|
Figure 1. Structure of the 2,2,7-trimethylguanosine cap.
|
|
Figure 2. Schematic representation of the m3G-CAP oligo construct bound to the Streptavidin-Alexa488 (STV) used in the nuclear transport assays by cytoplasmic microinjections in Xenopus oocytes and by PULSin protein delivery to the cytosol of a mammalian cell line. Image proportions are exaggerated for a better understanding.
|
|
Figure 3. Accumulation of Streptavidin complexes in Xenopus oocyte nuclei after cytoplasmic injections. Oocytes were injected in the cytoplasm with either STV-oligo2 or STV-oligo2CAP complexes and after 4 h incubation the nuclei were dissected and collected for western blot analysis. (A) Upper panel shows western blot using anti-Streptavidin probing. Each lane corresponds to one group of four nuclei pooled together. Lanes 1–2–7–8: nuclei dissected from oocytes injected with STV-oligo2CAP (m3G-CAP-PMO2); lanes 3–4–5–6: nuclei dissected from oocytes injected with STV-oligo2 (p-PMO2). Lower panel shows a protein loading control by staining the PAGE gel with comassie blue after transfer. (B) Graph showing the quantification of western blot results by densitometry with normalization to the loading controls using Fluor-S MultiImager and Quantity One software® (BioRad) (SDs for n = 4 are shown). ** P < 0.005.
|
|
Figure 4. Fluorescence imaging of the accumulation of Streptavidin complexes in Xenopus oocyte nuclei after cytoplasmic injections. Oocytes were injected in the cytoplasm with either STV-oligo2 or STV-oligo2CAP complexes and after 4 h incubation the nuclei were dissected. The nuclei were gently tapped with a glass rod, in order to release excess cytoplasm that might be still attached to the nuclear membrane after dissection. Photos were then taken with two nuclei side by side representing cytoplasmic injections of capped (nucleus 1) and non-capped (nucleus 2) STV-oligo constructs. (A) Photo taken with oblique visible light in order to get a phase contrast effect making it possible to see the almost transparent nuclear membrane; (B) same nuclei imaged with HBO light and fluorescence FITC filter; (C) same nuclei imaged both with low power visible light and with an HBO light with FITC filter. A number of 10 pairs of nuclei were analysed with all of them showing the same pattern as illustrated above.
|
|
Figure 5. Localization of fluorescent Streptavidin bound to 3–4 biotinylated 2′-O-methyl RNA oligos with (pictures A and B) or without (pictures C and D) m3G-CAP (p-PMO2 and m3G-CAP-PMO2 oligos, respectively). Streptavidin-oligo complexes were transfected into U2OS cell lines by the use of PULSin reagent (protein transfection reagent). After 4 h incubation at 37°C, cells were washed and incubated for 2 h at 37°C before fluorescent microscopy (B and D) and phase contrast pictures (A and C) were taken. Fluorescent microscopy photographs were used to count the cells according to the described criteria and results are presented in the lower table. N and C mark some nuclei and cytoplasm of cells, respectively, in the phase contrast pictures. Solid white arrows indicate examples of cells counted as positive for nuclear enrichment; open white arrows point to cells counted as negative for nuclear enrichment; the open white arrowhead points to aggregates of fluorescent STV complexes occurring during the transfection procedure; open rectangular boxes were drawn around the ‘STV-m3G-oligo nuclear bodies’ formed by increased accumulation of STV complexes in the nucleus.
|
|
Figure 6. Confocal imaging of mammalian U2OS cells with STV-oligo complexes transfected by PULSin. Same conditions as for Figure 5 were used except that for confocal imaging there was further processing such as nuclear staining by DRAQ5 (blue) after cell fixation by 3.7% paraformaldeheyde in PBS. The line trace profile shows the intensity of the DRAQ5 (blue line in graph) and fluorescent STV-oligo complexes (green line in graphs) along the drawn line (in red). The confocal pictures confirm the co-localization of the green fluorescence coming from the STV-Alexa488 with m3G-CAP and the localization of the STV-Alexa488 without the CAP only in the cytoplasm of the cell as can be further analysed in the traced line profile graphs (graph 1 shows the line trace profile over cells transfected with STV-CAP-oligo; graph 2 shows the line trace profiles over a cell transfected with STV-oligo).
|
|
Figure 7. (A) Fold-induction of splice-correction 24 h after transfection of HeLa/Luc705mut with 2′-O-methyl antisense (AS) oligos at different concentrations. Correction was measured by testing for luciferase activity after AS oligo treatment relative to mock treatment. Both oligos have an additional tri-nucleotide extension (AUA) on their 5′-ends to which an m3G-CAP was added in one of them. Error bars show standard deviations for at least n = 3. (B) RT–PCR. Total cellular RNA was subjected to RT–PCR. The upper band (268 bp) and lower band (142 bp) correspond to the aberrant and correct luciferase mRNA, respectively. Cp1 and p1 correspond to the antisense oligo (AS705) with (oligo Cp1) or without (oligo p1) m3G-CAP added. Cp2 and p2 correspond to the scrambled (control) antisense oligo with (oligo Cp2) or without (oligo p2) m3G-CAP.
|
|
Scheme 1. Synthesis of the m3G 5′-pyrophosphateimidazolide. Reagents and conditions: (i) a: Py/NH3aq sat., 3 h, r.t.,; b: MMTrCl, Py/DMF, 24 h, r.t.; c: BzCl, Py, r.t., 24 h, D 80% AcOH, r.t., 5 h; (ii) a: salicyl chlorophosphite, r.t., 15 min; b: tri-n-buthylammine pyrophosphate, DMF, r.t, 20 min; c: I2, Py/H2O, r.t, 15 min; d: ethylenediamine, r.t., E NH3aq sat., 0°C 48 h; (iii) MeI, DMF, 40°C, 5 h; (iv) imidazole, triphenylphosphine, di-2-pyridyldisulphide, DMF 24 h. Py, pyridine; MMTrCl, monometoxy trityl chloride; DMF, dimethylformamide; r.t. = room temperature; BzCl, benzoyl chloride; AcOH, acetic acid; MeI, methyl iodide.
|
