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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.

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Related in: MedlinePlus

Signal distribution of Alexa Fluor-labeled oligonucleotide probes, as indicated by fluorescence (A–C) and DIC (D–F) images of HDF cells 24 h after delivery of probes, including random-sequence linear oligonucleotide probes labeled with Alexa Fluor 488 (A and D) and Alexa Fluor 546 (B and E) respectively, and random-sequence MBs labeled with Alexa Fluor 546 (C and F). Alexa Fluor labeled probes showed perinuclear localization instead of mitochondria accumulation. Scale bar = 15 µm.
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Figure 5: Signal distribution of Alexa Fluor-labeled oligonucleotide probes, as indicated by fluorescence (A–C) and DIC (D–F) images of HDF cells 24 h after delivery of probes, including random-sequence linear oligonucleotide probes labeled with Alexa Fluor 488 (A and D) and Alexa Fluor 546 (B and E) respectively, and random-sequence MBs labeled with Alexa Fluor 546 (C and F). Alexa Fluor labeled probes showed perinuclear localization instead of mitochondria accumulation. Scale bar = 15 µm.

Mentions: For Cy3-labeled oligonucleotide probes used in our studies, oligonucleotide itself is negatively charged, while the Cy3 dye is positively charged. Thus, it is possible that the accumulation of fluorescence signals at mitochondria is mediated by the Cy3 dye, which belongs to the cyanine dye family. In fact, the dye we used for specific mitochondrial stain, DiOC6 is also a cyanine dye, and almost all dyes that show mitochondria-specific fluorescence are positively charged at physiological pH (33). To determine if other cyanine dyes induce mitochondrial accumulation of labeled oligonucleotide probed, we used Cy5 dye, which is also positively changed, to label random-sequence linear oligonucleotide probes instead of Cy3. Live HDF cells were imaged 24 hours after delivery of the Cy5 labeled linear ODN probed, and the images showed the same filament-like signal co-localized with mitochondrial staining as with Cy3-labeled probes (data not shown). We further used respectively Alexa Fluor 488 and Alexa Fluor 546 (with excitation and emission wavelengths similar to Cy3), which are negatively charged, to label random-sequence linear ODN probes instead of Cy3, and imaged the fluorescence emission 24 hours after probe delivery. However, as shown in Figure 5A and B, probes labeled respectively with Alexa Fluor 488 and Alexa Fluor 546 did not show signal accumulation at mitochondria; rather, the fluorescence signal from Alexa Fluor 488 and Alexa Fluor 546 labeled oligonucleotide probes showed a perinuclear localization with low signal intensity. Delivery of Alexa Fluor 546 labeled random MBs resulted in similar signal distribution 24 h after delivery (Figure 5C). These results clearly suggest that the accumulation of probe signal at mitochondria is dye-dependent, regardless of the structure of the ODN; cyanine dyes (positively charged) facilitate mitochondrial accumulation, whereas Alexa Fluor dyes (negatively charged) do not. Note that the results in Figure 5 were obtained after increasing the signal intensity of the fluorescence images significantly. With the same optics used for imaging Cy3 labeled ODN probes, we did not detect much signal.Figure 5.


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)

Signal distribution of Alexa Fluor-labeled oligonucleotide probes, as indicated by fluorescence (A–C) and DIC (D–F) images of HDF cells 24 h after delivery of probes, including random-sequence linear oligonucleotide probes labeled with Alexa Fluor 488 (A and D) and Alexa Fluor 546 (B and E) respectively, and random-sequence MBs labeled with Alexa Fluor 546 (C and F). Alexa Fluor labeled probes showed perinuclear localization instead of mitochondria accumulation. Scale bar = 15 µm.
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Figure 5: Signal distribution of Alexa Fluor-labeled oligonucleotide probes, as indicated by fluorescence (A–C) and DIC (D–F) images of HDF cells 24 h after delivery of probes, including random-sequence linear oligonucleotide probes labeled with Alexa Fluor 488 (A and D) and Alexa Fluor 546 (B and E) respectively, and random-sequence MBs labeled with Alexa Fluor 546 (C and F). Alexa Fluor labeled probes showed perinuclear localization instead of mitochondria accumulation. Scale bar = 15 µm.
Mentions: For Cy3-labeled oligonucleotide probes used in our studies, oligonucleotide itself is negatively charged, while the Cy3 dye is positively charged. Thus, it is possible that the accumulation of fluorescence signals at mitochondria is mediated by the Cy3 dye, which belongs to the cyanine dye family. In fact, the dye we used for specific mitochondrial stain, DiOC6 is also a cyanine dye, and almost all dyes that show mitochondria-specific fluorescence are positively charged at physiological pH (33). To determine if other cyanine dyes induce mitochondrial accumulation of labeled oligonucleotide probed, we used Cy5 dye, which is also positively changed, to label random-sequence linear oligonucleotide probes instead of Cy3. Live HDF cells were imaged 24 hours after delivery of the Cy5 labeled linear ODN probed, and the images showed the same filament-like signal co-localized with mitochondrial staining as with Cy3-labeled probes (data not shown). We further used respectively Alexa Fluor 488 and Alexa Fluor 546 (with excitation and emission wavelengths similar to Cy3), which are negatively charged, to label random-sequence linear ODN probes instead of Cy3, and imaged the fluorescence emission 24 hours after probe delivery. However, as shown in Figure 5A and B, probes labeled respectively with Alexa Fluor 488 and Alexa Fluor 546 did not show signal accumulation at mitochondria; rather, the fluorescence signal from Alexa Fluor 488 and Alexa Fluor 546 labeled oligonucleotide probes showed a perinuclear localization with low signal intensity. Delivery of Alexa Fluor 546 labeled random MBs resulted in similar signal distribution 24 h after delivery (Figure 5C). These results clearly suggest that the accumulation of probe signal at mitochondria is dye-dependent, regardless of the structure of the ODN; cyanine dyes (positively charged) facilitate mitochondrial accumulation, whereas Alexa Fluor dyes (negatively charged) do not. Note that the results in Figure 5 were obtained after increasing the signal intensity of the fluorescence images significantly. With the same optics used for imaging Cy3 labeled ODN probes, we did not detect much signal.Figure 5.

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