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  • Nonviral Transfer Technology
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Supramolecular structure and nuclear targeting efficiency determine the enhancement of transfection by modified polylysines

Gene Therapy volume 7, pages 1690–1697 (2000)Cite this article

Abstract

Polylysine (pLy) has been used as a DNA carrier in nonviral gene delivery systems because it forms complexes with plasmid DNA via charge interaction, and condenses it into a compact structure. We have recently shown that cross-linking nuclear localization sequences (NLSs) to pLy can enhance transfection by conferring specific recognition by the cellular nuclear import ‘receptor’, the NLS-binding importin α/β heterodimer. The present study examines and correlates for the first time the effect of the lysine/nucleotide (Ly/Nu) ratio on transfection, recognition by importin α/β, and structure as determined using electron microscopy (EM) and atomic force microscopy (AFM), for pLy–DNA complexes with and without NLSs or mutant versions thereof. Intriguingly, we observed two distinct peaks of transfection enhancement at Ly/Nu ratios of 0.4 and 4.0, attributable to specific NLS recognition by importins and DNA compaction, respectively. The results indicate a clear correlation between the pLy–DNA structure, importin α/β recognition, and gene transfer efficiency, thus underlining the importance of using pLy–DNA at the optimal Ly/Nu ratio.

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References

  1. Wagner E, Cotten M, Foisner R, Birnstiel ML . Transferrin-polycation–DNA complexes: the effect of polycations on the structure of the complex and DNA delivery to cells Proc Natl Acad Sci USA 1991 88: 4255–4259

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Wu GY, Wu CH . Receptor mediated gene delivery and expression in vivo J Biol Chem 1988 263: 14621–14624

    CAS  PubMed  Google Scholar 

  3. Hart SL et al. Gene delivery and expression mediated by an integrin-binding peptide Gene Therapy 1995 2: 552–554

    CAS  PubMed  Google Scholar 

  4. Rosenkranz AA et al. Receptor-mediated endocytosis and nuclear transport of a transfecting DNA construct Exp Cell Res 1992 199: 323–329

    Article  CAS  PubMed  Google Scholar 

  5. Ferkol T, Perales JC, Mularo F, Hanson RW . Receptor-mediated gene transfer into macrophages Proc Natl Acad Sci USA 1996 93: 101–105

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Sosnowski BA et al. Targeting DNA to cells with basic fibroblast growth factor (FGF2) J Biol Chem 1996 271: 33647–33653

    Article  CAS  PubMed  Google Scholar 

  7. Baeza I et al. Electron microscopy and biochemical properties of polyamine-compacted DNA Biochemistry 1987 26: 6387–6392

    Article  CAS  PubMed  Google Scholar 

  8. Perales JC et al. Gene transfer in vivo: sustained expression and regulation of genes introduced into the liver by receptor-targeted uptake Proc Natl Acad Sci USA 1994 91: 4086–4090

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Vitiello L et al. Condensation of plasmid DNA with polylysine improves liposome-mediated gene transfer into established and primary muscle cells Gene Therapy 1996 3: 396–404

    CAS  PubMed  Google Scholar 

  10. Perales JC et al. Biochemical and functional characterization of DNA complexes capable of targeting genes to hepatocytes via the asialoglycoprotein receptor J Biol Chem 1997 272: 7398–7407

    Article  CAS  PubMed  Google Scholar 

  11. Ziady AG et al. Chain length of the polylysine in receptor-targeted gene transfer complexes affects duration of reporter gene expression both in vitro and in vivo J Biol Chem 1999 274: 4908–4916

    Article  CAS  PubMed  Google Scholar 

  12. Chan CK, Jans DA . Enhancement of polylysine-mediated transferrinfection by nuclear localization sequences: polylysine does not function as a nuclear localization sequence Hum Gene Ther 1999 10: 1695–1702

    Article  CAS  PubMed  Google Scholar 

  13. Southern PJ, Berg P . Transformation of mammalian cells to antibiotic resistance with a bacteria gene under control of the SV40 early region promoter J Mol App Gen 1982 1: 327–341

    CAS  Google Scholar 

  14. Hall CV, Jacob PE, Ringold GM, Lee F . Expression and regulation of Escherichia coli lacZ1 gene fusions in mammalian cells J Mol Appl Genet 1983 2: 101–109

