A nucleic acid probe labeled with desmethyl thiazole orange: a new type of hybridization-sensitive fluorescent oligonucleotide for live-cell RNA imaging

Okamoto, Akimitsu; Sugizaki, Kaori; Yuki, Mizue; Yanagisawa, Hiroki; Ikeda, Shuji; Sueoka, Takuma; Hayashi, Gosuke; Wang, Dan Ohtan

doi:10.1039/c2ob26707a

Cited by 12 publications

(5 citation statements)

References 37 publications

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“…A special form of such multicolor probe design consists of co‐labeling a single oligonucleotide with two fluorophores that can engage in energy transfer. Similarly to MB‐FRET, multilabeled ECHO probes, dual‐fluorophore‐labeled PNA FIT, and TO‐JO (oxazolopyridine)‐labeled DNA FIT probes possess greater responsiveness and thus better target discriminating ability, as efficient energy transfer only occurs between bright‐state fluorescent molecules. The high responsiveness of such probes allows wash‐free, single‐step RNA FISH, greatly decreasing the amount of time and labor required to develop the signal—even in large specimens—making these methods ideal tools for high‐throughput quantitative RNA localization studies …”

Section: Fluorogenic Approaches Of In Situ Rna Detectionmentioning

confidence: 99%

Strength in numbers: quantitative single‐molecule RNA detection assays

Gáspár

Ephrussi

2015

WIREs Developmental Biology

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Gene expression is a fundamental process that underlies development, homeostasis, and behavior of organisms. The fact that it relies on nucleic acid intermediates, which can specifically interact with complementary probes, provides an excellent opportunity for studying the multiple steps—transcription, RNA processing, transport, translation, degradation, and so forth—through which gene function manifests. Over the past three decades, the toolbox of nucleic acid science has expanded tremendously, making high‐precision in situ detection of DNA and RNA possible. This has revealed that many—probably the vast majority of—transcripts are distributed within the cytoplasm or the nucleus in a nonrandom fashion. With the development of microscopy techniques we have learned not only about the qualitative localization of these molecules but also about their absolute numbers with great precision. Single‐molecule techniques for nucleic acid detection have been transforming our views of biology with elementary power: cells are not average members of their population but are highly distinct individuals with greatly and suddenly changing gene expression, and this behavior of theirs can be measured, modeled, and thus predicted and, finally, comprehended. WIREs Dev Biol 2015, 4:135–150. doi: 10.1002/wdev.170For further resources related to this article, please visit the WIREs website.Conflict of interest: The authors have declared no conflicts of interest for this article.

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Section: Fluorogenic Approaches Of In Situ Rna Detectionmentioning

confidence: 99%

Strength in numbers: quantitative single‐molecule RNA detection assays

Gáspár

Ephrussi

2015

WIREs Developmental Biology

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“…These methods are based on binding of a marker conjugated specific probes on an mRNA in living cells. Recently, techniques involving interaction with small hybridizing sensors been developed (Ikeda et al, ; Okamoto et al, ) and the obtained data revealed the kinetics of mRNA localization as well as spatiotemporal regulation of gene expression in living cells.…”

Section: Introductionmentioning

confidence: 99%

Immuno‐detection of mRNA‐binding protein complex in human cells under transmission electron microscopy

Tatsuno

Nakamura

et al. 2019

Microscopy Res & Technique

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Transit from the nuclear complex to the cytoplasm through the nuclear pore complex permits modification of mRNA, including processing such as splicing, capping, and polyadenylation, etc. At each of these events, mRNA interacts with various proteins to form mRNA‐protein complex. Visualizing the mRNA is crucial for understanding the mechanisms underlying mRNA processing and elucidating its structure and recent advances in mRNA imaging allow detection of real‐time mRNA localization in living cells. However, these techniques revealed only the location of mRNA but cannot visualize the conformation of mRNA‐protein complex in cells. On the other hand, transmission electron microscopy has been used to visualize the structure of the Balbiani ring‐derived large mRNA, but their observations were limited to the insect cells. In this study, we visualized the structure of mRNA‐protein complex in human culture cells by using immuno‐electron microscopy. Through immuno‐detection, an mRNA exon junction binding complex Y14, and its binding protien Upf2, different gold particle patterns were imaged with transmission electron microscopy and analyzed. Characteristic linear and stacked particle orientation were observed. Across the nuclear membrane, only linear aggregation pattern was observed, whereas the stacked aggregation pattern was detected in the cytoplasm. Our method is able to visualize mRNA‐conformation and applicable to many cell types, including mammalian cells, where genes can easily be manipulated.

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“…TO undergoes forced intercalation upon hybridization of the probe with target RNA, resulting in a significant increase in fluorescence ( Figure 3D ; Bethge et al, 2010). Both ECHO and FIT probes are suitable for in vivo mRNA imaging upon delivery into living eukaryotic cells (Kubota et al, 2009, 2010; Hövelmann et al, 2013, 2014; Okamoto et al, 2013). Interestingly, injection of approximately four FIT probes gives comparable signals as those obtained with the MS2 system in living Drosophila oocytes (Hövelmann et al, 2014).…”

Section: Introductionmentioning

confidence: 99%

Illuminating Messengers: An Update and Outlook on RNA Visualization in Bacteria

Gijtenbeek

2017

Front. Microbiol.

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To be able to visualize the abundance and spatiotemporal features of RNAs in bacterial cells would permit obtaining a pivotal understanding of many mechanisms underlying bacterial cell biology. The first methods that allowed observing single mRNA molecules in individual cells were introduced by Bertrand et al. (1998) and Femino et al. (1998). Since then, a plethora of techniques to image RNA molecules with the aid of fluorescence microscopy has emerged. Many of these approaches are useful for the large eukaryotic cells but their adaptation to study RNA, specifically mRNA molecules, in bacterial cells progressed relatively slow. Here, an overview will be given of fluorescent techniques that can be used to reveal specific RNA molecules inside fixed and living single bacterial cells. It includes a critical evaluation of their caveats as well as potential solutions.

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A nucleic acid probe labeled with desmethyl thiazole orange: a new type of hybridization-sensitive fluorescent oligonucleotide for live-cell RNA imaging

Cited by 12 publications

References 37 publications

Strength in numbers: quantitative single‐molecule RNA detection assays

Strength in numbers: quantitative single‐molecule RNA detection assays

Immuno‐detection of mRNA‐binding protein complex in human cells under transmission electron microscopy

Illuminating Messengers: An Update and Outlook on RNA Visualization in Bacteria

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