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A novel transfecting peptide comprising a tetrameric nuclear localization sequence

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Abstract

The transport of exogenous DNA into the nucleus of eukaryotic cells is a prerequisite for successful gene delivery. To favor nuclear transport we synthesized a tetramer of the nuclear localization signal (NLS) of the SV40 large T-antigen as a novel nonviral gene delivery vector. This 4.4-kDa lysine-rich peptide (NLSV404) binds and compacts DNA by electrostatic interaction and forms stable polyplexes. Apart from its sequence-specific potency to mediate nuclear accumulation of conjugated albumin, NLSV404 also displays properties of nuclear transport for plasmid DNA as confirmed by fluorescence in situ hybridization. Further, NLSV404 polyplexes are shown to efficiently transfect various cell lines such as 16HBE14o–, HeLa S6, and Cos7 cells. NLSV404 polyplexes displayed at least 20-fold higher transfection rates than analogous polyplexes formed by the nuclear transport-deficient mutant sequence cNLS. Using growth-arrested cells, NLSV404 complexes were at least 100-fold more efficient than cNLS complexes. Combination of NLSV404 peptide but not of cNLS peptide with preformed polyethylenimine and dendrimer DNA complexes resulted in a strong increase in transfection efficiency. Incubation of cells prior to transfection with NLSV404 polyplexes with excess free peptide NLSV404 but not with cNLS resulted in a dose-dependent dramatic decrease in the transfection rate, suggesting a sequence-specific competitive inhibition. These results indicate that NLSV404 mediates nuclear accumulation of transfected plasmid DNA and that it can be a highly useful component of nonviral gene vectors.

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Abbreviations

BSA-BODIPY :

Fluorescence-labeled bovine serum albumin

EGFP :

Enhanced green fluorescent protein

FISH :

Fluorescent in situ hybridization

NLS :

Nuclear localization signal

PEI :

Polyethylenimine

SV40 :

Simian virus 40

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Acknowledgements

We thank the Förderverein für mukoviszidosekranke Kinder und Jugendliche der Region Ulm e.V. for financial support to purchase a fluorescence microscope. This work was supported by grants from the Bundesministerium für Bildung und Forschung, Bonn, Germany (01 GE9617/9, 01 GE0002, and 01 KV9554/1) and by the Deutsche Forschungsgemeinschaft (RO994/2–1).

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Authors and Affiliations

  1. Division of Molecular Pulmonology, Department of Pediatrics, Ludwig-Maximilians-Universität, Lindwurmstrasse 4, 80336, Munich, Germany

    Wolfgang Ritter, James Lausier, Carsten Rudolph, Dietrich Reinhardt & Joseph Rosenecker

  2. Institute of Experimental Oncology, Technische Universität, Munich, Germany

    Christian Plank

  3. Institute of Anthropology and Human Genetics, Ludwig-Maximilians-Universität, Munich, Germany

    Daniela Zink

Authors
  1. Wolfgang Ritter
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  2. Christian Plank
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  3. James Lausier
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  4. Carsten Rudolph
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  5. Daniela Zink
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  6. Dietrich Reinhardt
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  7. Joseph Rosenecker
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Correspondence to Joseph Rosenecker.

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Ritter, W., Plank, C., Lausier, J. et al. A novel transfecting peptide comprising a tetrameric nuclear localization sequence. J Mol Med 81, 708–717 (2003). https://doi.org/10.1007/s00109-003-0483-2

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  • DOI: https://doi.org/10.1007/s00109-003-0483-2

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