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Real time monitoring of endogenous cytoplasmic mRNA using linear antisense 2'-O-methyl RNA probes in living cells.

Okabe K, Harada Y, Zhang J, Tadakuma H, Tani T, Funatsu T - Nucleic Acids Res. (2010)

Bottom Line: Visualization and monitoring of endogenous mRNA in the cytoplasm of living cells promises a significant comprehension of refined post-transcriptional regulation.Fluorescently labeled linear antisense oligonucleotides can bind to natural mRNA in a sequence-specific way and, therefore, provide a powerful tool in probing endogenous mRNA.Thus, our approach provides a basis for real time monitoring of endogenous cytoplasmic mRNA in living cells.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo Bunkyo-ku, Tokyo 113-0033, Japan.

ABSTRACT
Visualization and monitoring of endogenous mRNA in the cytoplasm of living cells promises a significant comprehension of refined post-transcriptional regulation. Fluorescently labeled linear antisense oligonucleotides can bind to natural mRNA in a sequence-specific way and, therefore, provide a powerful tool in probing endogenous mRNA. Here, we investigated the feasibility of using linear antisense probes to monitor the variable and dynamic expression of endogenous cytoplasmic mRNAs. Two linear antisense 2'-O-methyl RNA probes, which have different interactive fluorophores at the 5'-end of one probe and at the 3'-end of the other, were used to allow fluorescence resonance energy transfer (FRET) upon hybridization to the target mRNA. By characterizing the formation of the probe-mRNA hybrids in living cells, we found that the probe composition and concentration are crucial parameters in the visualization of endogenous mRNA with high specificity. Furthermore, rapid hybridization (within 1 min) of the linear antisense probe enabled us to visualize dynamic processes of endogenous c-fos mRNA, such as fast elevation of levels after gene induction and the localization of c-fos mRNA in stress granules in response to cellular stress. Thus, our approach provides a basis for real time monitoring of endogenous cytoplasmic mRNA in living cells.

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Antisense 2′OMeRNA probes can form a stable complex with mRNA in living cells whereas antisense ODN probes cannot. (A–C) PC images and fluorescence images of the mRNA complex that was hybridized with probes in vitro and then microinjected into the cytoplasm of living COS7 cells; Antisense ODN probes (A), antisense 2′OMeRNA probes (B) and sense 2′OMeRNA probes (C). D, F and A in each image indicate donor image, FRET image and acceptor image, respectively. Scale bars, 10 µm.
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Figure 2: Antisense 2′OMeRNA probes can form a stable complex with mRNA in living cells whereas antisense ODN probes cannot. (A–C) PC images and fluorescence images of the mRNA complex that was hybridized with probes in vitro and then microinjected into the cytoplasm of living COS7 cells; Antisense ODN probes (A), antisense 2′OMeRNA probes (B) and sense 2′OMeRNA probes (C). D, F and A in each image indicate donor image, FRET image and acceptor image, respectively. Scale bars, 10 µm.

Mentions: Cells in which the mRNA-antisense ODN probe complexes were introduced showed FRET fluorescence in the cytoplasm immediately after microinjection (see the middle fluorescence image of the upper panel of Figure 2A), but the FRET signal soon disappeared. Ten minutes after injection, no FRET signal was detected in the cytoplasm; a weak FRET fluorescence observed in the nucleus was at the background noise level. Donor and acceptor images in the lower panel of Figure 2A show that both probes moved into the nucleus. Free oligonucleotides accumulate in the nucleus when they are introduced into the cytoplasm (12), therefore, ODN probes might exist alone and redistribute as free oligonucleotides 10 min after microinjection. This suggests that ODN probes, even though once hybridized to c-fos mRNA in vitro, were dissociated from the mRNA in living cells and that free ODN probes moved into the nucleus. Since the mRNA-ODN complex is unstable in living cells, linear ODN probes were inappropriate for the visualization of mRNAs in living cells.Figure 2.


