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. 2010 Apr;20(2):103-9.
doi: 10.1089/oli.2009.0211.

The biological effect of an antisense oligonucleotide depends on its route of endocytosis and trafficking

Md Rowshon Alam  1 , Xin Ming, Vidula Dixit, Michael Fisher, Xiaoyuan Chen, Rudolph L Juliano
Affiliations

The biological effect of an antisense oligonucleotide depends on its route of endocytosis and trafficking

Md Rowshon Alam et al. Oligonucleotides. 2010 Apr.

Abstract

We demonstrate that the biological effect of an oligonucleotide is influenced by its route of cellular uptake. Utilizing a splice-switching antisense oligonucleotide (SSO) and a sensitive reporter assay involving correction of RNA splicing, we examined induction of luciferase in cells treated either with various concentrations of an unconjugated ("free") SSO or an SSO conjugated to a bivalent RGD ligand that promotes binding to the alphavbeta3 integrin (RGD-SSO). Under conditions of equal accumulation in cells, the RGD-SSO consistently had a greater effect on luciferase induction than the unconjugated SSO. We determined that the RGD-SSO and the unconjugated SSO were internalized by distinct endocytotic pathways, suggesting that the route of internalization affects the magnitude of the biological response.

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Figures

FIG. 1.
FIG. 1.
Diagram of the RGD-conjugated splice-shifting oligonucleotide (RGD-623). The RGD moiety is conjugated at the 5′ position, while a Tamra fluorophore is at the 3′ position. The oligonucleotide is a 2′-O-Me-phosphorothioate.
FIG. 2.
FIG. 2.
Uptake vs. effect. A375SM-Luc705-B cells (150,000/well) were treated with varying concentrations of 623-Tamra, or RGD-623-Tamra at 37°C for 4 hours in OPTI-MEM. At that point, 1% serum was added for 24 hours; the medium was then changed to 1% FBS/DMEM for another 24 hours. Cells were washed, lysed in reporter lysis buffer, freeze-thawed, vortexed, and centrifuged (14,000 rpm, 10 minutes); luciferase activity, protein concentration, and cell uptake of fluorescent oligonucleotides were measured as described in Materials and Methods. Luciferase activity was expressed as relative luminescent units (RLU)/μg protein, while cell uptake was expressed as relative fluorescent units (RFU)/μg protein. Results are the means of triplicate determinations. Graphs were produced with a curve-fitting program.
FIG. 3.
FIG. 3.
Effects of actin inhibitors. A375SM-Luc705-B cells (150,000/well) were preincubated with (A) cytochalasin D or (B) latrunculin A at the indicated concentrations for 1 hour at 37°C in OPTI-MEM containing 1% FBS. At that point, 623-Tamra (100 nM) or RGD-623-Tamra (100 nM) were added to the wells, and the cells were treated for 24 hours. The cells were washed with PBS, and lysed with 1× reporter lysis buffer (150 μL). The cell extracts were frozen and thawed, vortexed, and centrifuged. Relative fluorescent units (RFUs) were measured using a microfluorimeter. The uptake data was normalized on protein concentration. Results represent means and standard deviations of triplicate determinations. Uptake was considered as 100% where no inhibitor was added.
FIG. 4.
FIG. 4.
Effects of dynamin. A375SM-Luc705-B cells (1 million) were transfected at 90% confluence with 2 μg of DN dynamin-GFP plasmid using an Amaxa Nucleoporation system. Cells were then cultured in DMEM plus 10% FBS for 24 hours to allow protein expression. The cells were then treated with 100 nM of 623-Tamra or 50 nM of RGD-623-Tamra for 4 hours. Cells were then trypsinized and seeded in 35-mm glass bottom microwell dishes previously coated with 5 μg/cm2 of fibronectin for 1 hour at room temperature. Confocal fluorescent images were obtained as described in Materials and Methods. Cells expressing high levels of DN dynamin-GFP are marked by green arrows while untransfected cells are marked by orange arrows and cells with weaker dynamin-GFP expression are not marked. (A) RGD-623-Tamra. (B) 623-Tamra. Results are typical of multiple microscopic fields observed.
FIG. 5.
FIG. 5.
Endocytosis vs. direct delivery to the cytosol. (A) RGD-623-Tamra or 623-Tamra (50 nM) was directly delivered to the cytosol of A375SM-Luc705-B cells by electroporation (Amaxa Nucleoporation system) or scrape-loading (see B below). The cells were incubated in DMEM plus 1% FBS for 24 hours and then the cells were harvested and analyzed for protein content and luciferase activity. Results are the means and standard deviations of 3 determinations and are expressed as RLU/μg protein. (B) Cells were treated with 100-nM RGD-623-Tamra in the presence or absence of 2-μM cytochalasin D. In one set of experiments, the intact cells were simply incubated with the oligonucleotide and inhibitor in DMEM plus 1% FBS. In the other case, the cells were “scrape-loaded” in the presence of oligonucleotide and inhibitor by dislodging them from the substratum with a spatula. The cells were then allowed to re-attach. After 24 hours, the cells were harvested, lysed, and analyzed for protein content and luciferase activity as described in Materials and Methods. Results are means and standard deviations of triplicate determinations and are expressed as RLU/μg protein. The hatched bar represents cells treated with cytochalasin D, while the solid bar represents control cells. For the intact cell case, the difference between control- and cytochalasin D-treated cells is significant at the P < 0.01 level.
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