Oligonucleotide-conjugated Thiazole Orange Probes as “Light-up” Probes for Messenger Ribonucleic Acid Molecules in Living Cells¶

Privat, Eric; Melvin, T.; Asseline, Ulysse; Vigny, Paul

doi:10.1562/0031-8655(2001)0740532octopa2.0.co2

Cited by 13 publications

(4 citation statements)

References 38 publications

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“…For each directed movement of either the LacZ-24-hGH or the LacZ-24-SV mRNA particles, the mean velocity was calculated. The corresponding histograms show a peak at 1.0-1.5 mm/s (Knowles et al 1996(Knowles et al , 1997Bertrand et al 1998;Theurkauf et al 1998;Politz et al 1999;Lorenz et al 2000;Dirks et al 2001;Perlette et al 2001;Privat et al 2001)…”

Section: Resultsmentioning

confidence: 96%

“…Furthermore, the future will yield new developments in imaging cells in live animals (Farina et al 1998) and will eventually unveil the details of the RNA lifecycle. Existing technology for the visualization of mRNA in living cells, including images obtained using those technologies (Knowles et al 1996(Knowles et al , 1997Bertrand et al 1998;Theurkauf et al 1998;Politz et al 1999;Lorenz et al 2000;Dirks et al 2001;Perlette et al 2001;Privat et al 2001) Dynamics of mRNA molecules in the cytoplasm of mammalian cells (reproduced from Fusco et al 2003). All images were obtained at a rate of nine images per second, for periods of 22 s, and were deconvolved.…”

Section: Resultsmentioning

confidence: 99%

“…Oligonucleotides are delivered across the plasma membrane and hybridize with the target sequence to identify RNA directly in living cells (Politz et al 1995(Politz et al , 1999. Six categories of fluorescent oligonucleotide probes exist, including conventional DNA probes (Politz et al 1995;Lorenz et al 2000), caged DNA probes (Politz et al 1999;Pederson 2001), linear 2′ O methyl (2′OMe) RNA probes (CarmoFonseca et al 1991;Dirks et al 2001;Molenaar et al 2001), peptide nucleic acid (PNA) probes , molecular beacons (Tyagi et al 1996(Tyagi et al , 1998Sokol et al 1998;Molenaar et al 2001), and probes with a linked fluorophore whose fluorescence properties change upon association with the target mRNA strand (Thuong et al 1987;Privat et al 2001). Advantages of oligodeoxynucleotide (ODN) probes are their ability to detect endogenous mRNA, their relative stability (as long as 18 h, Politz et al 1995), and the ease with which they are introduced into the cell.…”

Section: Fluorescent Oligonucleotide Probes (Fivh-fluorescent In Vivomentioning

confidence: 99%

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Imaging of Single mRNAs in the Cytoplasm of Living Cells

Fusco

Bertrand

Singer

2008

RNA Trafficking and Nuclear Structure Dynamics

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Section: Resultsmentioning

confidence: 96%

Section: Resultsmentioning

confidence: 99%

Section: Fluorescent Oligonucleotide Probes (Fivh-fluorescent In Vivomentioning

confidence: 99%

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Imaging of Single mRNAs in the Cytoplasm of Living Cells

Fusco

Bertrand

Singer

2008

RNA Trafficking and Nuclear Structure Dynamics

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“…Apart from PNA-conferred improvements in probe composition, novel probe chemistries have also been devised for both DNA-and PNA-based probes. These include molecular beacons (MBs) (50,55), adjacent fluorescence resonance energy transfer probes (9,49), light-up probes (38,46), and quenched autoligation probes (40,41). The first DNA MB probes were developed by Tyagi and Kramer (50).…”

mentioning

confidence: 99%

Quantitative rRNA-Targeted Solution-Based Hybridization Assay Using Peptide Nucleic Acid Molecular Beacons

