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

Fluorescence images of Cy3-labeled random-sequence linear ODN probe showing that the ODN probes are not inside the mitochondrial inner membrane. After delivery of 1 µM of Cy3-labeled random-sequence linear ODN probes, HDF cells were incubated for 24 h. Cells were further incubated without (A and C) or with (C and D) 2 µM of quencher-conjugated linear complementary target, and imaged at 1 h (A and C) or 24 h (B and D) after target delivery. The same exposure time was used for all images.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 7: Fluorescence images of Cy3-labeled random-sequence linear ODN probe showing that the ODN probes are not inside the mitochondrial inner membrane. After delivery of 1 µM of Cy3-labeled random-sequence linear ODN probes, HDF cells were incubated for 24 h. Cells were further incubated without (A and C) or with (C and D) 2 µM of quencher-conjugated linear complementary target, and imaged at 1 h (A and C) or 24 h (B and D) after target delivery. The same exposure time was used for all images.

Mentions: Why fluorescence signal from cyanine dye-labeled ODN probes accumulated at mitochondria 24 h after delivery? There are a few possible reasons: (i) charge of the dye, (ii) ODN structure, (iii) degradation of the probe inside mitochondria. Since the chemical environment inside the inner mitochondrial membrane (mitochondrial matrix) is different from that of cytosol, it is possible that cyanine dye-labeled ODN probes are degraded in the mitochondrial matrix. To determine if the probes were inside or outside the mitochondria inner membrane (the outer membrane of mitochondria is permeable to biomolecules <5 KD, while the inner membrane of mitochondria is impermeable to ions and macromolecules), we designed a linear target oligonucleotide (sense strand, Table 1) conjugated with a BHQ-2 quencher (Figure 1F), which has a sequence complementary to the random-sequence linear oligonucleotide probe (antisense, Table 1). Upon hybridization with the Cy3-labeled random-sequence linear ODN probe, the fluorescence of Cy3 is quenched by this target (data not shown). As discussed earlier, our results indicate that the BHQ-2 quencher is not involved in the mitochondrial localization of the ODN probes. Therefore, the BHQ-2 labeled ODN target by itself should not accumulate at mitochondria. We delivered the Cy3-labeled random-sequence linear ODN probes into live HDF cells, incubated the cells at 37°C for 24 h to allow the probes accumulate at mitochondria (as indicated by the bright signal from the Cy3-labeled linear ODN probe at this time point), then delivered the BHQ-2 labeled targets into these cells and incubated the cells at 37°C for up to 24 h. As shown in Figure 7, the fluorescence signal from Cy3-labeled ODN probes accumulated at mitochondria was decreased significantly by the BHQ-2 labeled target ODN just 1 h after target ODN delivery. 24 h after target ODN delivery, most of the fluorescence signals disappeared, presumably due to quenching by the ODN target delivered.


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)

Fluorescence images of Cy3-labeled random-sequence linear ODN probe showing that the ODN probes are not inside the mitochondrial inner membrane. After delivery of 1 µM of Cy3-labeled random-sequence linear ODN probes, HDF cells were incubated for 24 h. Cells were further incubated without (A and C) or with (C and D) 2 µM of quencher-conjugated linear complementary target, and imaged at 1 h (A and C) or 24 h (B and D) after target delivery. The same exposure time was used for all images.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 7: Fluorescence images of Cy3-labeled random-sequence linear ODN probe showing that the ODN probes are not inside the mitochondrial inner membrane. After delivery of 1 µM of Cy3-labeled random-sequence linear ODN probes, HDF cells were incubated for 24 h. Cells were further incubated without (A and C) or with (C and D) 2 µM of quencher-conjugated linear complementary target, and imaged at 1 h (A and C) or 24 h (B and D) after target delivery. The same exposure time was used for all images.
Mentions: Why fluorescence signal from cyanine dye-labeled ODN probes accumulated at mitochondria 24 h after delivery? There are a few possible reasons: (i) charge of the dye, (ii) ODN structure, (iii) degradation of the probe inside mitochondria. Since the chemical environment inside the inner mitochondrial membrane (mitochondrial matrix) is different from that of cytosol, it is possible that cyanine dye-labeled ODN probes are degraded in the mitochondrial matrix. To determine if the probes were inside or outside the mitochondria inner membrane (the outer membrane of mitochondria is permeable to biomolecules <5 KD, while the inner membrane of mitochondria is impermeable to ions and macromolecules), we designed a linear target oligonucleotide (sense strand, Table 1) conjugated with a BHQ-2 quencher (Figure 1F), which has a sequence complementary to the random-sequence linear oligonucleotide probe (antisense, Table 1). Upon hybridization with the Cy3-labeled random-sequence linear ODN probe, the fluorescence of Cy3 is quenched by this target (data not shown). As discussed earlier, our results indicate that the BHQ-2 quencher is not involved in the mitochondrial localization of the ODN probes. Therefore, the BHQ-2 labeled ODN target by itself should not accumulate at mitochondria. We delivered the Cy3-labeled random-sequence linear ODN probes into live HDF cells, incubated the cells at 37°C for 24 h to allow the probes accumulate at mitochondria (as indicated by the bright signal from the Cy3-labeled linear ODN probe at this time point), then delivered the BHQ-2 labeled targets into these cells and incubated the cells at 37°C for up to 24 h. As shown in Figure 7, the fluorescence signal from Cy3-labeled ODN probes accumulated at mitochondria was decreased significantly by the BHQ-2 labeled target ODN just 1 h after target ODN delivery. 24 h after target ODN delivery, most of the fluorescence signals disappeared, presumably due to quenching by the ODN target delivered.

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