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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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Hybridization of antisense probes with the target mRNA. (A) Schematic drawing of mRNA detection. When two kinds of fluorescently labeled antisense probes are hybridized to adjacent sequences of the target mRNA, the distance between the two fluorophores becomes short and FRET occurs. (B) The fluorescence spectra of fluorescently labeled probes containing 2′OMeRNA or DNA backbones in the absence (gray line) or presence (red line) of the target c-fos mRNA, prepared by an in vitro transcription system. FRET was observed when mRNA hybridized with antisense probes; in contrast, FRET was not detected when using linear sense probes.
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Figure 1: Hybridization of antisense probes with the target mRNA. (A) Schematic drawing of mRNA detection. When two kinds of fluorescently labeled antisense probes are hybridized to adjacent sequences of the target mRNA, the distance between the two fluorophores becomes short and FRET occurs. (B) The fluorescence spectra of fluorescently labeled probes containing 2′OMeRNA or DNA backbones in the absence (gray line) or presence (red line) of the target c-fos mRNA, prepared by an in vitro transcription system. FRET was observed when mRNA hybridized with antisense probes; in contrast, FRET was not detected when using linear sense probes.

Mentions: c-fos mRNA, which encodes the transcription factor c-Fos, was chosen as the target endogenous mRNA. Linear antisense probes were designed (Table 1) according to a previous report (20). Cy3 and Cy5 were selected as a pair of fluorophores for the detection of FRET. The donor and acceptor probes were labeled with Cy3 at the 5′-end and Cy5 at the 3′-end, respectively, as illustrated in Figure 1A. Nucleic acid probes having sense sequences of c-fos mRNA (sense probes) were also prepared for negative control experiments. The concentration and the molar ratio of fluorescent dye to oligonucleotide were measured using UV–VIS photospectroscopy (V-570; JASCO, Hachioji, Japan). The molar ratios of dyes to oligonucleotides were in the range from 0.72 to 1.03. 2′OMeRNA MBs for c-fos mRNA were also prepared (Table 1). These MBs consisted of the same antisense sequences as the linear antisense donor probe and contained self-complementary sequences (stem) on either end that were labeled by Cy3 and BHQ2 (22). Pairs of antisense probes bearing a 2′OMeRNA or a DNA backbone and 2′OMeRNA MBs were purchased from Proligo (Kyoto, Japan) and FASMAC (Atsugi, Japan).Figure 1.


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)

Hybridization of antisense probes with the target mRNA. (A) Schematic drawing of mRNA detection. When two kinds of fluorescently labeled antisense probes are hybridized to adjacent sequences of the target mRNA, the distance between the two fluorophores becomes short and FRET occurs. (B) The fluorescence spectra of fluorescently labeled probes containing 2′OMeRNA or DNA backbones in the absence (gray line) or presence (red line) of the target c-fos mRNA, prepared by an in vitro transcription system. FRET was observed when mRNA hybridized with antisense probes; in contrast, FRET was not detected when using linear sense probes.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 1: Hybridization of antisense probes with the target mRNA. (A) Schematic drawing of mRNA detection. When two kinds of fluorescently labeled antisense probes are hybridized to adjacent sequences of the target mRNA, the distance between the two fluorophores becomes short and FRET occurs. (B) The fluorescence spectra of fluorescently labeled probes containing 2′OMeRNA or DNA backbones in the absence (gray line) or presence (red line) of the target c-fos mRNA, prepared by an in vitro transcription system. FRET was observed when mRNA hybridized with antisense probes; in contrast, FRET was not detected when using linear sense probes.
Mentions: c-fos mRNA, which encodes the transcription factor c-Fos, was chosen as the target endogenous mRNA. Linear antisense probes were designed (Table 1) according to a previous report (20). Cy3 and Cy5 were selected as a pair of fluorophores for the detection of FRET. The donor and acceptor probes were labeled with Cy3 at the 5′-end and Cy5 at the 3′-end, respectively, as illustrated in Figure 1A. Nucleic acid probes having sense sequences of c-fos mRNA (sense probes) were also prepared for negative control experiments. The concentration and the molar ratio of fluorescent dye to oligonucleotide were measured using UV–VIS photospectroscopy (V-570; JASCO, Hachioji, Japan). The molar ratios of dyes to oligonucleotides were in the range from 0.72 to 1.03. 2′OMeRNA MBs for c-fos mRNA were also prepared (Table 1). These MBs consisted of the same antisense sequences as the linear antisense donor probe and contained self-complementary sequences (stem) on either end that were labeled by Cy3 and BHQ2 (22). Pairs of antisense probes bearing a 2′OMeRNA or a DNA backbone and 2′OMeRNA MBs were purchased from Proligo (Kyoto, Japan) and FASMAC (Atsugi, Japan).Figure 1.

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
Related in: MedlinePlus