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

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.

References

  1. 1

    Kircheis, R et al. Coupling of cell-binding ligands to polyethylenimine for targeted gene delivery. Gene Ther. 4,409–418 (1997).

    CAS  Article  Google Scholar 

  2. 2

    Boussif, O. et al. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethyleneimine. Proc. Natl. Acad. Sci. USA 92, 7297–7301 (1995).

    CAS  Article  Google Scholar 

  3. 3

    Nielsen, P.E., Egholm, M., Koch, T., Christensen, J.B. & Buchardt, O. Photolytic cleavage of DNA by nitrobenzamido ligands linked to 9-aminoacridines gives DNA polymerase substrates in a wavelength-dependent reaction. Bioconjug. Chem. 2, 57–66 (1991).

    CAS  Article  Google Scholar 

  4. 4

    Bukrinsky, M.I. & Haffar, O.K. HIV-1 nuclear import: matrix protein is back on center stage, this time together with Vpr. Mol. Med. 4, 138–143 (1998).

    CAS  Article  Google Scholar 

  5. 5

    Knudsen, H. & Nielsen, P.E. Antisense properties of duplex- and triplex-forming PNAs. Nucleic Acids Res. 24, 494–500 (1996).

    CAS  Article  Google Scholar 

  6. 6

    Sebestyen, M.G. et al. DNA vector chemistry: the covalent attachment of signal peptides to plasmid DNA. Nat. Biotechnol. 16, 80–85 (1998).

    CAS  Article  Google Scholar 

  7. 7

    Yoneda, Y. et al. A long synthetic peptide containing a nuclear localization signal and its flanking sequences of SV40 T-antigen directs the transport of IgM into the nucleus efficiently. Exp. Cell. Res. 201, 313–320 (1992).

    CAS  Article  Google Scholar 

  8. 8

    Zanta, M.A., Belguise-Valladier, P. & Behr, J. Gene delivery: a single nuclear localization signal peptide is sufficient to carry DNA to the cell nucleus. Proc. Natl. Acad. Sci. USA 96, 91–96 (1999).

    CAS  Article  Google Scholar 

  9. 9

    Mujumdar, R.B., Ernst, L.A., Mujumdar, S.R., Lewis, C.J. & Waggoner, A.S. Cyanine dye labeling reagents: sulfoindocyanine succinimidyl esters. Bioconjug. Chem. 4, 105–111 (1993).

    CAS  Article  Google Scholar 

  10. 10

    Miyamoto, Y. et al. Differential modes of nuclear localization signal (NLS) recognition by three distinct classes of NLS receptors. J. Biol. Chem. 272, 26375–26381 (1997).

    CAS  Article  Google Scholar 

  11. 11

    Michaud, N. & Goldfarb, D.S. Most nuclear proteins are imported by a single pathway. Exp. Cell Res. 208, 128–136 (1993).

    CAS  Article  Google Scholar 

  12. 12

    Xiao, C.Y., Hubner, S. & Jans, D.A. SV40 large tumor antigen nuclear import is regulated by the double-stranded DNA-dependent protein kinase site (serine 120) flanking the nuclear localization sequence. J. Biol. Chem. 272, 22191–22198 (1997).

    CAS  Article  Google Scholar 

  13. 13

    http://www.pbio.com/cat/synth/pna/pnacycle.htm. PerSeptive Biosystems, Inc., Peptide Nucleic Acid (PNA) Synthesis: Custom PNA Synthesis.

  14. 14

    Mobility shift assay using low-ionic-strength PAGE. Short protocols in molecular biology 2nd edn (eds Ausubel, F.M. et al.). 12–5-12–6. 2nd ed. Greene Publishing Associates and John Wiley and Sons, NY. (1992).

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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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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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