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Methods for Evaluating Cell-Specific, Cell-Internalizing RNA Aptamers.

Hernandez LI, Flenker KS, Hernandez FJ, Klingelhutz AJ, McNamara JO, Giangrande PH - Pharmaceuticals (Basel) (2013)

Bottom Line: Here we describe methods to monitor for cellular uptake of aptamers.These include: (1) antibody amplification microscopy, (2) microplate-based fluorescence assay, (3) a quantitative and ultrasensitive internalization method ("QUSIM") and (4) a way to monitor for cytoplasmic delivery using the ribosome inactivating protein-based (RNA-RIP) assay.Collectively, these methods provide a toolset that can expedite the development of aptamer ligands to target and deliver therapeutic siRNAs in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, University of Iowa, Iowa City, IA 52242, USA.

ABSTRACT
Recent clinical trials of small interfering RNAs (siRNAs) highlight the need for robust delivery technologies that will facilitate the successful application of these therapeutics to humans. Arguably, cell targeting by conjugation to cell-specific ligands provides a viable solution to this problem. Synthetic RNA ligands (aptamers) represent an emerging class of pharmaceuticals with great potential for targeted therapeutic applications. For targeted delivery of siRNAs with aptamers, the aptamer-siRNA conjugate must be taken up by cells and reach the cytoplasm. To this end, we have developed cell-based selection approaches to isolate aptamers that internalize upon binding to their cognate receptor on the cell surface. Here we describe methods to monitor for cellular uptake of aptamers. These include: (1) antibody amplification microscopy, (2) microplate-based fluorescence assay, (3) a quantitative and ultrasensitive internalization method ("QUSIM") and (4) a way to monitor for cytoplasmic delivery using the ribosome inactivating protein-based (RNA-RIP) assay. Collectively, these methods provide a toolset that can expedite the development of aptamer ligands to target and deliver therapeutic siRNAs in vivo.

No MeSH data available.


Fluorescence microscopy. (A) Direct fluorescence method. FAM labeled anti-PSMA RNA aptamer (A9g) was incubated with either PC3(PSMA+) or PC3(PSMA-) cells. A high salt wash step was performed to remove unbound or surface bound RNA. Internalized RNA was visualized using fluorescence microscopy. A scrambled, non-internalizing aptamer (Scr) was used as a negative control in these experiments. Florescence images were overlaid with DAPI and P/C (phase contrast) channels. Arrows indicate perinuclear localization of internalized A9g aptamer. (B) Antibody amplification method. FAM-labeled anti-TrkB RNA aptamer (C4-3) was incubated with either TrkB expressing or non-expressing HEK293 cells at 37 °C. FAM-labeled control aptamer (Scr) was used as a control for specificity. Unbound and surface bound RNA was removed as described above. Internalized RNA signal was amplified by incubation with an anti-FITC antibody and Alexa488 secondary antibody. Vehicle treated cells (No RNA) or cells subjected to incubation with antibodies alone (Ab control) were used as controls.
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pharmaceuticals-06-00295-f002: Fluorescence microscopy. (A) Direct fluorescence method. FAM labeled anti-PSMA RNA aptamer (A9g) was incubated with either PC3(PSMA+) or PC3(PSMA-) cells. A high salt wash step was performed to remove unbound or surface bound RNA. Internalized RNA was visualized using fluorescence microscopy. A scrambled, non-internalizing aptamer (Scr) was used as a negative control in these experiments. Florescence images were overlaid with DAPI and P/C (phase contrast) channels. Arrows indicate perinuclear localization of internalized A9g aptamer. (B) Antibody amplification method. FAM-labeled anti-TrkB RNA aptamer (C4-3) was incubated with either TrkB expressing or non-expressing HEK293 cells at 37 °C. FAM-labeled control aptamer (Scr) was used as a control for specificity. Unbound and surface bound RNA was removed as described above. Internalized RNA signal was amplified by incubation with an anti-FITC antibody and Alexa488 secondary antibody. Vehicle treated cells (No RNA) or cells subjected to incubation with antibodies alone (Ab control) were used as controls.

Mentions: We evaluated the sub-cellular localization of FAM-labeled aptamer A9g using fluorescence microscopy (Figure 2A). FAM-labeled A9g was incubated with either PC3 cells lacking PSMA [PC3(PSMA-)] or PC3 cells expressing PSMA [PC3(PSMA+)] at 37 °C for 30 min (Figure 2A). A high salt wash step was used to remove unbound or surface bound RNA. As expected, fluorescence signal was observed only in PC3(PSMA+) cells. The signal was mostly localized in the perinuclear region, as shown by the arrow heads in the merged (FAM and DAPI) micrographs (Figure 2A). No signal was seen in PC3 cells lacking PSMA expression. Furthermore, a control, scrambled FAM-RNA (Scr) did not bind/internalize to PC3(PSMA+) cells, suggesting that the fluorescence signal is specific to A9g. Samples treated with buffer only (No RNA) were used to control for background fluorescence. We verified that the fluorescence signal seen in PC3(PSMA+) cells was mostly due to internalized RNA by performing the incubation of RNA on cells at 4 °C, which inhibits active transport (Supplementary Figure 1). As seen in Supplementary Figure 1, when the high salt wash step is applied to PC3(PSMA+) cells following incubation with FAM-A9g at 4 °C, the majority of the fluorescence signal is abrogated resulting only in a residual, low-intensity membrane signal (Supplementary Figure 1; right panel). This signal corresponds to surface bound FAM-A9g confirming that at 4 °C no active transport is occurring. Together, these data suggest that the fluorescence signal observed at 37 °C, in PC3(PSMA+) cells incubated with FAM-A9g, is due to internalized RNA.


