2'-Fluoro-modified phosphorothioate oligonucleotide can cause rapid degradation of P54nrb and PSF

Abstract

Synthetic oligonucleotides are used to regulate gene expression through different mechanisms. Chemical modifications of the backbone of the nucleic acid and/or of the 2' moiety of the ribose can increase nuclease stability and/or binding affinity of oligonucleotides to target molecules. Here we report that transfection of 2'-F-modified phosphorothioate oligonucleotides into cells can reduce the levels of P54nrb and PSF proteins through proteasome-mediated degradation. Such deleterious effects of 2'-F-modified oligonucleotides were observed in different cell types from different species, and were independent of oligonucleotide sequence, positions of the 2'-F-modified nucleotides in the oligonucleotides, method of delivery or mechanism of action of the oligonucleotides. Four 2'-F-modified nucleotides were sufficient to cause the protein reduction. P54nrb and PSF belong to Drosophila behavior/human splicing (DBHS) family. The third member of the family, PSPC1, was also reduced by the 2'-F-modified oligonucleotides. Preferential association of 2'-F-modified oligonucleotides with P54nrb was observed, which is partially responsible for the protein reduction. Consistent with the role of DBHS proteins in double-strand DNA break (DSB) repair, elevated DSBs were observed in cells treated with 2'-F-modified oligonucleotides, which contributed to severe impairment in cell proliferation. These results suggest that oligonucleotides with 2'-F modifications can cause non-specific loss of cellular protein(s).

Figure 1.

2′-F-modified oligonucleotides cause a reduction in levels of P54nrb and PSF proteins. (A) Gapmer PS-ASOs of the same sequence but modified with 2′-MOE (green), cEt (blue), LNA (orange) or 2′-F (red) on flanking nucleotides were transfected into HeLa cells at a final concentration of 30 nM. After 24 h, levels of indicated proteins were determined by western analysis. P32 served as a loading control. (B) 2′-F-PS-ASO (ISIS404130) was transfected into HeLa cells at a final concentration of 50 nM. P54nrb and PSF levels at the specified time were evaluated by western analysis. GAPDH served as a loading control. (C) 2′-F-PS-ASO (ISIS404130) was transfected into HeLa cells at specified concentration. After 24 h, P54nrb and PSF levels were determined by western analysis. P32 served as a loading control. (D) PS-oligonucleotides modified with 2′-MOE (ISIS116847), cEt (ISIS582801) or 2′-F (ISIS404130) were transfected into mouse MHT cells at a final concentration of 30 nM. Western analysis was performed 24 h after transfection. TCP1-β served as a loading control. (E) Levels of P54nrb were determined by western in HeLa cells 24 h after transfection of PS-ASOs with combined cEt and 2′-F modifications at a final concentration of 30 nM. GAPDH served as a loading control.

Figure 2.

2′-F-modified oligonucleotides designed to function through different mechanisms reduce P54nrb and PSF levels. (A) Sequences of the 2′-F-modified oligonucleotides tested are shown. The ASOs designed to modulate splicing and the gapmer ASO are fully PS modified. Both the single-stranded siRNA and double-stranded siRNA oligonucleotides have alternating PS (indicated by lower case ‘s’) and phosphodiester (indicated by lower case ‘o’) backbone residues. 2′-MOE, 2′-F and 2′-O-methyl are indicated in green, red and purple, respectively. (B–D) HeLa cells were transfected with a final concentration of 30-nM oligonucleotide, and western analysis was performed after 24 h, with γ-tubulin or GAPDH as loading control. (B) Effect of splicing modulators and gapmer on P54nrb and PSF levels compared to mock-transfected cells. (C) Effect of 2′-F-modified ss-siRNA on P54nrb, PSF, Drosha and Dicer levels compared to mock-transfected cells. (D) Effect of 2′-F-modified ds-siRNA on P54nrb and PSF levels compared to mock-transfected cells and cells treated with 2′-F-PS-ASO ISIS404130.

Figure 3.

