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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

Co-localization of fluorescence signal from random MB with lysosome staining. Twenty-four hours after random MB delivery, cells were incubated with LysoTracker to stain lysosomes, and the signal from Cy3 and LysoTracker were imaged using a deconvolution microscope. Some of the spot-like signal from random MB (A) is co-localized with signal from lysosome staining (B), as indicated by the merged images in (C).
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Figure 4: Co-localization of fluorescence signal from random MB with lysosome staining. Twenty-four hours after random MB delivery, cells were incubated with LysoTracker to stain lysosomes, and the signal from Cy3 and LysoTracker were imaged using a deconvolution microscope. Some of the spot-like signal from random MB (A) is co-localized with signal from lysosome staining (B), as indicated by the merged images in (C).

Mentions: The fluorescence signal from random MB with DNA backbone showed both filamentous signal and spot-like signal, as can be seen from the image in Figure 2C. While the filamentous signal is co-localized with mitochondria staining, the spot-like signal is not. To gain insight, we stained lysosomes in live HDF cells with a lysosome-specific dye LysoTracker 24 h after delivery of random MBs (with DNA backbone), and imaged the fluorescence signal from the random MB (Figure 4A) and LysoTracker (Figure 4B), respectively. Merging the images in Figure 4A and B indicated that most of the spot-like signal from random MBs was co-localized with lysosomes (Figure 4C). Since the spot-like signal from random MBs was observed only after at least 8 h after probe delivery, it is possible that some of the random MBs were internalized and trapped in the lysosomes and degraded there due to the endonuclease activity. This hypothesis is supported by the observation that random MBs with 2′-O-methyl backbone, which has higher resistance to nuclease degradation, did not show spot-like signal (Figure 2F).Figure 4.


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)

Co-localization of fluorescence signal from random MB with lysosome staining. Twenty-four hours after random MB delivery, cells were incubated with LysoTracker to stain lysosomes, and the signal from Cy3 and LysoTracker were imaged using a deconvolution microscope. Some of the spot-like signal from random MB (A) is co-localized with signal from lysosome staining (B), as indicated by the merged images in (C).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: Co-localization of fluorescence signal from random MB with lysosome staining. Twenty-four hours after random MB delivery, cells were incubated with LysoTracker to stain lysosomes, and the signal from Cy3 and LysoTracker were imaged using a deconvolution microscope. Some of the spot-like signal from random MB (A) is co-localized with signal from lysosome staining (B), as indicated by the merged images in (C).
Mentions: The fluorescence signal from random MB with DNA backbone showed both filamentous signal and spot-like signal, as can be seen from the image in Figure 2C. While the filamentous signal is co-localized with mitochondria staining, the spot-like signal is not. To gain insight, we stained lysosomes in live HDF cells with a lysosome-specific dye LysoTracker 24 h after delivery of random MBs (with DNA backbone), and imaged the fluorescence signal from the random MB (Figure 4A) and LysoTracker (Figure 4B), respectively. Merging the images in Figure 4A and B indicated that most of the spot-like signal from random MBs was co-localized with lysosomes (Figure 4C). Since the spot-like signal from random MBs was observed only after at least 8 h after probe delivery, it is possible that some of the random MBs were internalized and trapped in the lysosomes and degraded there due to the endonuclease activity. This hypothesis is supported by the observation that random MBs with 2′-O-methyl backbone, which has higher resistance to nuclease degradation, did not show spot-like signal (Figure 2F).Figure 4.

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