SIDT2 mediates gymnosis, the uptake of naked single-stranded oligonucleotides into living cells

Abstract

Single-stranded oligonucleotides (ssOligos) are efficiently taken up by living cells without the use of transfection reagents. This phenomenon called 'gymnosis' enables the sequence-specific silencing of target genes in various types of cells. Several antisense ssOligos are used for the treatment of human diseases. However, the molecular mechanism underlying the uptake of naked ssOligos into cells remains to be elucidated. Here, we show that systemic RNA interference deficient-1 (SID-1) transmembrane family 2 (SIDT2), a mammalian ortholog of the Caenorhabditis elegans double-stranded RNA channel SID-1, mediates gymnosis. We show that the uptake of naked ssOligos into cells is significantly downregulated by knockdown of SIDT2. Furthermore, knockdown of SIDT2 inhibited the effect of antisense RNA mediated by gymnosis. Overexpression of SIDT2 enhanced the uptake of naked ssOligos into cells, while a single amino acid mutation in SIDT2 abolished this effect. Our findings highlight the mechanism of extra- and intracellular RNA transport and may contribute to the further development of nucleic acid-based therapies.

Keywords: Gymnosis; RNA channel; SIDT2; membrane protein; single-stranded oligonucleotides.

Figure 1.

Uptake of naked ssOligos by HeLa cells. (A) Confocal imaging of HeLa cells. HeLa cells were incubated with or without 500 nM of the naked 15-nt 5′-Alexa568-labeled oligonucleotides (Alexa568-ssOligos) for 6 and 24 hours. Cells were washed with PBS and stained with Hoechst 33258. (B) HeLa cells were incubated with 500 nM of Alexa568-ssOligos for 0, 2, 4, 6 and 8 hours, and analyzed by confocal microscopy. Arrows indicate the 5′-Alexa568-labeled oligonucleotides in cells. (C and D) HeLa cells were incubated with or without 20 nM of naked antisense oligonucleotides targeting miR-16 (C) or naked negative control oligonucleotides (D) for 6 hours, and analyzed by qPCR analysis, miR-16 levels were normalized against β-actin mRNA levels. Error bars indicate s.d. (n = 3). *, P<0.05., n.s., not significant.

Figure 2.

Localization of SIDT2 to the plasma membrane. (A) mRNA levels in HeLa cells were analyzed by RT-PCR. Expression of SIDT2 mRNA was detected. In contrast, SIDT1 mRNA was not detected in Hela cells. (B) HeLa cells were incubated with (+) or without (−) biotin, and a biotin-streptavidin pull-down assay was performed to purify cell surface proteins as described in the Materials and Methods section. Proteins were analyzed by western blotting using anti-SIDT2 (Abnova), anti-β-actin (ACTB), anti-N-cadherin and anti-cathepsin D antibodies. (C) HeLa cells expressing EGFP-tagged SIDT2 were incubated with LysoTracker Red. Fluorescence images were visualized using a confocal laser-scanning microscope. Arrows indicate plasma membrane localization.

Figure 3.

Knockdown of SIDT2 reduces the uptake of naked ssOligos. (A) Experimental paradigm for the confocal microscopic analysis. (B and C) HeLa cells were transfected with 10 nM of control siRNA (siControl) or siRNA against SIDT2 (siSIDT2#1 or #2). At 72 hours after transfection, SIDT2 mRNA levels (B) and SIDT2 protein levels (C) were analyzed. In the qRT-PCR analysis, SIDT2 mRNA levels were normalized against β-actin mRNA levels (B). In the western blot analysis, anti-SIDT2 (custom antibody) and anti-β-actin antibodies were used (C). Error bars indicate s.d. (n = 3). *, P < 0.05. (D–F) HeLa cells were transfected with siControl or siSIDT2#1 (D and E) and siControl or siSIDT2#2 (F). Seventy-two hours after transfection, cells were washed with PBS, and incubated with or without 1000 nM (D) or 500 nM (E and F) of the naked Alexa568-ssOligos for 6 hours. Cells were washed with PBS again, and analyzed by confocal microscopy. Quantification of fluorescence intensity was performed with ImageJ. Error bars indicate s.d. (n = 3). *, P < 0.05.

Figure 4.

Knockdown of SIDT2 inhibits the antisense effect induced by gymnosis.(A) Experimental paradigm for the qRT-PCR analysis. (B and C) HeLa cells were transfected with siControl or siSIDT2#1 (B) and siControl or siSIDT2#2 (C). Seventy-two hours after transfection, cells were washed with PBS, incubated with (oligo+) or without (oligo–) 20 nM of the naked antisense oligonucleotides targeting miR–16 for 6 hours, and RNA levels were analyzed by qPCR. The miR-16 levels were normalized against β-actin mRNA levels. The miR-16 levels were expressed as percentages compared to the levels of oligo– samples (expressed as 100%). Error bars indicate s.d. (n = 6). *, P < 0.05.

Figure 5.

Effect of overexpression of SIDT2 on the uptake of naked ssRNA. (A) Experimental paradigm for the confocal microscope analysis. (B) HeLa cells were transfected with empty vector, pCI-neo/mouse SIDT2 or pCI-neo/human SIDT2 plasmids. β-actin was used as a loading control. Overexpression of SIDT2 was confirmed by western blot analysis using an anti-SIDT2 antibody (Abnova). (C) HeLa cells were transfected as indicated and cultured for 48 hours. Cells were washed with PBS, incubated with or without 500 nM of the naked Alexa568-ssOligos, washed with PBS again, and analyzed by confocal microscopy. Quantification of fluorescence intensity was performed. Error bars indicate s.d. (n = 3). *, P < 0.05.

Figure 6.

Effect of mutations of SIDT2 on the uptake of naked ssOligos. (A) HeLa cells were transfected with empty vector, pCI-neo/WT SIDT2, pCI-neo/F154T SIDT2, or pCI-neo/S564A SIDT2 plasmids. Overexpression of SIDT2 variants was confirmed by western blot analysis using an anti-SIDT2 antibody (Abnova). β-actin was used as a loading control. (B) HeLa cells were transfected as indicated and cultured for 48 hours. Cells were washed with PBS, incubated with or without 500 nM of the naked Alexa568-ssOligos, washed with PBS again, and analyzed by confocal microscopy. Quantification of fluorescence intensity was performed. Error bars indicate s.d. (C: n = 3, D: n = 6). *, P < 0.05. n.s., not significant. (C) Hela cells expressing EGFP-tagged SIDT2 (S564A or F154T) were incubated with LysoTracker Red. Fluorescence images were acquired using a confocal laser-scanning microscope. Arrows indicate plasma membrane localization.

Figure 7.

Effect of overexpression of SIDT2 on endocytosis. (A) Experimental paradigm for the confocal microscope analysis. (B and C) HeLa cells were transfected with empty vector, pCI-neo/mouse SIDT2 or pCI-neo/human SIDT2 plasmids, and cultured for 48 hours. Cells were washed with PBS, and incubated with or without 500 nM of Rhodamine B (B) or Rhodamine B isothiocyanate-dextran (C). Cells were washed with PBS again, and analyzed by confocal microscopy. Quantification of fluorescence intensity was performed. Error bars indicate s.d. (n = 3). *, n.s., not significant.