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A synthetic snRNA m3G-CAP enhances nuclear delivery of exogenous proteins and nucleic acids.

Moreno PM, Wenska M, Lundin KE, Wrange O, Strömberg R, Smith CI - Nucleic Acids Res. (2009)

Bottom Line: However, also for small oligonucleotides, achieving higher nuclear concentrations could be of great benefit.The cap is found in the small nuclear RNAs that are constitutive part of the small nuclear ribonucleoprotein complexes involved in nuclear splicing.The synthetic capping of oligos interfering with splicing may have immediate clinical applications.

View Article: PubMed Central - PubMed

Affiliation: Department of Laboratory Medicine, Clinical Research Center, Karolinska Institutet, Karolinska University Hospital, SE-141 86 Huddinge, Sweden. pedro.moreno@ki.se

ABSTRACT
Accessing the nucleus through the surrounding membrane poses one of the major obstacles for therapeutic molecules large enough to be excluded due to nuclear pore size limits. In some therapeutic applications the large size of some nucleic acids, like plasmid DNA, hampers their access to the nuclear compartment. However, also for small oligonucleotides, achieving higher nuclear concentrations could be of great benefit. We report on the synthesis and possible applications of a natural RNA 5'-end nuclear localization signal composed of a 2,2,7-trimethylguanosine cap (m(3)G-CAP). The cap is found in the small nuclear RNAs that are constitutive part of the small nuclear ribonucleoprotein complexes involved in nuclear splicing. We demonstrate the use of the m(3)G signal as an adaptor that can be attached to different oligonucleotides, thereby conferring nuclear targeting capabilities with capacity to transport large-size cargo molecules. The synthetic capping of oligos interfering with splicing may have immediate clinical applications.

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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.
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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.

Mentions: In Figure 3, a paired t-test with two-tailed P-value was used for analysis; In Figure 5, the 2 × 2 contingency table was analysed by the Fisher's exact test with two-tailed P-value.Figure 3.


A synthetic snRNA m3G-CAP enhances nuclear delivery of exogenous proteins and nucleic acids.

Moreno PM, Wenska M, Lundin KE, Wrange O, Strömberg R, Smith CI - Nucleic Acids Res. (2009)

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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2665231&req=5

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.
Mentions: In Figure 3, a paired t-test with two-tailed P-value was used for analysis; In Figure 5, the 2 × 2 contingency table was analysed by the Fisher's exact test with two-tailed P-value.Figure 3.

Bottom Line: However, also for small oligonucleotides, achieving higher nuclear concentrations could be of great benefit.The cap is found in the small nuclear RNAs that are constitutive part of the small nuclear ribonucleoprotein complexes involved in nuclear splicing.The synthetic capping of oligos interfering with splicing may have immediate clinical applications.

View Article: PubMed Central - PubMed

Affiliation: Department of Laboratory Medicine, Clinical Research Center, Karolinska Institutet, Karolinska University Hospital, SE-141 86 Huddinge, Sweden. pedro.moreno@ki.se

ABSTRACT
Accessing the nucleus through the surrounding membrane poses one of the major obstacles for therapeutic molecules large enough to be excluded due to nuclear pore size limits. In some therapeutic applications the large size of some nucleic acids, like plasmid DNA, hampers their access to the nuclear compartment. However, also for small oligonucleotides, achieving higher nuclear concentrations could be of great benefit. We report on the synthesis and possible applications of a natural RNA 5'-end nuclear localization signal composed of a 2,2,7-trimethylguanosine cap (m(3)G-CAP). The cap is found in the small nuclear RNAs that are constitutive part of the small nuclear ribonucleoprotein complexes involved in nuclear splicing. We demonstrate the use of the m(3)G signal as an adaptor that can be attached to different oligonucleotides, thereby conferring nuclear targeting capabilities with capacity to transport large-size cargo molecules. The synthetic capping of oligos interfering with splicing may have immediate clinical applications.

Show MeSH