Limits...
Slow non-specific accumulation of 2'-deoxy and 2'-O-methyl oligonucleotide probes at mitochondria in live cells.

Rhee WJ, Bao G - Nucleic Acids Res. (2010)

Bottom Line: Molecular beacons (MBs) have the potential to provide a powerful tool for rapid RNA detection in living cells, as well as monitoring the dynamics of RNA expression in response to external stimuli.To exploit this potential, it is necessary to distinguish true signal from background signal due to non-specific interactions.These results may help design and optimize fluorescence imaging probes for long-time RNA detection and monitoring in living cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA.

ABSTRACT
Molecular beacons (MBs) have the potential to provide a powerful tool for rapid RNA detection in living cells, as well as monitoring the dynamics of RNA expression in response to external stimuli. To exploit this potential, it is necessary to distinguish true signal from background signal due to non-specific interactions. Here, we show that, when cyanine-dye labeled 2'-deoxy and 2'-O-methyl oligonucleotide probes are inside living cells for >5 h, most of their signals co-localize with mitochondrial staining. These probes include random-sequence MB, dye-labeled single-strand linear oligonucleotide and dye-labeled double-stranded oligonucleotide. Using carbonyl cyanide m-chlorophenyl hydrazone treatment, we found that the non-specific accumulation of oligonucleotide probes at mitochondria was driven by mitochondrial membrane potential. We further demonstrated that the dye-labeled oligonucleotide probes were likely on/near the surface of mitochondria but not inside mitochondrial inner membrane. Interestingly, oligonucleotides probes labeled respectively with Alexa Fluor 488 and Alexa Fluor 546 did not accumulate at mitochondria, suggesting that the non-specific interaction between dye-labeled ODN probes and mitochondria is dye-specific. These results may help design and optimize fluorescence imaging probes for long-time RNA detection and monitoring in living cells.

Show MeSH

Related in: MedlinePlus

The effect of mitochondrial membrane potential on the accumulation of fluorescence signal from: (A) random MB, (B) random-sequence linear oligonucleotide probe, (C) DSO probe labeled with a dye-quencher pair, (D) dye-labeled DSO probe without quencher. Upper panel: fluorescence images of Cy3-labeled random-sequence oligonucleotide probes (upper left panel) and mitochondrial staining (upper middle panel) in normal HDF cells. The merge of the images in upper left and upper middle panels is shown in the upper right panel. Lower panel: fluorescence images of dye-labeled ODN probes (lower left panel) and mitochondria staining (lower middle panel) in CCCP-treated HDF cells. The merge of the images in lower left panel and lower middle panel is shown in the lower right panel. After the delivery of dye-labeled oligonucleotide probes, cells were incubated for 24 h and then treated with CCCP for 30 min. Cells were further incubated in DiOC6- and CCCP-containing medium for 30 min before imaged using a deconvolution microscope with the same exposure time for dye-labeled ODN probe and DiOC6.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2875028&req=5

Figure 3: The effect of mitochondrial membrane potential on the accumulation of fluorescence signal from: (A) random MB, (B) random-sequence linear oligonucleotide probe, (C) DSO probe labeled with a dye-quencher pair, (D) dye-labeled DSO probe without quencher. Upper panel: fluorescence images of Cy3-labeled random-sequence oligonucleotide probes (upper left panel) and mitochondrial staining (upper middle panel) in normal HDF cells. The merge of the images in upper left and upper middle panels is shown in the upper right panel. Lower panel: fluorescence images of dye-labeled ODN probes (lower left panel) and mitochondria staining (lower middle panel) in CCCP-treated HDF cells. The merge of the images in lower left panel and lower middle panel is shown in the lower right panel. After the delivery of dye-labeled oligonucleotide probes, cells were incubated for 24 h and then treated with CCCP for 30 min. Cells were further incubated in DiOC6- and CCCP-containing medium for 30 min before imaged using a deconvolution microscope with the same exposure time for dye-labeled ODN probe and DiOC6.

Mentions: The non-specific fluorescence signal of the oligonucleotide probes 24 h after delivery all showed filament-like localization, similar to the staining of mitochondria (30). To confirm this, we used DiOC6, a mitochondrial staining dye to stain mitochondria in live HDF cells 24 h after delivery of oligonucleotide probes (including random MB, dye-labeled ODN and DSO probes), and imaged the fluorescence signals from Cy3 (excitation at 545 nm and emission detection at 570 nm) and DiOC6 (excitation at 470 nm and emission detection at 525 nm), respectively. As shown in Figure 3A, the filament-like signal from the deoxynucleotide random MB (red) co-localizes with that from mitochondrial staining (green), as indicated by the merged images. The use of a different dye, MitoFluor Green to stain mitochondria gave the same result (data not shown). For dye-labeled ODN and DSO probes with DNA backbone, the filamentous signals (red) were also found to co-localize with the signal from mitochondrial staining (green), as shown by the images in Figure 3B and C, respectively. These results provide strong evidence that, after long-time (24 h) incubation, the Cy3-labeled ODN probes accumulated at mitochondria in live HDF cells.Figure 3.


