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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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(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.
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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.

Mentions: The oligonucleotide used for this study was a 2′-O-methyl oligoribonucleotide based on the same sequence as reported by Kang et al. (43) but with a 3-nt addition (AUA) to its 5′-end. The oligonucleotide included an overhanging phosphate group at this same 5′-end that was used later for the addition of the m3G-CAP (m3GpppAUA-). After transfection of both oligo versions, with (oligo1CAP) or without (oligo1) m3G-CAP addition, we found that the increase of luciferase activity relatively to untreated HeLa/Luc705 was dose-dependent and that the capped antisense oligo had an increasingly higher activity across the concentration range tested. At the highest concentration tested there was an ∼7.9-fold luciferase activity increase for the m3G-capped oligo version and only 2.3-fold for the uncapped oligo version (Figure 7A). Thus, we observed a maximum of 3.4-fold increase in efficiency when using the m3G-CAP signal.Figure 7.


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)

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

License
Show All Figures
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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.
Mentions: The oligonucleotide used for this study was a 2′-O-methyl oligoribonucleotide based on the same sequence as reported by Kang et al. (43) but with a 3-nt addition (AUA) to its 5′-end. The oligonucleotide included an overhanging phosphate group at this same 5′-end that was used later for the addition of the m3G-CAP (m3GpppAUA-). After transfection of both oligo versions, with (oligo1CAP) or without (oligo1) m3G-CAP addition, we found that the increase of luciferase activity relatively to untreated HeLa/Luc705 was dose-dependent and that the capped antisense oligo had an increasingly higher activity across the concentration range tested. At the highest concentration tested there was an ∼7.9-fold luciferase activity increase for the m3G-capped oligo version and only 2.3-fold for the uncapped oligo version (Figure 7A). Thus, we observed a maximum of 3.4-fold increase in efficiency when using the m3G-CAP signal.Figure 7.

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