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A peptide nucleic acid–nuclear localization signal fusion that mediates nuclear transport of DNA

Nature Biotechnology volume 17, pages 784–787 (1999)Cite this article

Abstract

We have combined a peptide nucleic acid (PNA) with the SV40 core nuclear localization signal (NLS), to create a bifunctional PNA–NLS peptide. The PNA–NLS peptide increased the nuclear uptake of oligonucleotides and enhanced the transfection efficacy of plasmids. Gene expression from an enhanced green fluorescent protein plasmid and a lacZ plasmid was preserved when hybridized to PNA–NLS. In combination with the transfection agent polyethyleneimine, we have improved both the nuclear translocation of fluorescence-marked oligonucleotides, and the efficacy of plasmid transfection, up to eightfold. The technique obviates the use of cumbersome coupling procedures of the vector due to DNA–PNA duplex formation or displacement of the antisense plasmid DNA strand by a PNA molecule.

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Figure 1: Schematic representation of the target site in the antisense Cy-5 oligonucleotide hybridizing to the sense PNA–NLS peptide.
Figure 2: (A) Nuclear translocation of fluorescence-labeled oligonucleotides. The antisense Cy-5 labeled oligonucleotide is shown in red.
Figure 3: Shift assay of antisense and sense oligonucleotides with PNA–NLS dual-function peptide.
Figure 4: (A) Cy-3–labeled oligonucleotide hybridized to PNA–NLS and subsequently translocated to the nuclei of exposed cells.
Figure 5: Fold increase in transfection efficiency of lacZ and EGFP plasmids with or without the addition of PNA–NLS.

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Acknowledgements

This work was supported by the Swedish Medical Research Council. The skillful technical assistance of research student Wolfgang Köhnlein is gratefully acknowledged. The microscopy work was done at the Department of Immunology, Microbiology, Pathology and Infectious Diseases with the skillful aid of Associate Professor Birger Christensson, MD, PhD.

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Authors and Affiliations

  1. Department of Biosciences, Center for BioTechnology, Karolinska Institutet, NOVUM, Huddinge, SE-14157, Sweden

    Lars J. Brandén, Abdalla J. Mohamed & C. I. Edvard Smith

  2. Department of Clinical Immunology, Microbiology, Pathology, and Infectious Diseases, Huddinge Hospital, Huddinge, Sweden

    Lars J. Brandén & C. I. Edvard Smith

  3. University College of South Stockholm, Stockholm, SE-14104, Sweden

    Abdalla J. Mohamed

  4. Gene Therapy Research Centre, Clinical Research Centre, Huddinge Hospital, Karolinska Institutet, Huddinge, SE-14186, Sweden

    C. I. Edvard Smith

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  1. Lars J. Brandén
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Correspondence to Lars J. Brandén.

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Brandén, L., Mohamed, A. & Smith, C. A peptide nucleic acid–nuclear localization signal fusion that mediates nuclear transport of DNA. Nat Biotechnol 17, 784–787 (1999). https://doi.org/10.1038/11726

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