Morgenroth

Raskin

2008

Appl Environ Microbiol

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The potential of a solution-based hybridization assay using peptide nucleic acid (PNA) molecular beacon (MB) probes to quantify 16S rRNA of specific populations in RNA extracts of environmental samples was evaluated by designing PNA MB probes for the genera Dechloromonas and Dechlorosoma. In a kinetic study with 16S rRNA from pure cultures, the hybridization of PNA MB to target 16S rRNA exhibited a higher final hybridization signal and a lower apparent rate constant than the hybridizations to nontarget 16S rRNAs. A concentration of 10 mM NaCl in the hybridization buffer was found to be optimal for maximizing the difference between final hybridization signals from target and nontarget 16S rRNAs. Hybridization temperatures and formamide concentrations in hybridization buffers were optimized to minimize signals from hybridizations of PNA MB to nontarget 16S rRNAs. The detection limit of the PNA MB hybridization assay was determined to be 1.6 nM of 16S rRNA. To establish proof for the application of PNA MB hybridization assays in complex systems, target 16S rRNA from Dechlorosoma suillum was spiked at different levels to RNA isolated from an environmental (bioreactor) sample, and the PNA MB assay enabled effective quantification of the D. suillum RNA in this complex mixture. For another environmental sample, the quantitative results from the PNA MB hybridization assay were compared with those from clone libraries.Accurate quantification of specific populations in complex microbial communities is important in many areas of microbiology. Today, real-time PCR-based techniques are often used for this purpose (45). Other PCR-based techniques, such as denaturing gradient gel electrophoresis (30) and terminal restriction fragment length polymorphism (29), have also been used to study microbial population dynamics, but they are less suitable as quantitative techniques (52). Oligonucleotide probe-based microbial quantification methods that do not rely on PCR usually target the small subunit rRNA and include quantitative membrane hybridization, quantitative fluorescence in situ hybridization (FISH), and phylogenetic microarrays. While membrane hybridization has been used successfully to study microbial population dynamics in a variety of complex microbial systems (39, 58), this technique is time consuming and labor intensive. Quantitative FISH also has been used effectively to quantify microbial populations (34, 59). However, in some applications, traditional FISH methods do not provide satisfactory quantitative results because of poor cell permeability for oligonucleotide probes (10), low accessibility of target sites in rRNA (18), or poor sensitivity when the cellular rRNA content is low (20). Microarray techniques have the potential to quantify rRNA extracted from environmental samples (16, 23), but reproducibility and specificity issues have not been addressed satisfactorily (37,43).The peptide nucleic acid (PNA) oligomer probe was studied for its hybridization properties for the first time about 15 years ago (15). PNA di...

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Fluorescence to Study Nucleic Acids

Asseline

2008

Wiley Encyclopedia of Chemical Biology

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The power of fluorescence spectroscopy in the study of nucleic acids relies on the availability of hundreds of different fluorophores that span the visible spectrum of light and the possibility of using them to label oligonucleotides (short fragments of DNA or RNA obtained by chemical synthesis) specifically. Any sequence can be obtained with one or more fluorophores and fluorophore quenchers attached at preselected positions by linkages with suitable parameters to ensure the optimal properties for the fluorescent oligonucleotides. Fluorescent labeling of long DNA or RNA fragments is also possible by enzymatic methods. Fluorescence‐based studies of nucleic acids are widely used in different areas such as basic research, molecular and clinical diagnostics, disease monitoring, therapeutic development, food technology, environmental sciences, and biotechnology. After a few words on biological and fluorescence backgrounds, the main labeling strategies will be developed and examples of biological questions that can be addressed with fluorescent oligonucleotides and the major types of fluorescence spectroscopy techniques will be reported.

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Oligonucleotide-conjugated Thiazole Orange Probes as “Light-up” Probes for Messenger Ribonucleic Acid Molecules in Living Cells¶

Cited by 13 publications

References 38 publications

Imaging of Single mRNAs in the Cytoplasm of Living Cells

Imaging of Single mRNAs in the Cytoplasm of Living Cells

Quantitative rRNA-Targeted Solution-Based Hybridization Assay Using Peptide Nucleic Acid Molecular Beacons

Fluorescence to Study Nucleic Acids

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