Real time monitoring of endogenous cytoplasmic mRNA using linear antisense 2'-O-methyl RNA probes in living cells.

Okabe K, Harada Y, Zhang J, Tadakuma H, Tani T, Funatsu T - Nucleic Acids Res. (2010)

Antisense 2′OMeRNA probes can form a stable complex with mRNA in living cells whereas antisense ODN probes cannot. (A–C) PC images and fluorescence images of the mRNA complex that was hybridized with probes in vitro and then microinjected into the cytoplasm of living COS7 cells; Antisense ODN probes (A), antisense 2′OMeRNA probes (B) and sense 2′OMeRNA probes (C). D, F and A in each image indicate donor image, FRET image and acceptor image, respectively. Scale bars, 10 µm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3045578&req=5

Figure 2: Antisense 2′OMeRNA probes can form a stable complex with mRNA in living cells whereas antisense ODN probes cannot. (A–C) PC images and fluorescence images of the mRNA complex that was hybridized with probes in vitro and then microinjected into the cytoplasm of living COS7 cells; Antisense ODN probes (A), antisense 2′OMeRNA probes (B) and sense 2′OMeRNA probes (C). D, F and A in each image indicate donor image, FRET image and acceptor image, respectively. Scale bars, 10 µm.
Mentions: Cells in which the mRNA-antisense ODN probe complexes were introduced showed FRET fluorescence in the cytoplasm immediately after microinjection (see the middle fluorescence image of the upper panel of Figure 2A), but the FRET signal soon disappeared. Ten minutes after injection, no FRET signal was detected in the cytoplasm; a weak FRET fluorescence observed in the nucleus was at the background noise level. Donor and acceptor images in the lower panel of Figure 2A show that both probes moved into the nucleus. Free oligonucleotides accumulate in the nucleus when they are introduced into the cytoplasm (12), therefore, ODN probes might exist alone and redistribute as free oligonucleotides 10 min after microinjection. This suggests that ODN probes, even though once hybridized to c-fos mRNA in vitro, were dissociated from the mRNA in living cells and that free ODN probes moved into the nucleus. Since the mRNA-ODN complex is unstable in living cells, linear ODN probes were inappropriate for the visualization of mRNAs in living cells.Figure 2.

Bottom Line: Visualization and monitoring of endogenous mRNA in the cytoplasm of living cells promises a significant comprehension of refined post-transcriptional regulation.Fluorescently labeled linear antisense oligonucleotides can bind to natural mRNA in a sequence-specific way and, therefore, provide a powerful tool in probing endogenous mRNA.Thus, our approach provides a basis for real time monitoring of endogenous cytoplasmic mRNA in living cells.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo Bunkyo-ku, Tokyo 113-0033, Japan.

ABSTRACT
Visualization and monitoring of endogenous mRNA in the cytoplasm of living cells promises a significant comprehension of refined post-transcriptional regulation. Fluorescently labeled linear antisense oligonucleotides can bind to natural mRNA in a sequence-specific way and, therefore, provide a powerful tool in probing endogenous mRNA. Here, we investigated the feasibility of using linear antisense probes to monitor the variable and dynamic expression of endogenous cytoplasmic mRNAs. Two linear antisense 2'-O-methyl RNA probes, which have different interactive fluorophores at the 5'-end of one probe and at the 3'-end of the other, were used to allow fluorescence resonance energy transfer (FRET) upon hybridization to the target mRNA. By characterizing the formation of the probe-mRNA hybrids in living cells, we found that the probe composition and concentration are crucial parameters in the visualization of endogenous mRNA with high specificity. Furthermore, rapid hybridization (within 1 min) of the linear antisense probe enabled us to visualize dynamic processes of endogenous c-fos mRNA, such as fast elevation of levels after gene induction and the localization of c-fos mRNA in stress granules in response to cellular stress. Thus, our approach provides a basis for real time monitoring of endogenous cytoplasmic mRNA in living cells.

Show MeSH