. We show that these sensors can detect a variety of different small molecules
in vitro and in living cells. These RNAs constitute a versatile approach for fluorescence imaging of small molecules and have
the potential to detect essentially any cellular biomolecule.

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    • "Biosensors capable of sensing and responding to small molecules in vivo have wide-ranging applica- tions in biological research and biotechnology, including metabolic pathway regulation (Zhang et al., 2012), biosynthetic pathway optimization (Raman et al., 2014;Tang and Cirino, 2011), metabolite concentration measurement and imaging (Paige et al., 2012), environmental toxin detection (Gil et al., 2000), and small molecule-triggered therapeutic response (Ye et al., 2013). Despite such broad utility, no single strategy for the construction of biosensors has proven sufficiently generalizable to gain widespread use. "
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    ABSTRACT: Biosensors for small molecules can be used in applications that range from metabolic engineering to orthogonal control of transcription. Here, we produce biosensors based on a ligand-binding domain (LBD) by using a method that, in principle, can be applied to any target molecule. The LBD is fused to either a fluorescent protein or a transcriptional activator and is destabilized by mutation such that the fusion accumulates only in cells containing the target ligand. We illustrate the power of this method by developing biosensors for digoxin and progesterone. Addition of ligand to yeast, mammalian or plant cells expressing a biosensor activates transcription with a dynamic range of up to ~100-fold. We use the biosensors to improve the biotransformation of pregnenolone to progesterone in yeast and to regulate CRISPR activity in mammalian cells. This work provides a general methodology to develop biosensors for a broad range of molecules in eukaryotes.
    Full-text · Article · Dec 2015 · eLife Sciences
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    • "Spinach resembles enhanced GFP emitting a green fluorescence that is remarkably resistant to photobleaching (51). Trafficking of Spinach-fused RNAs was successfully imaged in live cells without nonspecific fluorescence or cytotoxicity in cells (51,52). "
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    ABSTRACT: In situ detection of RNAs is becoming increasingly important for analysis of gene expression within and between intact cells in tissues. International genomics efforts are now cataloging patterns of RNA transcription that play roles in cell function, differentiation, and disease formation, and they are demonstrating the importance of coding and noncoding RNA transcripts in these processes. However, these techniques typically provide ensemble averages of transcription across many cells. In situ hybridization-based analysis methods complement these studies by providing information about how expression levels change between cells within normal and diseased tissues, and they provide information about the localization of transcripts within cells, which is important in understanding mechanisms of gene regulation. Multi-color, single-molecule fluorescence in situ hybridization (smFISH) is particularly useful since it enables analysis of several different transcripts simultaneously. Combining smFISH with immunofluorescent protein detection provides additional information about the association between transcription level, cellular localization, and protein expression in individual cells. [BMB Reports 2013; 46(2): 065-072].
    Full-text · Article · Feb 2013 · BMB reports
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    • "Several alternative approaches to intracellular pH measurement have been proposed including surface enhanced Raman scattering (SERS) based sensors (Kneipp et al., 2010), green florescent protein (GFP) based sensors (Kneen et al., 1998), and RNA based sensors (Paige et al., 2012). However, the most widely implemented approach utilizes pH-sensitive fluorophores. "
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    ABSTRACT: Measurement of intracellular acidification is important for understanding fundamental biological pathways as well as developing effective therapeutic strategies. Fluorescent pH nanosensors are an enabling technology for real-time monitoring of intracellular acidification. The physicochemical characteristics of nanosensors can be engineered to target specific cellular compartments and respond to external stimuli. Therefore, nanosensors represent a versatile approach for probing biological pathways inside cells. The fundamental components of nanosensors comprise a pH-sensitive fluorophore (signal transducer) and a pH-insensitive reference fluorophore (internal standard) immobilized in an inert non-toxic matrix. The inert matrix prevents interference of cellular components with the sensing elements as well as minimizing potentially harmful effects of some fluorophores on cell function. Fluorescent nanosensors are synthesized using standard laboratory equipment and are detectable by non-invasive widely accessible imaging techniques. The outcomes of studies employing this technology are dependent on reliable methodology for performing measurements. In particular, special consideration must be given to conditions for sensor calibration, uptake conditions and parameters for image analysis. We describe procedures for: (1) synthesis and characterization of polyacrylamide and silica based nanosensors, (2) nanosensor calibration and (3) performing measurements using fluorescence microscopy.
    Full-text · Article · Jan 2013 · Frontiers in Physiology
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