Fluorescence Imaging of Cellular Metabolites with RNA
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.
Full-textDOI: · Available from: Wenjiao Song, Sep 28, 2015
- SourceAvailable from: PubMed Central
[Show abstract] [Hide abstract]
- "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). "
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].BMB reports 02/2013; 46(2):65-72. DOI:10.5483/BMBRep.2013.46.2.016 · 2.60 Impact Factor
[Show abstract] [Hide abstract]
- "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. "
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.Frontiers in Physiology 01/2013; 4:401. DOI:10.3389/fphys.2013.00401 · 3.53 Impact Factor
[Show abstract] [Hide abstract]
- "The application of aptamers for molecular imaging is being used in clinics for the evaluation of several diseases (Cibiel et al., 2012) with the great advantage of not being an invasive methodology and may be helpfully applied also in parasitology. An exciting use of the technique for cell biology is the imaging of cellular metabolites with RNA-based sensors recently described, where the intracellular levels of ADP and S-adenosylmethionine were measured (Paige et al., 2012). Since RNA aptamers to any molecule can be rapidly selected, a rapid expansion of the methodology to other small molecules is envisaged. "
ABSTRACT: Parasite infections are largely dependent on interactions between pathogen and different host cell populations to guarantee a successful infectious process. This is particularly true for obligatory intracellular parasites as Plasmodium, Toxoplasma, Leishmania, to name a few. Adhesion to and entry into the cell are essential steps requiring specific parasite and host cell molecules. The large amount of possible involved molecules poses additional difficulties for their identification by the classical biochemical approaches. In this respect, the search for alternative techniques should be pursued. Among them two powerful methodologies can be employed, both relying upon the construction of highly diverse combinatorial libraries of peptides or oligonucleotides that randomly bind with high affinity to targets on the cell surface and are selectively displaced by putative ligands. These are, respectively, the peptide-based phage display and the oligonucleotide-based aptamer techniques. The phage display technique has been extensively employed for the identification of novel ligands in vitro and in vivo in different areas such as cancer, vaccine development and epitope mapping. Particularly, phage display has been employed in the investigation of pathogen-host interactions. Although this methodology has been used for some parasites with encouraging results, in trypanosomatids its use is, as yet, scanty. RNA and DNA aptamers, developed by the SELEX process (Systematic Evolution of Ligands by Exponential Enrichment), were described over two decades ago and since then contributed to a large number of structured nucleic acids for diagnostic or therapeutic purposes or for the understanding of the cell biology. Similarly to the phage display technique scarce use of the SELEX process has been used in the probing of parasite-host interaction. In this review, an overall survey on the use of both phage display and aptamer technologies in different pathogenic organisms will be discFrontiers in Immunology 01/2012; 3:419. DOI:10.3389/fimmu.2012.00419