    CAS  PubMed  Google Scholar 

  15. Hubner S, Xiao CY, Jans DA . The protein kinase CK2 site (Ser111/112) enhances recognition of the simian virus 40 large T-antigen nuclear localization sequence by importin J Biol Chem 1997 272: 17191–17195

    Article  CAS  PubMed  Google Scholar 

  16. Bustamante C et al. Circular DNA molecules imaged in air by scanning force microscopy Biochemistry 1992 31: 22–26

    Article  CAS  PubMed  Google Scholar 

  17. Hansma HG et al. DNA condensation for gene therapy as monitored by atomic force microscopy Nucleic Acids Res 1998 26: 2481–2487

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Feldherr CM, Akin D . EM visualization of nucleocytoplasmic transport processes Electron Microsc Rev 1990 3: 73–86

    Article  CAS  PubMed  Google Scholar 

  19. Feldherr CM, Akin D . The location of the transport gate in the nuclear pore complex J Cell Sci 1997 110: 3065–3070

    CAS  PubMed  Google Scholar 

  20. Page RL et al. Transgenesis in mice by cytoplasmic injection of polylysine/DNA mixtures Transgenic Res 1995 4: 353–360

    Article  CAS  PubMed  Google Scholar 

  21. Pollard H et al. Polyethylenimine but not cationic lipids promotes transgene delivery to the nucleus in mammalian cells J Biol Chem 1998 273: 7507–7511

    Article  CAS  PubMed  Google Scholar 

  22. Goldberg MW, Allen TD . High resolution scanning electron microscopy of the nuclear envelope: demonstration of a new, regular, fibrous lattice attached to the baskets of the nucleoplasmic face of the nuclear pores J Cell Biol 1992 119: 1429–1440

    Article  CAS  PubMed  Google Scholar 

  23. Akey CW . Visualization of transport-related configurations of the nuclear pore transporter Biophys J 1990 58: 341–355

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Wagner E et al. Transferrin–polycation conjugates as carriers for DNA uptake into cells Proc Natl Acad Sci USA 1990 87: 3410–3414

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Ma C, Bloomfield VA . Condensation of supercoiled DNA induced by MnCl2 Biophys J 1994 67: 1678–1681

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Okayama H, Chen C . In M Ej (ed) Methods in Molecular Biology, Vol 7: Gene Transfer and Expression protocols Humana Press: Clifton, NJ 1991 pp 15–21

    Chapter  Google Scholar 

  27. Chan CK, Hubner S, Hu W, Jans DA . Mutual exclusivity of DNA binding and nuclear localization signal recognition by the yeast transcription factor GAL4: implications for nonviral DNA delivery Gene Therapy 1998 5: 1204–1212

    Article  CAS  PubMed  Google Scholar 

  28. Xiao CY, Hubner S, Jans DA . 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 1997 272: 22191–22198

    Article  CAS  PubMed  Google Scholar 

  29. Jans DA et al. p34cdc2-mediated phosphorylation at T124 inhibits nuclear import of SV40 T-antigen proteins J Cell Biol 1991 115: 1203–1212

    Article  CAS  PubMed  Google Scholar 

  30. Xiao CY et al. A consensus cAMP-dependent protein kinase (PK-A) site in place of the CcN motif caesin kinase II site of Simian virus 40 large T-antigen confers PK-A mediated regulation of nuclear import J Biol Chem 1996 271: 6451–6457

    Article  CAS  PubMed  Google Scholar 

  31. Tyler JM, Branton D . Rotary shadowing of extended molecules dried from glycerol J Ultrastruct Res 1980 71: 95–102

    Article  CAS  PubMed  Google Scholar 

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Authors and Affiliations

  1. Division of Biochemistry and Molecular Biology, Nuclear Signalling Laboratory, John Curtin School of Medical Research, Australia

    C-K Chan

  2. Research School of Physical Sciences and Engineering, Australian National University, Canberra, Australia

    T Senden

  3. Division of Biochemistry and Molecular Biology, Nuclear Signalling Laboratory, John Curtin School of Medical Research, Australian National University, Canberra, 0200, Australia

    D A Jans

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  1. C-K Chan
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  2. T Senden
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  3. D A Jans
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Chan, CK., Senden, T. & Jans, D. Supramolecular structure and nuclear targeting efficiency determine the enhancement of transfection by modified polylysines. Gene Ther 7, 1690–1697 (2000). https://doi.org/10.1038/sj.gt.3301275

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  • DOI: https://doi.org/10.1038/sj.gt.3301275

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