Limits...
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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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.
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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.

Mentions: Streptavidin-Alexa488 (Molecular Probes) + oligonucleotide constructs (Figure 2) were formed by incubating 3 µg STV (Streptavidin) reconstituted in 1× phosphate buffer saline (PBS, pH 7.4) with 2–4 times molar amount of biotinylated oligo constructs. The volume was adjusted with nuclease free water (Qiagen) to have a final concentration of 329 ng STV/µl and the incubation proceeded for 2 h at r.t. and if not used the same day the constructs were stored at 4°C.Figure 2.


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)

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

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

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.
Mentions: Streptavidin-Alexa488 (Molecular Probes) + oligonucleotide constructs (Figure 2) were formed by incubating 3 µg STV (Streptavidin) reconstituted in 1× phosphate buffer saline (PBS, pH 7.4) with 2–4 times molar amount of biotinylated oligo constructs. The volume was adjusted with nuclease free water (Qiagen) to have a final concentration of 329 ng STV/µl and the incubation proceeded for 2 h at r.t. and if not used the same day the constructs were stored at 4°C.Figure 2.

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