Methods for Evaluating Cell-Specific, Cell-Internalizing RNA Aptamers.

Hernandez LI, Flenker KS, Hernandez FJ, Klingelhutz AJ, McNamara JO, Giangrande PH - Pharmaceuticals (Basel) (2013)

Fluorescence microscopy. (A) Direct fluorescence method. FAM labeled anti-PSMA RNA aptamer (A9g) was incubated with either PC3(PSMA+) or PC3(PSMA-) cells. A high salt wash step was performed to remove unbound or surface bound RNA. Internalized RNA was visualized using fluorescence microscopy. A scrambled, non-internalizing aptamer (Scr) was used as a negative control in these experiments. Florescence images were overlaid with DAPI and P/C (phase contrast) channels. Arrows indicate perinuclear localization of internalized A9g aptamer. (B) Antibody amplification method. FAM-labeled anti-TrkB RNA aptamer (C4-3) was incubated with either TrkB expressing or non-expressing HEK293 cells at 37 °C. FAM-labeled control aptamer (Scr) was used as a control for specificity. Unbound and surface bound RNA was removed as described above. Internalized RNA signal was amplified by incubation with an anti-FITC antibody and Alexa488 secondary antibody. Vehicle treated cells (No RNA) or cells subjected to incubation with antibodies alone (Ab control) were used as controls.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3722562&req=5

pharmaceuticals-06-00295-f002: Fluorescence microscopy. (A) Direct fluorescence method. FAM labeled anti-PSMA RNA aptamer (A9g) was incubated with either PC3(PSMA+) or PC3(PSMA-) cells. A high salt wash step was performed to remove unbound or surface bound RNA. Internalized RNA was visualized using fluorescence microscopy. A scrambled, non-internalizing aptamer (Scr) was used as a negative control in these experiments. Florescence images were overlaid with DAPI and P/C (phase contrast) channels. Arrows indicate perinuclear localization of internalized A9g aptamer. (B) Antibody amplification method. FAM-labeled anti-TrkB RNA aptamer (C4-3) was incubated with either TrkB expressing or non-expressing HEK293 cells at 37 °C. FAM-labeled control aptamer (Scr) was used as a control for specificity. Unbound and surface bound RNA was removed as described above. Internalized RNA signal was amplified by incubation with an anti-FITC antibody and Alexa488 secondary antibody. Vehicle treated cells (No RNA) or cells subjected to incubation with antibodies alone (Ab control) were used as controls.
Mentions: We evaluated the sub-cellular localization of FAM-labeled aptamer A9g using fluorescence microscopy (Figure 2A). FAM-labeled A9g was incubated with either PC3 cells lacking PSMA [PC3(PSMA-)] or PC3 cells expressing PSMA [PC3(PSMA+)] at 37 °C for 30 min (Figure 2A). A high salt wash step was used to remove unbound or surface bound RNA. As expected, fluorescence signal was observed only in PC3(PSMA+) cells. The signal was mostly localized in the perinuclear region, as shown by the arrow heads in the merged (FAM and DAPI) micrographs (Figure 2A). No signal was seen in PC3 cells lacking PSMA expression. Furthermore, a control, scrambled FAM-RNA (Scr) did not bind/internalize to PC3(PSMA+) cells, suggesting that the fluorescence signal is specific to A9g. Samples treated with buffer only (No RNA) were used to control for background fluorescence. We verified that the fluorescence signal seen in PC3(PSMA+) cells was mostly due to internalized RNA by performing the incubation of RNA on cells at 4 °C, which inhibits active transport (Supplementary Figure 1). As seen in Supplementary Figure 1, when the high salt wash step is applied to PC3(PSMA+) cells following incubation with FAM-A9g at 4 °C, the majority of the fluorescence signal is abrogated resulting only in a residual, low-intensity membrane signal (Supplementary Figure 1; right panel). This signal corresponds to surface bound FAM-A9g confirming that at 4 °C no active transport is occurring. Together, these data suggest that the fluorescence signal observed at 37 °C, in PC3(PSMA+) cells incubated with FAM-A9g, is due to internalized RNA.

Bottom Line: Here we describe methods to monitor for cellular uptake of aptamers.These include: (1) antibody amplification microscopy, (2) microplate-based fluorescence assay, (3) a quantitative and ultrasensitive internalization method ("QUSIM") and (4) a way to monitor for cytoplasmic delivery using the ribosome inactivating protein-based (RNA-RIP) assay.Collectively, these methods provide a toolset that can expedite the development of aptamer ligands to target and deliver therapeutic siRNAs in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, University of Iowa, Iowa City, IA 52242, USA.

ABSTRACT
Recent clinical trials of small interfering RNAs (siRNAs) highlight the need for robust delivery technologies that will facilitate the successful application of these therapeutics to humans. Arguably, cell targeting by conjugation to cell-specific ligands provides a viable solution to this problem. Synthetic RNA ligands (aptamers) represent an emerging class of pharmaceuticals with great potential for targeted therapeutic applications. For targeted delivery of siRNAs with aptamers, the aptamer-siRNA conjugate must be taken up by cells and reach the cytoplasm. To this end, we have developed cell-based selection approaches to isolate aptamers that internalize upon binding to their cognate receptor on the cell surface. Here we describe methods to monitor for cellular uptake of aptamers. These include: (1) antibody amplification microscopy, (2) microplate-based fluorescence assay, (3) a quantitative and ultrasensitive internalization method ("QUSIM") and (4) a way to monitor for cytoplasmic delivery using the ribosome inactivating protein-based (RNA-RIP) assay. Collectively, these methods provide a toolset that can expedite the development of aptamer ligands to target and deliver therapeutic siRNAs in vivo.

No MeSH data available.