Coupling of Nuclear Localization Signals to Plasmid DNA and Specific Interaction of the Conjugates with Importin α

Ciolina, Carole; Byk, Gérardo; Blanche, Francis; Thuillier, Vincent; Scherman, Daniel; Wils, Pierre

doi:10.1021/bc980061a

Cited by 113 publications

(92 citation statements)

References 23 publications

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“…Several methods for covalent attachment of molecules to nucleic acids have been developed and reported so far [11,12,15,16]. Among them, the use of photoactive molecules leads to e¤cient but non-speci¢c covalent modi¢cation of DNA [12,17].…”

Section: Discussionmentioning

confidence: 99%

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Coupling of a targeting peptide to plasmid DNA by covalent triple helix formation

et al. 1999

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The nuclear localization signal (NLS) of the SV40 large T antigen efficiently induces nuclear entry of proteins. We have developed a strategy for covalent coupling of one or a controlled number of NLS peptides to plasmid DNA at a specific site by triple helix formation. A psoralen-oligonucleotide-NLS peptide conjugate was synthesized and characterized by proteolysis with trypsin. This conjugate was used to covalently associate one NLS peptide to plasmid DNA by triple helix formation and photoactivation. The oligonucleotide-NLS peptide conjugate interacted with the NLS-receptor importin K K. The reporter gene was expressed after transfection of the modified plasmid in NIH 3T3 cells, indicating no loss of the gene expression functionality of the plasmid. On the other hand, no increase in expression was observed as a result of the NLS peptide. This site-specific coupling technology can be used to couple to a plasmid other ligands targeting to a specific receptor.z 1999 Federation of European Biochemical Societies.

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Section: Discussionmentioning

confidence: 99%

“…Non-speci¢c covalent linkage of multiple NLS peptides on linearized or circular DNA has been described [11,12]. However, in these strategies, NLS peptides were associated with non-speci¢c sites on DNA, leading to the loss of gene expression.…”

Section: Introductionmentioning

confidence: 99%

Coupling of a targeting peptide to plasmid DNA by covalent triple helix formation

et al. 1999

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“…One hypothesis was that the NLS-modified DNA is sequestered in the cytoplasm of microinjected cells, as has been found for naked DNA; nevertheless, these results cause a severe limitation to such experimental approach. Similar approaches have been developed in several other systems, but with lower enhancement of transfection (Ciolina et al, 1999;Ludtke et al, 1999).…”

Section: Linkage Of Nls Sequence Directly To Dnamentioning

confidence: 95%

Improvement of exogenous DNA nuclear importation by nuclear localization signal‐bearing vectors: a promising way for non‐viral gene therapy?

Hebert

2003

Biology of the Cell

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Several vectors have been developed in order to target genes to specific cells. Virus-based vectors lead to a high transfection efficiency in vitro, but display important disadvantages such as pathological risks, which they expose to patients. Plasmid-associated chemical vectors lack these disadvantages, but allow only a very low efficiency of transgene expression. Most of the non-viral-based gene transfer techniques developed until now mainly focused their efforts to overcome the problem of DNA entry into the cell. Some recent works, however, have begun to investigate the nucleus entry problem and suggest that the trafficking of DNA from cytosol to the nucleus may be improved by using the nuclear localization signal (NLS) found in some nuclear proteins. If the vector contains one or several NLS, either as covalently or non-covalently DNA-linked peptides, a competition may take place between the rate of dissociation of the DNA-vector complexes and the rate of loading of the complexes to the NLS-mediated nucleus importation machinery. This equilibrium may be displaced towards the importation pathway by the use of NLS-bearing proteins instead of peptides. The possibility of recruiting normal endogenous cellular pathways of nuclear uptake to promote entry of exogenously applied DNA through the nuclear pore complex would, thus, seem promising. Nevertheless, attempts to improve the transport of DNA to the nucleus through the use of NLSs have achieved limited success. Although these systems show improved transgene expression, little is known about how they function in transfected cells, and the optimal formulation for gene expression is yet to be determined.

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“…It is conceivable that a combination of NLS with plasmid DNA might improve efficiency of gene delivery (see reviews 78,88,89 ). Attempts have been made to either add nucleoproteins or NLS into cocktails of plasmid solution or to covalently link NLS to plasmid DNA, either directly 90 or indirectly through DNA-binding agents such as cationic polymers and peptides. 91 Collas and Alestrom in experiments involving micro-injection of plasmid into the cytoplasm of zebrafish eggs, reported that use of NLS increased nuclear accumulation of plasmid DNA and expression of the transgene.…”

Section: Proteins and Polypeptidesmentioning

confidence: 99%

Non-viral gene delivery in skeletal muscle: a protein factory

Lu¹,

Bou‐Gharios²,

Partridge³

2003

Gene Ther

170

109

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Ever since the publication of the first reports in 1990 using skeletal muscle as a direct target for expressing foreign transgenes, an avalanche of papers has identified a variety of proteins that can be synthesized and correctly processed by skeletal muscle. The impetus to the development of such applications is not only amelioration of muscle diseases, but also a range of therapeutic applications, from immunization to delivery of therapeutic proteins, such as clotting factors and hormones. Although the most efficient way of introducing transgenes into muscle fibres has been by a variety of recombinant viral vectors, there are potential benefits in the use of non-viral vectors. In this review we assess the recent advances in construction and delivery of naked plasmid DNA to skeletal muscle and highlight the options available for further improvements to raise efficiency to therapeutic levels.

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Coupling of Nuclear Localization Signals to Plasmid DNA and Specific Interaction of the Conjugates with Importin α

Cited by 113 publications

References 23 publications

Coupling of a targeting peptide to plasmid DNA by covalent triple helix formation

Coupling of a targeting peptide to plasmid DNA by covalent triple helix formation

Improvement of exogenous DNA nuclear importation by nuclear localization signal‐bearing vectors: a promising way for non‐viral gene therapy?

Non-viral gene delivery in skeletal muscle: a protein factory

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