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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 time course studies of antisense probes with mRNA. Cases using linear antisense 2′OMeRNA probes in a 1×SSC solution (A), MB in a 1×SSC solution (B), streptavidin-bound linear antisense 2′OMeRNA probes in living COS7 cells (C) and streptavidin-bound MB in living COS7 cells (D) are presented. A c-fos mRNA prepared by in vitro transcription and the endogenously expressed c-fos mRNA in the cytoplasm of living COS7 cells was targeted in (A and B) and (C and D), respectively. The concentration of mRNA and antisense probes was 50 nM in a 1×SSC solution (A and B). In living cell studies, 3 µM of probes (in a microinjection needle) were introduced into the cell (C and D). The estimated concentration of probes inside the cell is ∼0.5 µM. The time course plots were fitted to a single-exponential function of the time constant (t) for the hybridization reaction in each case (red fit line for linear antisense probe and blue fit line for MB). Error bas in (C and D) represent SD.
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Figure 4: Hybridization time course studies of antisense probes with mRNA. Cases using linear antisense 2′OMeRNA probes in a 1×SSC solution (A), MB in a 1×SSC solution (B), streptavidin-bound linear antisense 2′OMeRNA probes in living COS7 cells (C) and streptavidin-bound MB in living COS7 cells (D) are presented. A c-fos mRNA prepared by in vitro transcription and the endogenously expressed c-fos mRNA in the cytoplasm of living COS7 cells was targeted in (A and B) and (C and D), respectively. The concentration of mRNA and antisense probes was 50 nM in a 1×SSC solution (A and B). In living cell studies, 3 µM of probes (in a microinjection needle) were introduced into the cell (C and D). The estimated concentration of probes inside the cell is ∼0.5 µM. The time course plots were fitted to a single-exponential function of the time constant (t) for the hybridization reaction in each case (red fit line for linear antisense probe and blue fit line for MB). Error bas in (C and D) represent SD.

Mentions: A highly advantageous characteristic of linear antisense probes for real time monitoring of endogenous mRNA in living cells would be a fast hybridization reaction with target mRNA. However, the intracellular kinetics of linear antisense probe hybridization has been unclear. Here, hybridization time course studies were performed in vitro and in living cells by measuring the intensities of FRET fluorescence and resultant time constants were calculated. These were compared with those obtained with an MB that has an identical antisense sequence for the target (Supplementary Figure S3). Results obtained in 1×SSC solution are shown in Figure 4A and B. The time constants for hybridization of the linear antisense 2′OMeRNA probes and the MB were 3.97 min and 4.72 h, respectively. Linear antisense probes were, therefore, shown to hybridize more quickly than the MB, which had a time constant 71.3 times larger than that of the linear antisense probes. The hybridization kinetics of MB strongly depends on nucleotide composition and stem length (16). Therefore, MBs that possess different composition (ODN) and shorter stem (4 nt) were also tested (Supplementary Table S1 and Figure S4). Time constants for hybridization of ODN MB and 2′OMeRNA MB with short stem in 1×SSC solution were 2.35 and 2.57 h, respectively (Supplementary Figure S5), suggesting that these alterations of probe chemistry result in a better MB kinetics.Figure 4.


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 time course studies of antisense probes with mRNA. Cases using linear antisense 2′OMeRNA probes in a 1×SSC solution (A), MB in a 1×SSC solution (B), streptavidin-bound linear antisense 2′OMeRNA probes in living COS7 cells (C) and streptavidin-bound MB in living COS7 cells (D) are presented. A c-fos mRNA prepared by in vitro transcription and the endogenously expressed c-fos mRNA in the cytoplasm of living COS7 cells was targeted in (A and B) and (C and D), respectively. The concentration of mRNA and antisense probes was 50 nM in a 1×SSC solution (A and B). In living cell studies, 3 µM of probes (in a microinjection needle) were introduced into the cell (C and D). The estimated concentration of probes inside the cell is ∼0.5 µM. The time course plots were fitted to a single-exponential function of the time constant (t) for the hybridization reaction in each case (red fit line for linear antisense probe and blue fit line for MB). Error bas in (C and D) represent SD.
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Related In: Results  -  Collection

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Figure 4: Hybridization time course studies of antisense probes with mRNA. Cases using linear antisense 2′OMeRNA probes in a 1×SSC solution (A), MB in a 1×SSC solution (B), streptavidin-bound linear antisense 2′OMeRNA probes in living COS7 cells (C) and streptavidin-bound MB in living COS7 cells (D) are presented. A c-fos mRNA prepared by in vitro transcription and the endogenously expressed c-fos mRNA in the cytoplasm of living COS7 cells was targeted in (A and B) and (C and D), respectively. The concentration of mRNA and antisense probes was 50 nM in a 1×SSC solution (A and B). In living cell studies, 3 µM of probes (in a microinjection needle) were introduced into the cell (C and D). The estimated concentration of probes inside the cell is ∼0.5 µM. The time course plots were fitted to a single-exponential function of the time constant (t) for the hybridization reaction in each case (red fit line for linear antisense probe and blue fit line for MB). Error bas in (C and D) represent SD.
Mentions: A highly advantageous characteristic of linear antisense probes for real time monitoring of endogenous mRNA in living cells would be a fast hybridization reaction with target mRNA. However, the intracellular kinetics of linear antisense probe hybridization has been unclear. Here, hybridization time course studies were performed in vitro and in living cells by measuring the intensities of FRET fluorescence and resultant time constants were calculated. These were compared with those obtained with an MB that has an identical antisense sequence for the target (Supplementary Figure S3). Results obtained in 1×SSC solution are shown in Figure 4A and B. The time constants for hybridization of the linear antisense 2′OMeRNA probes and the MB were 3.97 min and 4.72 h, respectively. Linear antisense probes were, therefore, shown to hybridize more quickly than the MB, which had a time constant 71.3 times larger than that of the linear antisense probes. The hybridization kinetics of MB strongly depends on nucleotide composition and stem length (16). Therefore, MBs that possess different composition (ODN) and shorter stem (4 nt) were also tested (Supplementary Table S1 and Figure S4). Time constants for hybridization of ODN MB and 2′OMeRNA MB with short stem in 1×SSC solution were 2.35 and 2.57 h, respectively (Supplementary Figure S5), suggesting that these alterations of probe chemistry result in a better MB kinetics.Figure 4.

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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