2′-F-modified oligonucleotides reduce P54nrb and PSF levels by decreasing protein stability. (A) 2′-F-oligonucleotides did not reduce levels of P54nrb or PSF mRNAs. HeLa cells were transfected with 2′-F-PS-ASO (ISIS404130) at a final concentration of 30 nM. After 24 h, RNA was isolated and indicated mRNAs were quantified by qRT-PCR. On-target reduction of PTEN mRNA was evaluated as a positive control for transfection. The error bars represent standard deviations from three experiments. (B) 2′-F oligonucleotides have no effect on translation of P54nrb and PSF. Nascent ³⁵S-methionine-labeled P54nrb and its heterodimer partner PSF were co-immunoprecipitated from mock-transfected cells or from HeLa cells transfected with 20-nM final concentration of 2′-F-PS-ASO (ISIS404130) and visualized by direct autoradiography. Autoradiography of 5% of the total cell lysate used for immunoprecipitation served as a loading control. (C) 2′-F oligonucleotides reduce P54nrb and PSF stability. Western analysis of P54nrb and PSF from mock-transfected cells or from HeLa cells transfected with 20-nM final concentration of 2′-F-PS-ASO (ISIS404130) treated with CHX (50 μg/ml) for 0, 6, 9 and 12 h. Silver-staining of total cell lysate served as a loading control. (D) 2′-F oligonucleotides cause degradation of P54nrb and PSF through the proteasome-mediated pathway. Western analysis of P54nrb and PSF from HeLa cells treated with DMSO or MG132 (10 μM) with or without transfection of 2′-F-PS-ASO (ISIS404130). GADPH served as a loading control. pCyclin D1_Thr286 served as a positive control for proteasome inhibition.

Figure 4.

PSPC1 levels are reduced by 2′-F-modified oligonucleotides. (A) Indicated oligonucleotides were transfected into HeLa cells at a final concentration of 30 nM. After 24 h, levels of PSPC1 were evaluated by western. GAPDH served as a loading control. (B) A431 cells were incubated with ISIS404130 by free uptake for 40 h at the indicated concentrations. Levels of P54nrb, PSF and PSF proteins were evaluated by western. GAPDH served as a loading control.

Figure 5.

P54nrb preferentially associates with 2′-F-modified oligonucleotides. (A) Oligonucleotides used in this experiment. (B) ASO-binding proteins were isolated from HeLa cell extracts using a biotinylated gapmer 2′-F-PS-ASO (ISIS623496). Proteins were eluted from beads by competition with indicated gapmers. The affinity-selected proteins were analyzed by western blot. Ku70 served as a loading control. (C) Western analysis following ASO-pull down as described in panel (A) indicated that P54nrb has higher affinity for oligonucleotides with the 2′-F modification on the 3′ side. Ku70 served as a loading control. (D) Transfection of 2′-F-oligonucleotides reduced P54nrb but not FUS protein levels. HeLa cells were transfected with 2′-F-PS-ASO (ISIS404130) at a final concentration of 30 nM, and protein levels were determined by western analysis. (E) Western analysis of proteins isolated from HeLa cells following treatment of cells with siRNAs targeting P54nrb or PSPC1 mRNA. Cells were transfected with siRNA at 3-nM final concentration and harvested after 36 h.

Figure 6.

2′-F-modified oligonucleotides cause severe cell death via induction of DNA damage. (A) HeLa cell viability was analyzed using a WST-8 assay 24 h after mock transfection or transfection of cells with the indicated concentrations of 2′-F-PS-ASO (ISIS404130) or 2′-MOE-PS-ASO (ISIS116847). The error bars represent standard deviations from four independent experiments. (B) The chemical modifications, sequences and numbers of the 2′-F-modified nucleotides of the tested oligonucleotides are shown. (C) HeLa cell viability was analyzed using a WST-8 assay 18 h after transfection of cells with the indicated concentrations of different oligonucleotides. The error bars represent standard deviations from four independent experiments. (D) HeLa cells were mock transfected or were transfected with 2′-F-PS-ASO (ISIS404130) or 2′-MOE-PS-ASO (ISIS116847). Cells were analyzed after 16 h using a neutral comet assay. Representative images are shown. Severity of DNA damage was reflected by the intensity of comet tail relative to the head. Scale bars, 150 μM.