Slow non-specific accumulation of 2'-deoxy and 2'-O-methyl oligonucleotide probes at mitochondria in live cells.

Rhee WJ, Bao G - Nucleic Acids Res. (2010)

The effect of mitochondrial membrane potential on the accumulation of fluorescence signal from: (A) random MB, (B) random-sequence linear oligonucleotide probe, (C) DSO probe labeled with a dye-quencher pair, (D) dye-labeled DSO probe without quencher. Upper panel: fluorescence images of Cy3-labeled random-sequence oligonucleotide probes (upper left panel) and mitochondrial staining (upper middle panel) in normal HDF cells. The merge of the images in upper left and upper middle panels is shown in the upper right panel. Lower panel: fluorescence images of dye-labeled ODN probes (lower left panel) and mitochondria staining (lower middle panel) in CCCP-treated HDF cells. The merge of the images in lower left panel and lower middle panel is shown in the lower right panel. After the delivery of dye-labeled oligonucleotide probes, cells were incubated for 24 h and then treated with CCCP for 30 min. Cells were further incubated in DiOC6- and CCCP-containing medium for 30 min before imaged using a deconvolution microscope with the same exposure time for dye-labeled ODN probe and DiOC6.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2875028&req=5

Figure 3: The effect of mitochondrial membrane potential on the accumulation of fluorescence signal from: (A) random MB, (B) random-sequence linear oligonucleotide probe, (C) DSO probe labeled with a dye-quencher pair, (D) dye-labeled DSO probe without quencher. Upper panel: fluorescence images of Cy3-labeled random-sequence oligonucleotide probes (upper left panel) and mitochondrial staining (upper middle panel) in normal HDF cells. The merge of the images in upper left and upper middle panels is shown in the upper right panel. Lower panel: fluorescence images of dye-labeled ODN probes (lower left panel) and mitochondria staining (lower middle panel) in CCCP-treated HDF cells. The merge of the images in lower left panel and lower middle panel is shown in the lower right panel. After the delivery of dye-labeled oligonucleotide probes, cells were incubated for 24 h and then treated with CCCP for 30 min. Cells were further incubated in DiOC6- and CCCP-containing medium for 30 min before imaged using a deconvolution microscope with the same exposure time for dye-labeled ODN probe and DiOC6.
Mentions: The non-specific fluorescence signal of the oligonucleotide probes 24 h after delivery all showed filament-like localization, similar to the staining of mitochondria (30). To confirm this, we used DiOC6, a mitochondrial staining dye to stain mitochondria in live HDF cells 24 h after delivery of oligonucleotide probes (including random MB, dye-labeled ODN and DSO probes), and imaged the fluorescence signals from Cy3 (excitation at 545 nm and emission detection at 570 nm) and DiOC6 (excitation at 470 nm and emission detection at 525 nm), respectively. As shown in Figure 3A, the filament-like signal from the deoxynucleotide random MB (red) co-localizes with that from mitochondrial staining (green), as indicated by the merged images. The use of a different dye, MitoFluor Green to stain mitochondria gave the same result (data not shown). For dye-labeled ODN and DSO probes with DNA backbone, the filamentous signals (red) were also found to co-localize with the signal from mitochondrial staining (green), as shown by the images in Figure 3B and C, respectively. These results provide strong evidence that, after long-time (24 h) incubation, the Cy3-labeled ODN probes accumulated at mitochondria in live HDF cells.Figure 3.

Bottom Line: Molecular beacons (MBs) have the potential to provide a powerful tool for rapid RNA detection in living cells, as well as monitoring the dynamics of RNA expression in response to external stimuli.To exploit this potential, it is necessary to distinguish true signal from background signal due to non-specific interactions.These results may help design and optimize fluorescence imaging probes for long-time RNA detection and monitoring in living cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA.

ABSTRACT
Molecular beacons (MBs) have the potential to provide a powerful tool for rapid RNA detection in living cells, as well as monitoring the dynamics of RNA expression in response to external stimuli. To exploit this potential, it is necessary to distinguish true signal from background signal due to non-specific interactions. Here, we show that, when cyanine-dye labeled 2'-deoxy and 2'-O-methyl oligonucleotide probes are inside living cells for >5 h, most of their signals co-localize with mitochondrial staining. These probes include random-sequence MB, dye-labeled single-strand linear oligonucleotide and dye-labeled double-stranded oligonucleotide. Using carbonyl cyanide m-chlorophenyl hydrazone treatment, we found that the non-specific accumulation of oligonucleotide probes at mitochondria was driven by mitochondrial membrane potential. We further demonstrated that the dye-labeled oligonucleotide probes were likely on/near the surface of mitochondria but not inside mitochondrial inner membrane. Interestingly, oligonucleotides probes labeled respectively with Alexa Fluor 488 and Alexa Fluor 546 did not accumulate at mitochondria, suggesting that the non-specific interaction between dye-labeled ODN probes and mitochondria is dye-specific. These results may help design and optimize fluorescence imaging probes for long-time RNA detection and monitoring in living cells.

Show MeSH
Related in: MedlinePlus